Sirex noctilio F., a Eurasian horntail woodwasp recently introduced into North America, oviposits in pines and other conifers and in the process spreads a phytopathogenic fungus that serves as a food source for its larvae. During oviposition the woodwasp also deposits a mucus produced in its acid (venom) gland that alters pine defense responses and facilitates infection by the fungus. A 26,496-feature loblolly pine cDNA microarray was used to survey gene expression of pine tissue responding to S. noctilio venom. Six genes were selected for further assessment by qRT-PCR, including one that encoded an apparent PR-4 protein and another that encoded a thaumatin-like protein. Expression of both was strongly induced in response to venom, while expression of an apparent actin gene (ACT1) was stable in response to the venom. The pattern of gene response was similar in Pinus taeda L. and P. radiata D. Don, but the magnitude of response in P. radiata was significantly stronger for each of the induced genes. The magnitude of biomarker gene response to venom also varied according to genotype within these two species. The qRT-PCR assay was used to demonstrate that the primary bioactive component in S. noctilio venom is a polypeptide.

We are generating a new mouse model for breast cancer heterogenaity using lentiviral infection to integrate the sporatically transforming EF1α-PyMT10C or Muc-PyMT10C lentivirus into mouse mammary epithelial cell genomes. We then transplant those cells into cleared mammary fat pads of recipient mice, allowing tumors to develop from luminal , myoepithelial, stem and progenitor cell lineages. We developed a wide variety of tumors including rare histologies such as squamous, tubular, spindloid and lipid rich. We used microarray analysis to compare our mouse model with a microarray analysis of 9 established mouse models (Herschkowitz, J.I. et al. Genome Biology, 2007). Heirarchal clustering was used to establish the molecular subtype of tumors developed through the lentiviral-PyMT mouse model. In addition, micrarray analysis was used in conjunction with GeneSifter and GO ontology to identify unique pathways for each of the rare tumor types.

To explore the mechanism underlying antioxidant activity of extracts from black soybean sprouts 0.5 cm long, Agilent-016772 G. max (Soybean) Oligo Microarray 4x44K was used to compare mRNA expression between the black soybean sprouts 0.5 cm long (n=4) and the black soybean sprouts 5 cm long (n=4). GO term enrichment analysis showed ten up-regulated genes (BE823689.1_567, GMFL01-02-F14-R_381, GMFL01-03-G22-R_364, GMFL01-14-M12-R_553, GMFL01-51-M23-R_265, AW757007.1_297, AW761420.1_260, BI788389.1_501, BQ273202.1_332 and GMFL01-10-I14-F_701) in the 0.5 cm seedlings were associated with response to oxidative stress. qRT-PCR assay confirmed the up-regulation of these ten genes in sprouts 0.5 cm long. In conclusion, these ten genes may contribute to antioxidant activity of sprout extract.

To better understanding the genetic and physiological changes behind the dormancy process in tree peony, we performed customized cDNA microarray to investigate gene expression profiling in tree peony ‘Feng Dan Bai’ buds during chilling induced dormancy release. Endo-dormant tree peony plants were exposed to 0-4°C from 5 November to 30 December 2009 in Qingdao, Shandong, China. Buds were collected after 0 d, 6 d, 12 d, 15 d, 18 d and 24 d chilling endured. DNA microarrays were customized using Agilent eArray 5.0 program, containing spots with 14,957 gene-specific 60-mer oligonucleotides representing 14,957 non abundant ESTs obtained from 454 sequencing normalized cDNA of tree peony buds during chilling duration (TSA, 65,217). Total 3,174 significantly differentially-expressed genes (P<0.05) were observed through endo-dormancy release, and the number of up-regulated (1,611) and that of down-regulated (1,563) was almost same. Expression of differentially-expressed genes associated with GA biosynthesis and signaling, cell growth and development was confirmed by quantitative RT-PCR, which displayed similar trends pattern in expression.

Cytokines are implicated in the development of inflammatory diseases such as endometriosis. This project was designed to test the hypothesis that specific cytokines that are secreted by macrophages, such as tumor necrosis factor α (TNFα) and interleukin 1 beta (IL1β), cause gene expression changes in endometrial stromal cells. Telomerase-immortalized human endometrial stromal cells (T-HESC) were treated with TNFα (5ng/ml) ± IL1β (1ng/ml). DNA microarray and real time RT-PCR were used to study the gene expression changes in T-HESC cells. Two hundred and nineteen genes featuring in various gene ontologies were found to be differentially expressed in T-HESC cells treated with TNFα ± ILIβ. The gene ontologies included functions expected to be associated with the development of endometriosis such as peptidases, cell adhesion, cell death/apoptosis, cell cycle, growth factors, cytoskeletal organization, defense/immune system, signal transduction, and transcriptional regulation. The differential expression of 4 genes (interleukin 8 (IL8), interleukin 6 (IL6), IL1β and matrix metalloproteinase (MMP3) was confirmed by real time RT-PCR. All four genes were up-regulated in response to TNFα ± ILIβ in T-HESC cells. The effect of TNFα ± ILIβ on migration and invasion of T-HESC cells, as measured with Boyden chambers, was not affected by treatment with these cytokines.

By unbiased deep sequencing, we identified a novel, highly divergent polyomavirus, provisionally named MX polyomavirus (MXPyV), in stool samples from children. From Mexico, 12 samples (out of 96) were positive for MxPyV by MXPyV-specific PCR. We used the ViroChip microarray and PCR to screen these 12 samples for co-infection with common diarrheal viruses. Six of 12 MxPyV-positive diarrheal samples tested negative by the ViroChip and PCR, and the other 6 samples were positive for at least one known diarrheal virus.

Reactive astrocytes are typically studied in models that cause irreversible mechanical damage to axons, neuronal cell bodies, and glia. We evaluated the response of astrocytes in the optic nerve head to a subtle injury induced by a brief, mild elevation of the intraocular pressure. Astrocytes demonstrated reactive remodeling showing hypertrophy, process retraction and simplification of their shape. We used microarray to indentify differentially expressed genes and to investigate the molecular mechanisms of astrogliosis in response to this subtle injury.

Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle endpoints such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive endpoints has caused transcriptomics studies to be performed at low doses during short exposures, separately and independently from traditional ecotoxicity tests, making comparisons with life cycle endpoints indirect. Therefore we aimed to directly compare the effects on growth, survival and gene expression of the non-biting midge Chironomus riparius. To this purpose, we analyzed simultaneously life cycle and transcriptomics responses of chironomid larvae exposed to four model toxicants. We observed that already at the lowest test concentrations many transcripts were significantly differentially expressed, while the life cycle endpoints of C. riparius were hardly affected. Analysis of the differentially expressed transcripts showed that at the lowest test concentrations substantial and biologically relevant cellular stress was induced and that many transcripts responded already maximally at these lowest test concentrations. The direct comparison between molecular en life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle endpoints, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment. Cellular stress responses are frequently presumed to be more sensitive than traditional ecotoxicological life cycle endpoints such as survival and growth. Yet, the focus to reduce test duration and to generate more sensitive endpoints has caused transcriptomics studies to be performed at low doses during short exposures, separately and independently from traditional ecotoxicity tests, making comparisons with life cycle endpoints indirect. Therefore we aimed to directly compare the effects on growth, survival and gene expression of the non-biting midge Chironomus riparius. To this purpose, we analyzed simultaneously life cycle and transcriptomics responses of chironomid larvae exposed to four model toxicants. We observed that already at the lowest test concentrations many transcripts were significantly differentially expressed, while the life cycle endpoints of C. riparius were hardly affected. Analysis of the differentially expressed transcripts showed that at the lowest test concentrations substantial and biologically relevant cellular stress was induced and that many transcripts responded already maximally at these lowest test concentrations. The direct comparison between molecular en life cycle responses after fourteen days of exposure revealed that gene expression is more sensitive to toxicant exposure than life cycle endpoints, underlining the potential of transcriptomics for ecotoxicity testing and environmental risk assessment.

Introduction: Diagnosis of severe influenza pneumonia remains challenging because of the lack of correlation between presence of influenza virus and patient’s clinical status. We conducted gene expression profiling in the whole blood of critically ill patients to identify a gene signature that would allow clinicians to distinguish influenza infection from other causes of severe respiratory failure (e.g. bacterial pneumonia, non-infective systemic inflammatory response syndrome). Methods: Whole blood samples were collected from critically ill individuals and assayed on Illumina HT-12 gene expression beadarrays. Differentially expressed genes were determined by linear mixed model analysis and over-represented biological pathways determined using GeneGo MetaCore. Results: The gene expression profile of H1N1 influenza A pneumonia was distinctly different from bacterial pneumonia and systemic inflammatory response syndrome. The influenza gene expression profile is characterized by up-regulation of genes from cell cycle regulation, apoptosis and DNA-damage response pathways. In contrast, no distinctive gene-expression signature was found in patients with bacterial pneumonia or systemic inflammatory response syndrome. The gene expression profile of influenza infection persisted through five days of follow-up. Furthermore, in patients with primary H1N1 influenza A infection who subsequently developed bacterial co-infection, the influenza gene-expression signature remained unaltered, despite the presence of a super-imposed bacterial infection. Conclusions: The whole blood expression profiling data indicates that the host response to influenza pneumonia is distinctly different from that caused by bacterial pathogens. This information may speed up identification of the cause of infection in patients presenting with severe respiratory failure, allowing appropriate patient care to be undertaken more rapidly.

The objective of this study was analysis of the whole saliva transcriptome to search for biomarkers of psychosocial stressor exposure and substance use in young adults drawn from a population-based longitudinal cohort, the Oregon Youth Substance Use Project. We conducted genome-wide gene expression analysis on whole saliva RNA from 48 individuals stratified by psychosocial stressor exposure using an Affymetrix Gene ST 1.0 array. We applied Weighted Gene Co-expression Network Analysis (WCGNA) to characterize the high-level structure in the data and to relate expression patterns among samples to participant clinical characteristics. This approach finds clusters of correlated genes (modules) which can reflect clinical, histological, or intracellular organization and function. Using WCGNA, we constructed a gene expression network from saliva genome-wide gene expression data, which we found to be similar to a network we constructed from publicly available cell-free saliva genome-wide gene expression data. Functional characterization of the WGCNA modules suggested that the samples varied in composition between an expression profile similar to circulating antigen presenting cell types and a glycoprotein-containing expression profile. We identified a significant relation between one module of whole saliva genome-wide gene expression and smoking behavior, and one with psychosocial stressor exposure. The smoking-related module was related to mitochondrial gene expression, and ever-smokers were found to have reduced overall mitochondrial gene expression. To determine whether reduced expression could be explained by reduction in mitochondrial DNA, we measured mtDNA copy number variation within genomic DNA from 400 OYSUP participants. Surprisingly, we found that more frequent smoking is associated with increased mtDNA copy number. The network structure of whole saliva and its association with clinically assessed exposures and behaviors are reported here for the first time.

modENCODE_submission_2555 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

modENCODE_submission_2725 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

In colorectal cancer, increased expression of the CXC chemokine receptor 4 (CXCR4) has been shown to provoke metastatic disease due to the interaction with its ligand stromal cell-derived factor 1 (SDF-1). Recently, a second SDF-1 receptor, CXCR7, was found to enhance tumor growth in solid tumors. Albeit signaling cascades via SDF-1/CXCR4 have been intensively studied, the significance of the SDF-1/CXCR7-induced intracellular communication triggering malignancy is still only marginally understood. In tumor tissue of 52 colorectal cancer (CRC) patients, we observed that expression of CXCR7 and CXCR4 increased with tumor stage, tumor size, and lymph node infiltration. Asking whether activation of CXCR4 or CXCR7 might result in a similar expression pattern, we performed microarray expression analyses using lentivirally CXCR4- and/or CXCR7-overexpressing SW480 colon cancer cell lines with and without stimulation by SDF-1α. Gene regulation via SDF-1α/CXCR4 and SDF-1α/CXCR7 was completely different and partly antidromic. Expressions of the differentially expressed genes AKR1C3, AXL, EGFR, IGFBP7, IL24, TNNC1, TRIP6 were confirmed by qPCR. Differentially regulated genes were assigned by GO to migration and lipid metabolic processes. Furthermore, using the in silico gene set enrichment analysis we showed for the first time that expressions of miR-217 and miR-218 were increased in CXCR4 and reduced in CXCR7 cells after stimulation with SDF-1α. As expected, their putative target mRNAs were inversely expressed. Functional assays exerted that exposure to SDF-1α resulted in strongly amplified invasiveness and chemosensitivity of CXCR4-expressing cells. CXCR7 overexpression led to reduced invasiveness which could only be marginally increased by SDF-1α. The CXCR4 antagonist plerixafor significantly reduced invasiveness of CXCR4-overexpressing cells only. Similarly, compared to control cells, CXCR4 cells showed increased sensitivity against 5-FU, while CXCR7 cells were more chemoresistant. These opposing results for CXCR4- or CXCR7-overexpressing colon carcinoma cells demand an unexpected attention in the clinical application of chemokine receptor antagonists like Plerixafor.

Background: Synovial sarcoma (SS) occur in children as well as in adults, although metastatic events are much more common in the latter. Whereas the importance of the t(X;18) translocation in SS oncogenesis is well established, the genetic basis of SS metastasis is still poorly understood. We recently reported expression (CINSARC) and genomic (GI) prognostic signatures related to chromosome integrity in sarcomas and gastrointestinal stromal tumors (GISTs). Here we investigate whether these signatures can also predict outcomes in SS. Methods: One hundred primary untreated SS were selected for expression and genomic profiling in a training/validation approach. Results: CINSARC and GI have strong, independent and validated prognostic values (p<0.0001). Comparing expression profiles of tumors with or without metastasis, 14 genes common with the CINSARC signature were identified and the two top-ranked genes, KIF14 and CDCA2, were validated as prognostic markers in an independent cohort. Comparing genomic profiles of adult vs. pediatric SS, we show that metastasis is associated with genome complexity in both situations and that the adult genome is more frequently rearranged. Accordingly, pediatric patients with an even genomic profile do not develop metastasis. Conclusions: Metastasis development in SS is strongly associated with chromosome complexity, and CINSARC and GI are validated independent prognostic factors. The differences in metastasis frequency between adults and children are associated with genome instability, which is much more frequent in adults. GI is potentially the best overall biomarker, and clearly the most clinically relevant, considering that genome profiling from formalin fixed samples is already used in pathology.

Background: Human papillomavirus has been shown to have a causal role in the development of head and neck squamous cell carcinoma and represents a distinct and well-defined pathology. While HPV-positive HNSCC is associated with a better response to treatment and prognosis, a subset of patients do not respond favorably to current standard of care thus suffering unnecessary morbidity and delay to receive effective therapy. Methods: RNA from nineteen patients with HPV-positive HNSCC was subjected to gene expression analysis using Affymetrix microarrays. HPV-status was confirmed by detection of HPV16 E7 with RT-PCR. Results: In addition to specific genetic biomarkers (including LCE3D, KRTDAP, HMOX1, KRT19, MDK, TSPAN1), differentially expressed genes were highly represented in the cell processes of genomic stability, cell cycle, and DNA damage. Conclusions: This pilot study suggests possible biomarkers that predict response to chemoradiation therapy. These data can potentially lead to an assay that can be used clinically to predict HPV-positive HNSCC patients that will not benefit from chemoradiation, thus helping clinicians to lower morbidity and get selected patients to surgery faster.

Background: Synovial sarcoma (SS) occur in children as well as in adults, although metastatic events are much more common in the latter. Whereas the importance of the t(X;18) translocation in SS oncogenesis is well established, the genetic basis of SS metastasis is still poorly understood. We recently reported expression (CINSARC) and genomic (GI) prognostic signatures related to chromosome integrity in sarcomas and gastrointestinal stromal tumors (GISTs). Here we investigate whether these signatures can also predict outcomes in SS. Methods: One hundred primary untreated SS were selected for expression and genomic profiling in a training/validation approach. Results: CINSARC and GI have strong, independent and validated prognostic values (p<0.0001). Comparing expression profiles of tumors with or without metastasis, 14 genes common with the CINSARC signature were identified and the two top-ranked genes, KIF14 and CDCA2, were validated as prognostic markers in an independent cohort. Comparing genomic profiles of adult vs. pediatric SS, we show that metastasis is associated with genome complexity in both situations and that the adult genome is more frequently rearranged. Accordingly, pediatric patients with an even genomic profile do not develop metastasis. Conclusions: Metastasis development in SS is strongly associated with chromosome complexity, and CINSARC and GI are validated independent prognostic factors. The differences in metastasis frequency between adults and children are associated with genome instability, which is much more frequent in adults. GI is potentially the best overall biomarker, and clearly the most clinically relevant, considering that genome profiling from formalin fixed samples is already used in pathology.

Because PML-RARA-positive acute promyelocytic leukemia (APL) is a morphologically differentiated leukemia, much speculation has been made about whether its leukemic cell of origin might be committed myeloid precursor (e.g., a promyelocyte) vs. a hematopoietic stem/progenitor cell (HSPC). We originally targeted PML-RARA expression with CTSG regulatory elements, based on the early observation that this gene was maximally expressed in cells with promyelocyte morphology. Here, we show that both Ctsg and PML-RARA targeted to the Ctsg locus (in Ctsg-PML-RARA mice) are detected in the purified KLS cells of these mice (Kit+Lin-Sca+ cells, which are highly enriched for HSPCs), and this expression results in biological effects in multi-lineage competitive repopulation assays. Although PML-RARA is indeed expressed at high levels in the promyelocytes of Ctsg-PML-RARA mice, it does not significantly alter the transcriptional signature of these cells, or induce their self-renewal. In sum, these results suggest that in murine models, PML-RARA acts primarily to affect the function of multi-potent progenitor cells, rather than promyelocytes. Since PML/Pml is normally expressed in the HSPCs of both humans and mice, and since some human APL samples contain TCR rearrangements and express T lineage genes, we suggest that the very early hematopoietic expression of PML-RARA in our mouse model may closely mimic the physiologic expression pattern of PML-RARA in human APL patients.

The purpose of this study was to explore diurnal gene expression changes that occur in the RPE/Choroid/Sclera. Mice C57BL/6J were purchased from the Jackson Laboratory (Bar Harbor, ME) and raised to approximately 2 months of age and entrained to a 12hr/12hr light/dark cycle for two weeks. Eye cups from male mice were rapidly dissected and RPE/ choroid/sclera tissues were collected over three consecutive diurnal cycles at Zeitgeber time (ZT) 0.5, 1, 1.5, 4, 11, 13, 16, and 23 hrs, for a total of 24 time points. Three mice were used for each time point and dissections for dark time points were done under dim-red light. To generate a reference RNA for microarray hybridization, whole eyes from equal numbers of male and female mice were collected at ZT6 and ZT7. Time: Samples were collected at the indicated Zeitgeber time points

Chromosome rearrangements in small apes are up to 20 times more frequent than in most mammals. Because of their complexity, the full extent of chromosome evolution in these hominoids is not yet fully documented. However, previous work with array painting, BAC-FISH and selective sequencing in two of the four karyomorphs, has shown that high resolution methods can precisely define chromosome breakpoints and map the complex flow of evolutionary chromosome rearrangements. Here we use these tools to precisely define the rearrangements that have occurred in the remaining two karyomorphs, genera Symphalangus (2n=50), and Hoolock (2n=38). This research provides the most comprehensive insight into the evolutionary origins of chromosome rearrangements involved in transforming small apes genome. Bioinformatics analyses of the human-gibbon synteny breakpoints revealed association with transposable elements and segmental duplications providing some insight into the mechanisms that might have promoted rearrangements in small apes. In the near future, the comparison of gibbon genome sequences will provide novel insights to test hypotheses concerning the mechanisms of chromosome evolution. The precise definition of synteny block boundaries and orientation, chromosomal fusions, and centromere repositioning event presented here will facilitate genome sequence assembly for these close relatives of humans.

Prostate cancer (PCa) is the most common malignant carcinoma that develops in men in Western countries. Up to 30% of patients continue to suffer from disease progression following radical prostatectomy. Therefore, better prognostic markers and molecular targets for cancer treatment are needed. MicroRNA (miRNA) has the potential to be used as biomarkers and as a therapeutic target for the treatment of various cancers, including PCa. Here, to determine how miRNA is involved in PCa progression, we investigated the miRNA expression profiles of 3 PCa cell lines, namely PC3, DU145, and LNCaP, and 2 normal prostate cell lines, namely RWPE-1 and PrSc, using miRNA microarrays.

The understanding of molecular events occurring in Chlorella during heterotrophy to photoautotrophy transition as well as sudden light stress and glucose starvation, are still largely unknown. To well grasp its cellular metabolism, particularly the regulation of biosynthesis and degradation pathways of lipid, protein and carbohydrates, as well as the diverse trophic adaptation affecting carbon partitioning during heterotrophy to photoautotrophy transition process, we sequenced the transcriptome (RNA-seq) in six time points to discover how transcriptional changes in C. pyrenoidosa modulate metabolic flux trends leading to intracellular components dynamic reassortment.

Here we show that the phytochrome-less chlorophyte Chlamydomonas reinhardtii retains a functional pathway to synthesize the linear tetrapyrrole (bilin) precursor of the phytochrome chromophore. Reverse genetic, metabolic inactivation and bilin rescue experiments establish that this pathway is needed for heme iron acquisition and for the diurnal transition to phototrophic growth. RNA-Seq measurements reveal a bilin-dependent signaling network that is necessary for the heterotrophic to phototrophic transition. These results imply the presence of a novel bilin sensor pathway that may be widely distributed amongst oxygenic photosynthetic organisms.

Object: To study the difference of gene expression pattern of bone marrow mesenchymal stem cells (BMMSCs) between psoriatic patients, normal adults and aborted fetuses, and then to explore the influence of bone marrow mesenchymal stem cells to immune system. Methods: Bone marrow mononuclear cells (BMMNC) of 7 psoriatic patients, 4 healthy volunteers and 3 aborted fetuses were isolated and the BMMSCs were cultured using the adherent method. Gene expression of 14 samples was detected by gene microarray and the different expressed genes were analysised by SAM software. Results: 654 differentially expressed genes (66 up regulated, 588 down regulated) were detected between the psoriatic patients and normal adults, which were enriched in immune response, chemotaxis and cell adhesion etc. 2020 differentially expressed genes (888 up regulated, 1132 down regulated) were detected between the aborted fetuses and normal adults. These genes were enriched in cell cycle, cell division, immune response and MHC class II antigen etc. Conclusion: The gene expression pattern such as immune response, chemotaxis was aberrant in psoriatic BMMNCs, which was consistent with aborted fetuses in some immune related genes.

modENCODE_submission_747 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Streptococcus suis is an important zoonotic pathogen that can cause meningitis and sepsis in both pigs and humans. In this study,we evaluated the genetic difference of 40 Streptococcus suis strains belonging to various sequence types by comparative genomic hybridization to identify genes associated with the variation in pathogenicity using NimbleGen’s tilling microarray platform. Application of Comparative Phylogenomics to Identify Genetic Differences Relating to Pathogenicity of Streptococcus suis

modENCODE_submission_748 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

modENCODE_submission_750 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

modENCODE_submission_752 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

modENCODE_submission_754 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Hepatic fibrosis is a wound-healing response to chronic liver injury, which may result in cirrhosis and liver failure. The c-Jun N-terminal kinase-1 (JNK1) gene has been shown to be involved in liver fibrosis. Here, we aimed to investigate the molecular mechanism and identify the cell-type involved in mediating the JNK1-dependent effect on liver fibrogenesis Wild-type (WT), JNK1−/− and JNK1Δhepa (hepatocyte-specific deletion of JNK1) mice were subjected to bile duct ligation (BDL). Additionally, we performed bone marrow transplantations (BMT), isolated primary hepatic stellate cells (HSCs) and studied their activation in vitro. Serum markers of liver damage (liver transaminases, alkaline phosphatase and bilirubin) and liver histology revealed reduced injury in JNK1−/− compared to WT and JNK1Δhepa mice. Hepatocyte cell death and proliferation was reduced in JNK1−/− compared to WT and JNK1Δhepa. Parameters of liver fibrosis such as Sirius Red staining as well as Collagen IA1 and αSMA expression were down-regulated in JNK1−/− compared to WT and JNK1Δhepa livers, 4 weeks after BDL. To delineate the essential cell-type, we performed BMT of WT and JNK1-/- into JNK1-/- and WT mice, respectively. BMT experiments excluded bone marrow derived cells from having a major impact on the JNK1-dependent effect on fibrogenesis. Hence, we investigated primary HSCs from JNK1−/− livers showing reduced transdifferentiation compared with WT and JNK1Δhepa-derived HSCs. We conclude that JNK1 in HSCs plays a crucial role in hepatic fibrogenesis and thus represents a promising target for cell-directed treatment options for liver fibrosis.

modENCODE_submission_755 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

modENCODE_submission_757 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

DNA methylation is an epigenetic mark that silences transposable elements (TEs) and repeats. Whereas the establishment and maintenance of DNA methylation are relatively well understood, little is known on their dynamics and biological relevance in plant and animal innate immunity. Here, we show that some TEs are demethylated and transcriptionally reactivated during antibacterial defense in Arabidopsis. This effect is concomitant with the down-regulation of key transcriptional gene silencing factors as well as an active demethylation process. DNA demethylation restricts multiplication and vascular propagation of the bacterial pathogen Pseudomonas syringae in leaves and, accordingly, some immune-response genes, containing repeats in their promoters, are negatively regulated by DNA methylation. This study provides evidence that DNA demethylation is part of a plant-induced immune response, potentially acting to prime transcriptional activation of some defense genes linked to Tes/repeats. We have monitored the transcript changes in Arabidopsis plants treated with a flagellin-derived peptide.

modENCODE_submission_762 This submission comes from a modENCODE project of Eric Lai. For full list of modENCODE projects, see http://www.genome.gov/26524648 Project Goal: We plan to generate a comprehensive catalog of expressed and functional microRNAs, and generate biological evidence for their regulatory activity. We plan also to delineate the primary transcription units of microRNA genes. Finally, we plan to annotate other classes of non-miRNA expressed small RNAs, as least some of which may define novel classes of small RNA genes. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf

Purpose: The exosome plays major roles in RNA processing and surveillance but the in vivo target range and substrate acquisition mechanisms remain unclear. We applied an in vivo cross-linking technique coupled with deep sequencing (CRAC) that captures transcriptome-wide interactions between individual yeast exosome subunits and their targets in a living cell. Methods: We apply CRAC to HTP-tagged proteins (HTP: His6 – TEV cleavage site – two copies of the z-domain of Protein A): Two nucleases (Rrp44, Rrp6) and two structural subunits (Rrp41, Csl4) of the yeast exosome. At least two independent experiments were performed in each case and analyzed separately. We performed CRAC on wild-type (WT) Rrp44 and two catalytic mutants, rrp44-endo (D91N, E120Q, D171N, D198N) and rrp44-exo (D551N). We further developed CRAC using cleavable proteins (split-CRAC) to compare endonuclease and exonuclease targets of Rrp44. Plasmids designed for split-CRAC contain a PreScission protease cleavage site (PP) inserted between aa 241 and 242 in the RRP44 ORF to allow in vitro cleavage of purified protein, and a His6 tag to select the respective cleaved fragment. Results: Analysis of wild-type Rrp44 and catalytic mutants showed that both the CUT and SUT classes of noncoding RNA, snoRNAs and, most prominently, pre-tRNAs and other Pol III transcripts are targeted for oligoadenylation and exosome degradation. Unspliced pre-mRNAs were also identified as targets for Rrp44 and Rrp6. CRAC performed using cleavable proteins (split-CRAC) revealed that Rrp44 endonuclease and exonuclease activities cooperate on most substrates. Mapping oligoadenylated reads suggests that the endonuclease activity may release stalled exosome substrates. Rrp6 was preferentially associated with structured targets, which frequently did not associate with the core exosome. This indicates that substrates can follow multiple pathways to the nucleases. Conclusion: Our study represents the first transcriptome-wide map of substrates for the yeast exosome nuclease complex.

Twenty-four triple-negative breast cancer and 14 adjacent normal tissues were collected from breast cancer patients during surgeries at National Taiwan University Hospital (NTUH, Taipei, Taiwan). All triple-negative breast cancer samples were invasive ductal carcinomas (IDC) and were negative in immunohistochemical statuses of ER, PR, and HER2 receptors, as confirmed by professional pathologists. Treatment procedure of all patients followed the National Comprehensive Cancer Network (NCCN) guideline. All samples were neoadjuvant-free and were collected before systemic chemotherapy treatments. Written informed consent was obtained from all patients who participated in this study. Using human tissues for research in this study was approved by the institutional review board at NTUH. A novel set of 25-miRNA signature identified in this study was able to effectively distinguish between triple-negative breast cancer and adjacent normal tissues. Moreover, we documented the first evidence of seven polycistronic miRNA clusters preferentially harboring deregulated miRNA genes in triple-negative breast cancer.

The androgen receptor (AR) is a ligand-inducible transcription factor that mediates androgen action in target tissues. Upon ligand binding, the AR binds to thousands of genomic loci and activates a cell-type specific gene program. Prostate cancer growth and progression depend on androgen-induced AR signalling. Treatment of advanced prostate cancer through medical or surgical castration leads to initial response and durable remission, but resistance inevitably develops. In castration-resistant prostate cancer (CRPC), AR activity remains critical for tumor growth despite androgen deprivation. While previous studies have focused on ligand-dependent AR signalling, in this study we explore AR function under the androgen-deprived conditions characteristic of CRPC. Our data demonstrate that the AR persistently occupies a distinct set of genomic loci after androgen deprivation in CRPC. These androgen-independent AR occupied regions have constitutively open chromatin structures that lack the canonical androgen response element and are independent of FoxA1, a transcription factor involved in ligand-dependent AR targeting. Many AR binding events occur at proximal promoters, which can act as enhancers to augment transcriptional activities of other promoters through DNA looping. We further show that androgen-independent AR binding directs a distinct gene expression program in CRPC, which is necessary for the growth of CRPC after androgen withdrawal.

Piwi-interacting (pi) RNAs are a class of germline-expressed small RNAs that have been linked to epigenetic programming in metazoa. C. elegans piRNAs known as 21U-RNAs are defined by more than 15,000 genome-encoded species. To explore the origin of 21U-RNAs we employed methods to enrich the 5′ ends of Pol II transcripts. We show that a species of capped-short (cs) RNA is frequently expressed bidirectionally at Pol II loci in C. elegans. Interestingly, at annotated 21U-RNA loci, csRNAs originate precisely 2 nt upstream of the mature piRNA species suggesting that csRNAs are piRNA precursors. In addition, we show that csRNAs associated with TS sites genome-wide define a previously overlooked class of 21U-RNA loci, and nearly double the number of piRNA species available for genome surveillance. Our methods should be of general utility in TS site identification and 5′ anchored RNA-expression profiling.

We report the high-throughput profiling of H3K9ac and SRT1 in MH63 and SRT1 RNAi. By obtaining about 8 million pair-end reads from H3K9ac and 6 million pair-end reads from SRT1, we find that about 45% of rice genes are marked by H3K9ac in 11 day-old seedlings. There are 1824 genes may be bound directly by OsSRT1. OsSRT1 is a site-specific deacetylase involved in deacetylation of H3K9 over specific genes. Overlapping between increased H3K9ac peaks in the RNAi plants and that of OsSRT1-binding was found over 37 genes, suggesting that OsSRT1 directly targeted to these genes for H3K9 deacetylation. The observations that H3K9ac was enriched at the 5’ end of genes larger than 1.5 kb and OsSRT1 RNAi enhanced the enrichment at that position. However, the OsSRT1-binding was found to be enriched over gene bodies. While the meaning of this binding profile is not yet clear, it is speculated that OsSRT1 may contribute to maintain the low level of H3K9ac in the gene body. In addition, this work revealed that OsSRT1 is enriched over retrotransposons.

In this study we performed a systematic evaluation of functional miRNA-mRNA interactions associated with the aggressiveness of breast cancer cells using a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction, and functional assays. Analysis of the miRNA expression identified 11 miRNAs that were differentially expressed, including 7 down-regulated (miR-200c, miR-205, miR-203, miR-141, miR-34a, miR-183, and miR-375) and 4 up-regulated miRNAs (miR-146a, miR-138, miR-125b1 and miR-100), in aggressive cell lines when compared to normal and less aggressive cell lines. Transient overexpression of miR-200c, miR-205, and miR-375 in MDA-MB-231 cells led to the inhibition of cell migration and invasion. The integrated analysis of miRNA and mRNA expression identified 35 known and novel target genes of miR-200c, miR-205, and mir-375, including CFL2, LAMC1, TIMP2, ZEB1, CDH11, PRKCA, PTPRJ, PTPRM, LDHB, and SEC23A. Surprisingly, the majority of these genes (27 genes) were target genes of miR-200c, suggesting that it plays a more important role in regulating the aggressiveness of breast cancer cells. We characterized one of the target genes of miR-200c, CFL2, and demonstrated that CFL2 is overexpressed in aggressive breast cancer cell lines and can be significantly down-regulated by exogenous miR-200c. Tissue microarray analysis further revealed that CFL2 expression in primary breast cancer tissue correlated with tumor grade. To our knowledge, this study is the first systematic screening of functional miRNA target genes in aggressive breast cancer cells. The results obtained from this study may improve our understanding of the role of these candidate miRNAs and their target genes in relation to breast cancer aggressiveness and ultimately lead to the identification of novel biomarkers associated with prognosis.

In this study we performed a systematic evaluation of functional miRNA-mRNA interactions associated with the aggressiveness of breast cancer cells using a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction, and functional assays. Analysis of the miRNA expression identified 11 miRNAs that were differentially expressed, including 7 down-regulated (miR-200c, miR-205, miR-203, miR-141, miR-34a, miR-183, and miR-375) and 4 up-regulated miRNAs (miR-146a, miR-138, miR-125b1 and miR-100), in aggressive cell lines when compared to normal and less aggressive cell lines. Transient overexpression of miR-200c, miR-205, and miR-375 in MDA-MB-231 cells led to the inhibition of cell migration and invasion. The integrated analysis of miRNA and mRNA expression identified 35 known and novel target genes of miR-200c, miR-205, and mir-375, including CFL2, LAMC1, TIMP2, ZEB1, CDH11, PRKCA, PTPRJ, PTPRM, LDHB, and SEC23A. Surprisingly, the majority of these genes (27 genes) were target genes of miR-200c, suggesting that it plays a more important role in regulating the aggressiveness of breast cancer cells. We characterized one of the target genes of miR-200c, CFL2, and demonstrated that CFL2 is overexpressed in aggressive breast cancer cell lines and can be significantly down-regulated by exogenous miR-200c. Tissue microarray analysis further revealed that CFL2 expression in primary breast cancer tissue correlated with tumor grade. To our knowledge, this study is the first systematic screening of functional miRNA target genes in aggressive breast cancer cells. The results obtained from this study may improve our understanding of the role of these candidate miRNAs and their target genes in relation to breast cancer aggressiveness and ultimately lead to the identification of novel biomarkers associated with prognosis.

In this study we performed a systematic evaluation of functional miRNA-mRNA interactions associated with the aggressiveness of breast cancer cells using a combination of integrated miRNA and mRNA expression profiling, bioinformatics prediction, and functional assays. Analysis of the miRNA expression identified 11 miRNAs that were differentially expressed, including 7 down-regulated (miR-200c, miR-205, miR-203, miR-141, miR-34a, miR-183, and miR-375) and 4 up-regulated miRNAs (miR-146a, miR-138, miR-125b1 and miR-100), in aggressive cell lines when compared to normal and less aggressive cell lines. Transient overexpression of miR-200c, miR-205, and miR-375 in MDA-MB-231 cells led to the inhibition of cell migration and invasion. The integrated analysis of miRNA and mRNA expression identified 35 known and novel target genes of miR-200c, miR-205, and mir-375, including CFL2, LAMC1, TIMP2, ZEB1, CDH11, PRKCA, PTPRJ, PTPRM, LDHB, and SEC23A. Surprisingly, the majority of these genes (27 genes) were target genes of miR-200c, suggesting that it plays a more important role in regulating the aggressiveness of breast cancer cells. We characterized one of the target genes of miR-200c, CFL2, and demonstrated that CFL2 is overexpressed in aggressive breast cancer cell lines and can be significantly down-regulated by exogenous miR-200c. Tissue microarray analysis further revealed that CFL2 expression in primary breast cancer tissue correlated with tumor grade. To our knowledge, this study is the first systematic screening of functional miRNA target genes in aggressive breast cancer cells. The results obtained from this study may improve our understanding of the role of these candidate miRNAs and their target genes in relation to breast cancer aggressiveness and ultimately lead to the identification of novel biomarkers associated with prognosis.

Acidic tissue microenvironment is commonly found in a variety of pathophysiological conditions. GPR4 is a proton-sensing G protein-coupled receptor that is fully activated by acidic extracellular pH but has lesser activity at the physiological pH 7.4 and minimal activity at more alkaline pH. To determine the effects of GPR4 activation by acidosis on vascular endothelial cells, we examined the global gene expression of the acidosis response in primary human umbilical vein endothelial cells (HUVEC) with varying level of GPR4.

The CAMTA1 mutant and Col-0 were studied under water and drought condition. The camta1 showed stunted primary root growth under osmotic stress. The expression analysis revealed drought recovery as major indicative pathway along with membrane and chloroplast related protein in camta1 under drought stress. Large number of positively regulated genes were related to osmotic balance, transporters, AP2 and ABA. We used Affymetrix expression analysis to validate the role of CAMTA1 under drought stress.

The H2A variant H2AZ is essential for embryonic development and for proper execution of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions in H2AZ are likely key for its functional specialization, but we know little about how these differences contribute to chromatin regulation. Here, we show that the extended acidic patch, specifically the three divergent residues in the C-terminal docking domain, is necessary for lineage commitment during ESC differentiation and proper execution of gene expression programs during ESC differentiation. Surprisingly, disruption of the acidic patch domain has a distinct consequence on cellular specification compared to H2AZ depletion. This is consistent with differences in gene expression profiles of H2AZ –depleted and acidic patch (AP) mutant ESCs during early lineage commitment. Interestingly, the distinct consequence of AP mutant expression on gene regulation is coincidence with an altered destabilized chromatin state and high chromatin mobility dependent on active transcription. Collectively, our data shows that the divergent residues within the acidic patch domain are key structural determinants of H2AZ function and links chromatin structure and dynamics with gene regulation and cell fate specification.

The H2A variant H2AZ is essential for embryonic development and for proper execution of developmental gene expression programs in embryonic stem cells (ESCs). Divergent regions in H2AZ are likely key for its functional specialization, but we know little about how these differences contribute to chromatin regulation. Here, we show that the extended acidic patch, specifically the three divergent residues in the C-terminal docking domain, is necessary for lineage commitment during ESC differentiation and proper execution of gene expression programs during ESC differentiation. Surprisingly, disruption of the acidic patch domain has a distinct consequence on cellular specification compared to H2AZ depletion. This is consistent with differences in gene expression profiles of H2AZ –depleted and acidic patch (AP) mutant ESCs during early lineage commitment. Interestingly, the distinct consequence of AP mutant expression on gene regulation is coincidence with an altered destabilized chromatin state and high chromatin mobility dependent on active transcription. Collectively, our data shows that the divergent residues within the acidic patch domain are key structural determinants of H2AZ function and links chromatin structure and dynamics with gene regulation and cell fate specification.

This study investigates three radiation exposure scenarios in BALB/c and C57BL/6 mice: (1) low dose (LD) group — four weekly doses of 7.5 cGy, (2) high dose (HD) group — four weekly doses of 1.8 Gy, (3) unexposed group — four weekly sham exposures. We then used comparative expression profiles of the mouse mammary gland and cardiac blood to build a model of candidate tissue functions associated with LD cancer susceptibility in these strains and murine and human knowledgebases to characterize these tissue functions and their relevance to breast cancer.

T follicular helper (Tfh) cells play a pivotal role in germinal center reactions, which requires Bcl6 transcription factor. To analyze their relationships with other effector T cell lineages and their stability in vivo, we developed and analyzed a new Bcl6 reporter mouse alone or together with other lineage reporter systems. Assisted with genome-wide transcriptome analysis, we show substantial plasticity of T cell differentiation in the early phase of immune response. At this stage, CXCR5 appears to be expressed in a Bcl6-independent manner. Once Bcl6 is highly expressed, Tfh cells can persist in vivo and some of them develop into memory cells. Together, our results indicate Bcl6 as a bona fide marker for Tfh polarized program.

Hepcidin, a peptide hormone that decreases intestinal iron absorption and macrophage iron release, is a potential drug target for patients with iron overload syndromes because its levels are inappropriately low in these individuals. Endogenous stimulants of Hepcidin transcription include bone morphogenic protein 6 (BMP) and interleukin-6 (IL-6) via effects on Smad4 or Stat3, respectively. We conducted a small-scale chemical screen in zebrafish embryos to identify small molecules that modulate hepcidin expression. We found that treatment with the isoflavone genistein from 28−52 hours post-fertilization in zebrafish embryos enhanced Hepcidin transcript levels as assessed by whole mount in situ hybridization and quantitative realtime RT-PCR. Genistein’s stimulatory effect was conserved in human hepatocytes: genistein treatment of HepG2 cells increased both Hepcidin transcript levels and Hepcidin promoter activity. We found that genistein’s effect on Hepcidin expression did not depend on estrogen receptor signaling or increased cellular iron uptake, but was impaired by mutation of either the BMP response elements or the Stat3 binding site in the Hepcidin promoter. RNA-sequencing of transcripts from genistein-treated hepatocytes indicated that genistein upregulated 68% of the transcripts that were upregulated by BMP6, however genistein raised the levels of several transcripts involved in Stat3 signaling that were not upregulated by BMP6. Chromatin-immunoprecipitation and ELISA experiments revealed that genistein enhanced Stat3 binding to the Hepcidin promoter and increased phosphorylation of Stat3 in HepG2 cells. CONCLUSION: Genistein is the first small molecule experimental drug that stimulates Hepcidin expression in vivo and in vitro. These experiments demonstrate the feasibility of identifying and characterizing small molecules that increase Hepcidin expression. Genistein and other candidate molecules may subsequently be developed into new therapies for iron overload syndromes.

Purpose: To study the effects of drought at the transcriptomic level on two different actively dividing maize tissue: the ovaries, and the leaf meristem Methods: The Illumina reads were mapped to the maize B73 reference genome using Tophat followed by transcriptome reconstruction using Cufflinks. The FPKM valuse were extracted from cufflinks output and an R package called Limma was used to identify differentially expressed genes under drought under both tissues Results and Conclusions: Different processes which were differentially expressed under drought in both tissues were identified and analyzed in detail. A working hypothesis was formulated to account for the observed susceptibility of the reproductive tissue when compared to the robust response of the vegetative tissue. This analysis also servers as a basis for future study on drought-induced embryo abortion.

A small number of transcription factors, including Oct-3/4 and Sox2, constitute the transcriptional network that maintains pluripotency in embryonic stem (ES) cells. Previous reports suggested that some of these factors form a complex that binds the Oct-Sox element, a composite sequence consisting of closely juxtaposed Oct-3/4-binding and Sox2-binding sites. However, little is known regarding the components of the complex. In this study, we show that Sall4, a member of the Spalt-like family of proteins, directly interacts with Sox2 and Oct-3/4. Sall4 in combination with Sox2 or Oct-3/4 simultaneously occupies the Oct-Sox elements in mouse ES cells. Sall4 knockdown led to differentiation of ES cells. Overexpression of Sall4 in ES cells increased reporter activities in a luciferase assay when the Pou5f1- or Nanog-derived Oct-Sox element was included in the reporter. Microarray analyses revealed that Sall4 and Sox2 bound to the same genes in ES cells significantly more frequently than expected from random coincidence. These factors appeared to bind the promoter regions of a subset of the Sall4- and Sox2-double-positive genes in precisely similar distribution patterns along the promoter regions, suggesting that Sall4 and Sox2 associate with such Sall4/Sox2-overlapping genes as a complex. Importantly, gene ontology analyses indicated that the Sall4/Sox2-overlapping gene set is enriched for genes involved in maintaining pluripotency. Sall4/Sox2/Oct-3/4-triple-positive genes identified by referring to a previous study identifying Oct-3/4-bound genes in ES cells were further enriched for pluripotency genes than Sall4/Sox2-double-positive genes. These results demonstrate that Sall4 contributes to the transcriptional network operating in pluripotent cells, together with Oct-3/4 and Sox2.

Open chromatin regions have been shown to associate with the location of transcriptiotal enhancers, i.e., the binding locations of DNA-binding transcription factors. To investigate the effects of short-term treatment by the nuclear hormone 1α,25-dihydroxyvitamin D3 (VD), a specific ligand of the transcription factor vitamin D receptor, on chromatin accessibility, FAIRE-seq was utilized on the chromatin samples from THP-1 monocytic leukemia cells that were treated with 100 nM 1α,25-dihydroxyvitamin D3 for 20, 40, 60, 80, 100 and 120 min, or with vehicle (0.1% (v/v) ethanol) for 20 and 100 min.

In Oxytricha, the somatic genome is responsible for vegetative growth, while the germline contributes DNA to the next sexual generation. Somatic nuclear development eliminates all transposons and other so-called “junk DNA”, which constitute ~95% of the germline. We demonstrate that Piwi-interacting small RNAs (piRNAs) from the maternal nucleus can specify genomic regions for retention in this process. Oxytricha piRNAs map primarily to the somatic genome, representing the ~5% of the germline that is retained. Furthermore, injection of synthetic piRNAs corresponding to normally-deleted regions leads to their retention in subsequent generations. Our findings highlight small RNAs (sRNAs) as powerful transgenerational carriers of epigenetic information for genome programming. The backcross study here shows that the mating between an IES+ strain with the wild-type stain produces corresponding IES-containing sRNAs at 19 hr, and we provided the mapping to and the sequences of the specific loci of interest in the submission. As a control, wild-type cells do not produce such IES-containing sRNAs, and this analysis can be pulled out from the GSE35018 study since we provided mapping to the whole genome. The purpose of the 20 hr total sRNA sequencing study here is to show that the class of 27 nt sRNA is the major species of total sRNAs in Oxytricha at 20 hr, which we sequenced previously from Otiwi1-associated sRNAs at 12, 19, 23, and 30 hr (GSE35018). In addition, there is a less abundant class of small RNAs of 21-22 nt. These two classes are obvious by simply plotting the length distribution of the sRNA sequences.

Bile acids play multiple roles in vertebrate metabolism by facilitating lipid absorption in the intestine and acting as a signaling molecule in lipid and carbohydrate metabolism. Bile acids are also the main route to excrete excess cholesterol out of the body. Alpha-methyl-Coa racemase (Amacr) is one of the enzymes needed to produce bile acids from cholesterol. The mouse model lacking Amacr can produce only minor (less than 10%) amounts of bile acids, but still they are symptomless in normal laboratory conditions.

Chronic Otitis Media (OM) develops after sustained inflammation and is characterized by secretory middle ear epithelial metaplasia and effusion, most frequently mucoid. Non-typeable Haemophilus influenzae (NTHi), the most common acute OM pathogen, is known to activate inflammation and mucin expression in vitro and in animal models of OM. The goals of this study were to: examine expression profiling epithelial effects of NTHi challenge in murine middle ears. We used microarrays to detail examine the global programme of gene expression underlying epithelial effects of NTHi challenge in murine middle ears during this study.

Background: The 2009 pandemic H1N1 influenza virus emerged in swine and quickly became a major global health threat. In mouse, non-human primate, and swine infection models, the pH1N1 virus efficiently replicates in the lung and induces pro-inflammatory host responses; however, whether similar or different cellular pathways were impacted by pH1N1 virus across independent infection models remains to be further defined. To address this, we have performed a comparative transcriptomic analysis of acute host responses to a single pH1N1 influenza virus, A/California/04/2009 (CA04), in the lung of mice, macaques and swine. Results: Despite similarities in the clinical course, we observed differences in inflammatory molecules elicited, and the kinetics of their gene expression changes across all three species. The retinoid X receptor (RXR) signaling pathway controlling pro-inflammatory and metabolic processes was differentially regulated during infection in each species, though the heterodimeric RXR partner, pathway associated signaling molecules, and gene expression patterns differed in each species. Conclusions: By comparing transcriptional changes in the context of clinical and virological measures, we identified differences in the host transcriptional response to pH1N1 virus across independent models of acute infection. Antiviral resistance and the emergence of new influenza viruses have placed more focus on developing drugs that target the immune system. Underlying overt clinical disease are molecular events that suggest therapeutic targets identified in one host may not be appropriate in another.

Background: The 2009 pandemic H1N1 influenza virus emerged in swine and quickly became a major global health threat. In mouse, non-human primate, and swine infection models, the pH1N1 virus efficiently replicates in the lung and induces pro-inflammatory host responses; however, whether similar or different cellular pathways were impacted by pH1N1 virus across independent infection models remains to be further defined. To address this, we have performed a comparative transcriptomic analysis of acute host responses to a single pH1N1 influenza virus, A/California/04/2009 (CA04), in the lung of mice, macaques and swine. Results: Despite similarities in the clinical course, we observed differences in inflammatory molecules elicited, and the kinetics of their gene expression changes across all three species. The retinoid X receptor (RXR) signaling pathway controlling pro-inflammatory and metabolic processes was differentially regulated during infection in each species, though the heterodimeric RXR partner, pathway associated signaling molecules, and gene expression patterns differed in each species. Conclusions: By comparing transcriptional changes in the context of clinical and virological measures, we identified differences in the host transcriptional response to pH1N1 virus across independent models of acute infection. Antiviral resistance and the emergence of new influenza viruses have placed more focus on developing drugs that target the immune system. Underlying overt clinical disease are molecular events that suggest therapeutic targets identified in one host may not be appropriate in another.

Transcriptional profiling of BJAB cells expressing miR-K12-9 and BCBL cells treated with miR-K12-9 inhibitor. To identify host RNA targets of KSHV miRNAs, we took advantage of the observation that RNAs targeted by miRNAs often display small reductions in their steady-state levels, perhaps as a result of their impaired translation. Accordingly, we examined cellular transcript accumulation by array-based expression profiling under four sets of conditions in which KSHV miRNAs were expressed or inhibited. Cells transfected with negative control miRNA compared to miR-K12-9 or negative miRNA inhibitor compared to miR-K12-9 inhibitor.

The nuclear lamina (NL) interacts with hundreds of large genomic regions termed lamina-associated domains (LADs). The dynamics of these interactions and the relation to epigenetic modifications are poorly understood. We visualized the fate of LADs in single cells using a novel ‘molecular contact memory’ approach. In each interphase nucleus, only ~30% of LADs are positioned at the periphery; these LADs are in intermittent molecular contact with the NL but remain constrained to the periphery. Upon mitosis, LAD positioning is not detectably inherited but instead is stochastically reshuffled. Contact of individual LADs with the NL correlates with their degree of H3K9 dimethylation in single cells, and inactivation of the H3K9 methyltransferase G9a reduces the NL contact frequencies. These results indicate that nuclear positioning and histone modification of LADs are both stochastic yet linked in single cells. Collectively, these results highlight principles of the dynamic spatial architecture of chromosomes.

Genotype VIId NDV is characterized by severe tissue damage in chicken lymphoid organs compared to other virulent strains. However, biological basis of this unusual pathological phenotype is unknown. Host response is associated with pathogenicity of Newcastle Disease Virus (NDV). We aim to determine the contribution of host response to the severe tissue destruction in the lymphoid system caused by genotype VIId NDV. We used microarray analysis to evaluate the global transcriptional response in the spleen of chickens infected with genotype VIId NDV strain JS5/05 and genotype IV NDV Herts/33.

Cell-based models of many neurological and psychiatric diseases, established by reprogramming patient somatic cells into human induced pluripotent stem cells (hiPSCs), have now been reported. While numerous reports have demonstrated that neuronal cells differentiated from hiPSCs are electrophysiologically active mature neurons, the “age” of these cells relative to cells in the human brain remains unresolved. Comparisons of gene expression profiles of hiPSC-derived neural progenitor cells (NPCs) and neurons to the Allen BrainSpan Atlas indicate that hiPSC neural cells most resemble first trimester neural tissue. Consequently, we posit that hiPSC-derived neural cells may most accurately be used to model the early developmental defects that contribute to disease predisposition rather than the late features of the disease. Though the characteristic symptoms of schizophrenia (SCZD) generally appear late in adolescence, it is now thought to be a neurodevelopmental condition, often predated by a prodromal period that can appear in early childhood. Postmortem studies of SCZD brain tissue typically describe defects in mature neurons, such as reduced neuronal size and spine density in the prefrontal cortex and hippocampus, but abnormalities of neuronal organization, particularly in the cortex, have also been reported. We postulated that defects in cortical organization in SCZD might result from abnormal migration of neural cells. To test this hypothesis, we directly reprogrammed fibroblasts from SCZD patients into hiPSCs and subsequently differentiated these disorder-specific hiPSCs into NPCs. SCZD hiPSC differentiated into forebrain NPCs have altered expression of a number of cellular adhesion genes, reduced WNT signaling and aberrant cellular migration.

MLL-AF4 is a hallmark genomic aberration which arises prenatally in high-risk infant acute lymphoblastic leukemia (ALL). In human embryonic stem cells (hESCs), MLL-AF4 skewed hemato-endothelial specification but was not sufficient for transformation. Additional cooperating genetic insults seem required for MLL-AF4-mediated leukemogenesis. FLT3 is highly expressed in MLL-AF4+ ALL through activating mutations (FLT3-TKD or FLT3-ITD) or increased transcriptional expression, being therefore considered a potential cooperating event in MLL-AF4+ ALL. Here, we explored the developmental impact of FLT3 activation on its own or in cooperation with MLL-AF4 in the hematopoietic fate of hESCs. FLT3 activation did not impact specification of CD45-CD31+ hemogenic precursors but significantly enhanced the formation of CD45+CD34+ and CD45+ blood cells and blood progenitors with clonogenic potential. Importantly, FLT3 activation through FLT3 mutations or FLT3-WT overexpression completely abrogated hematopoietic differentiation from MLL-AF4-expressing hESCs, indicating that FLT3 activation cooperates with MLL-AF4 to inhibit human embryonic hematopoiesis. Cell cycle/apoptosis analyses suggest that FLT3 activation directly impacts hESC specification rather than selective proliferation/survival of hESC-emerging hematopoietic derivatives. Transcriptional profiling supported the limited impact of FLT3 activation on hESC specification towards CD45-hemogenic precursors and the enhanced hematopoiesis upon FLT3 activation, and inhibited hematopoiesis upon MLL-AF4 expression in FLT3-activated hESCs which was associated to large transcriptional changes and regulation of master early hematopoietic genes. Also, although FLT3 activation and MLL-AF4 cooperate to inhibit embryonic hematopoiesis the underlying molecular/genetic mechanisms differ depending on how FLT3 activation is achieved. Finally, FLT3 activation did not cooperate with MLL-AF4 to immortalize/transform hESC-derived hematopoietic cells.

We conducted RNA/RNA binding protein immunoprecipitation (RIP) followed by high throughput sequencing to identify mRNA targets of Brf1 (Zfp36l1) and Brf2 (Zfp36l2) in mouse embryonic stem cells. We collected over 190 million sequencing reads and identified many highly enriched protein coding mRNAs. Gene ontology analysis of enriched genes reveals that transcription factors and intercellular signaling proteins account for a large fraction of the targets, many of which are important for pluripotency and differentiation.

Serous borderline tumours (SBOT) are a challenging group of ovarian tumours positioned between benign and malignant disease. We have profiled the DNA methylomes of 12 low grade serous carcinoma (LGSC), 19 SBOT and 16 benign serous tumours (BST) across 27,578 CpG sites to further characterise the epigenomic relationship between these subtypes of ovarian tumours. Unsupervised hierarchical clustering of DNA methylation levels showed that LGSC differ distinctly from BST, however, not from SBOT. Gene ontology analysis of genes showing differential methylation at linked CpG sites between LGSC and BST revealed significant enrichment of gene groups associated with cell adhesion, cell-cell signalling and the extracellular region consistent with a more invasive phenotype of LGSC as compared to BST. Consensus clustering highlighted differences between SBOT methylomes and returned subgroups with malignant-like or benign-like methylation profiles. Furthermore, a two loci DNA methylation signature can distinguish between these SBOT subgroups with benign-like and malignant-like methylation characteristics. Our findings indicate striking similarities between SBOT and LGSC methylomes which supports a common origin and the view that LGSC may arise from SBOT. A subgroup of SBOT can be classified into tumours with a benign-like or a malignant-like methylation profile which may help in identifying tumours more likely to progress into LGSC.

To investigate Pten function in neonatal developing brain, we conditionally inactivated Pten in neural stem/progenitor cells at birth using a Nestin-CreER transgenic driver. Pten inactivation created a novel perivascular proliferative niche in the cerebellum that did not progress to malignancy during the lifespan of the mouse. Co-deletion of Pten and Trp53 synergized to cause fully penetrant medulloblastoma originating from a perivascular niche. The Pten and Trp53 double knock-out medulloblastomas showed an extensive and abnormal blood vessel network and advanced neuronal differentiation of tumor cells compared to medulloblastomas arising in Nestin-creER;Trp53fl/fl mice, suggesting that Pten loss promoted angiogenesis and neuronal differentiation in medulloblastoma. EdU pulse-chase experiments demonstrated a lineage hierarchy of the double knock-out medulloblastomas consistent with a perivascular cancer stem cell population. The Pten and Trp53 double knock-out medulloblastomas showed somatic loss of chromosomes 7, 13 and 16, and inactivating mutations in the tumor suppressor gene Ptch1. Gene expression profiles showed that this model recapitulated the subgroup of human medulloblastomas with de-regulated SHH signaling.

The use of alternative polyadenylation sites is common and affects the post-transcriptional fate of mRNA, including its stability, localization, and translation. Here we present a method for genome-wide and strand-specific mapping of poly(A) sites and quantification of RNA levels at unprecedented efficiency by using an on-cluster dark T-fill procedure on the Illumina sequencing platform. Our method outperforms former protocols in quality and throughput, and reveals new insights into polyadenylation in Saccharomyces cerevisiae.

The nuclear lamina (NL) interacts with hundreds of large genomic regions termed lamina-associated domains (LADs). The dynamics of these interactions and the relation to epigenetic modifications are poorly understood. We visualized the fate of LADs in single cells using a novel ‘molecular contact memory’ approach. In each interphase nucleus, only ~30% of LADs are positioned at the periphery; these LADs are in intermittent molecular contact with the NL but remain constrained to the periphery. Upon mitosis, LAD positioning is not detectably inherited but instead is stochastically reshuffled. Contact of individual LADs with the NL correlates with their degree of H3K9 dimethylation in single cells, and inactivation of the H3K9 methyltransferase G9a reduces the NL contact frequencies. These results indicate that nuclear positioning and histone modification of LADs are both stochastic yet linked in single cells. Collectively, these results highlight principles of the dynamic spatial architecture of chromosomes.

Recurrent hepatitis C virus (rHCV) is universal post-liver transplantation (LT), with accelerated fibrosis rates compared to non-transplanted patients. rHCV is associated with increased mortality and morbidity post-transplant and is a leading indication for re-transplantation. We hypothesized that miRNA expression profiles from liver grafts can distinguish severity of HCV recurrence and differentiate this from acute cellular rejection (ACR). Methods Using microarrays, we characterized global microRNA (miRNA) expression from patients with slow HCV fibrosis progression (F<2 Ishak), fast HCV fibrosis progression (F≥2 Ishak), ACR and non-HCV transplanted patients. Selected miRNA were analysed by quantitative PCR (qPCR) using both liver tissue and serum samples. Results We demonstrated changes in miRNA expression in patients with slow HCV fibrosis progression that were anti-fibrogenic, anti-angiogenic and anti-inflammatory in comparison to patients with fast HCV fibrosis progression. miRNA-146a, miRNA-19a, miRNA- 20a and miRNA-let-7e expression were increased in the slow HCV fibrosis progression group. In addition, comparison of patients with fast HCV progression against patients with ACR identified pro-fibrogenic pathways. qPCR analysis on liver tissue and serum confirmed the up-regulation of miRNAs in the slow HCV fibrosis progression group. Conclusion We demonstrate specific miRNA expression signatures that distinguish rate of progression of HCV recurrence and ACR post –liver transplantation. Pathway analysis indicates that specific miRNA may play a regulatory role in these processes. The miRNAs identified may act as potential biomarkers for HCV recurrence post-LT and help distinguish between ACR and recurrent HCV.

Background: The ZNF217 gene, encoding a C2H2 zinc finger protein, is located at 20q13 and found amplified and overexpressed in greater than 20% of breast tumors. Current studies indicate ZNF217 drives tumorigenesis, yet the regulatory mechanisms of ZNF217 are largely unknown. Because ZNF217 associates with chromatin modifying enzymes, we postulate that ZNF217 functions to regulate specific gene signaling networks. Here, we present a large-scale functional genomic analysis of ZNF217, which provides insights into the regulatory role of ZNF217 in MCF7 breast cancer cells. Results: ChIP-seq analysis reveals that the majority of ZNF217 binding sites are located at distal regulatory regions associated with the chromatin marks H3K27ac and H3K4me1. Analysis of ChIPseq transcription factor binding sites shows clustering of ZNF217 with FOXA1, GATA3 and ERalpha binding sites, supported by the enrichment of corresponding motifs for the ERalpha-associated cisregulatory sequences. ERalpha expression highly correlates with ZNF217 in lysates from breast tumors (n=15), and ERalpha co-precipitates ZNF217 and its binding partner CtBP2 from nuclear extracts. Transcriptome profiling following ZNF217 depletion identifies differentially expressed genes co-bound by ZNF217 and ERalpha; gene ontology suggests a role for ZNF217-ERalpha in expression programs associated with ER+ breast cancer studies found in the Molecular Signature Database. Data-mining of expression data from breast cancer patients correlates ZNF217 with reduced overall survival in multiple subtypes. Conclusions: Our genome-wide ZNF217 data suggests a functional role for ZNF217 at ERalpha target genes. Future studies will investigate whether ZNF217 expression contributes to aberrant ERalpha regulatory events in ER+ breast cancer and hormone resistance

Differences in global levels of histone acetylation occur in normal and cancer cells, although the reason cells regulate these levels has remained unclear. Here we demonstrate a role for histone acetylation in regulating intracellular pH (pHi). As pHi decreases, histones are globally deacetylated by histone deacetylases (HDACs) and the released acetate anions are co-exported with protons out of the cell by monocarboxylate transporters (MCTs), preventing further reductions in pHi. Conversely, global histone acetylation increases at more alkaline pHi, such as when resting cells are induced to proliferate. Inhibition of HDACs or MCTs decreases acetate export and lowers pHi, particularly compromising pHi maintenance in acidic environments. Global deacetylation at low pH is reflected at a genomic level by decreased abundance and extensive redistribution of acetylation at promoters and intergenic regions. Thus acetylation of chromatin functions as a rheostat to regulate pHi with important implications for therapeutic use of HDAC inhibitors.

Pathogenic germline mutations in BRCA1 or BRCA2 are detected in less than one third of families with a strong history of breast cancer. It is therefore expected that mutations still remain undetected by currently used screening methods. In addition, a growing number of BRCA1/2 sequence variants of unclear pathogen significance are found in the families, constituting an increasing clinical challenge. New methods are therefore needed to improve the detection rate and aid the interpretation of the clinically uncertain variants. In this study we analyzed a series of 33 BRCA1, 22 BRCA2, and 128 sporadic tumors by RNA profiling to investigate the classification potential of RNA profiles to predict BRCA1/2 mutation status. We found that breast tumors from BRCA1 and BRCA2 mutation carriers display characteristic RNA expression patterns, allowing them to be distinguished from sporadic tumors. The majority of BRCA1 tumors were basal-like while BRCA2 tumors were mainly luminal B. Using RNA profiles, we were able to distinguish BRCA1 tumors from sporadic tumors among basal-like tumors with 83% accuracy and BRCA2 from sporadic tumors among luminal B tumors with 89% accuracy. Furthermore, subtype-specific BRCA1/2 gene signatures were successfully validated in two independent data sets with high accuracies. Although additional validation studies are required, indication of BRCA1/2 involvement (“BRCAness”) by RNA profiling could potentially be valuable as a tool for distinguishing pathogenic mutations from benign variants, for identification of undetected mutation carriers, and for selecting patients sensitive to new therapeutics such as PARP inhibitors.

Background. Pneumococcus is a major human pathogen and the polysaccharide capsule is considered its main virulence factor. Nevertheless, strains lacking a capsule, named non-typeable pneumococcus (NT), are maintained in nature and frequently colonise the human nasopharynx. Interest in these strains, not targeted by any of the currently available pneumococcal vaccines, has been rising as they seem to play an important role in the evolution of the species. Currently, there is a paucity of data regarding this group of pneumococci. Also, questions have been raised on whether they are true pneumococci. We aimed to obtain insights in the genetic content of NT and the mechanisms leading to non-typeability and to genetic diversity. Methods. A collection of 52 NT isolates representative of the lineages circulating in Portugal between 1997 and 2007, as determined by pulsed-field gel electrophoresis and multilocus sequence typing, was analysed. The capsular region was sequenced and comparative genomic hybridisation (CGH) using a microarray covering the genome of 10 pneumococcal strains was carried out. The presence of mobile elements was investigated as source of intraclonal variation. Results. NT circulating in Portugal were found to have similar capsular regions, of cps type NCC2, i.e., having aliB-like ORF1 and aliB-like ORF2 genes. The core genome of NT was essentially similar to that of encapsulated strains. Also, competence genes and most virulence genes were present. The few virulence genes absent in all NT were the capsular genes, type-I and type-II pili, choline-binding protein A (cbpA/pspC), and pneumococcal surface protein A (pspA). Intraclonal variation could not be entirely explained by the presence of prophages and other mobile elements. Conclusions. NT circulating in Portugal are a homogeneous group belonging to cps type NCC2. Our observations support the theory that they are bona-fide pneumococcal isolates that do not express the capsule but are otherwise essentially similar to encapsulated pneumococci. Thus we propose that NT should be routinely identified and reported in surveillance studies.

Diagnosis of inflamed human orbit tissue with standard clinical and histopathology evaluation data is imprecise. A large number of these patients are diagnosed with the catch-all classification of nonspecific orbital inflammation (NSOI). We utilized gene expression analysis of orbit biopsies to assist in the classification of sarcoidosis, granulomatosis with polyangiitis (GPA), thyroid eye disease (TED), IgG4-associated disease, and subdivisions of NSOI. As part of this process, we are investigating correlations between gene expression levels and disease characteristics.

mTOR regulates mRNA translation. Whereas ribosome-profiling suggested that mTOR exclusively stimulates translation of TOP (containing a 5’-terminal oligopyrimidine [5’TOP] motif) and TOP-like mRNAs, polysome-profiling implied that mTOR also modulates translation of non-TOP mRNAs. We show that ribosome-, but not polysome-profiling, is biased towards identification of TOP mRNAs as differentially translated while obscuring detection of changes in non-TOP mRNA translation. Transcription start site profiling by Nano-Cap Analysis of Gene Expression (nanoCAGE) revealed that many mTOR-sensitive mRNAs do not have 5’TOP motifs. Moreover, nanoCAGE showed that 5’ UTR features distinguish two functionally and translationally distinct subsets of mTOR-sensitive mRNAs: i) those with short 5’ UTRs enriched for mitochondrial functions such as respiration, that are translated in an eIF4E, but not eIF4A1-dependent manner and ii) mRNAs encoding proliferation- and survival-promoting proteins, that harbor long 5’ UTRs, and require both eIF4E and eIF4A1 for their efficient translation. Selective inhibition of translation of mRNAs harboring long 5’ UTRs via suppression of eIF4A leads to uncoupling of expression of proteins involved in respiration (e.g. ATP5O) from those protecting mitochondrial integrity (e.g. BCL-2) ultimately resulting in apoptosis. Conversely, simultaneous translational downregulation of both long and short 5’ UTR mRNAs by mTOR inhibitors results in suppression of mitochondrial respiration and predominantly cytostatic effects. Therefore, 5’ UTR features define differential modes of translation of functionally distinct mTOR-sensitive mRNAs, which explains discrepancies between the effects of mTOR and eIF4A inhibitors on neoplastic cells.

Previous results suggest that Bmh might inhibit the activity of the transcription factor Adr1 after binding to Adr1-dependent promoters. In a strain lacking the two major histone deacetylases, Hda1 and Rpd3 (hdac∆), Adr1 is bound to its target promoters recruiting what appears to be an inactive RNA ploymerase II preinitiation complex (PIC). To determine whether Bmh activity inhibits this inactive PIC and the generality of this effect on glucose-repressed gene expression, the mRNA profiles of wild type, bmh mutant, hdac mutant, and bmh hdac mutant cells grown in high glucose medium were compared.

For assessing the cancer-causing potential for humans of a chemical compound, the conventional approach is the use of the 2-year rodent carcinogenicity bioassay, thus alternatives such as in vitro toxicogenomics are highly desired. In the present study, the transcriptomics responses following exposure to genotoxic (GTX) and non-genotoxic (NGTX) hepatocarcinogens and non-carcinogens (NC) in five liver-based in vitro models, namely conventional and epigenetically-stabilized cultures of primary rat hepatocytes, the human hepatoma-derived HepaRG and HepG2 cell lines and the human embryonic stem cell-derived hepatocyte-like cells hES-Heps are examined and compared.

Transposable elements (TEs) occupy large fraction of metazoan genomes and pose constant threat to genomic integrity. This threat is particularly critical in germ cells, as changes in the genome that are induced by TEs will be transmitted to the next generation. Small non-coding piwi interacting (pi)RNAs recognize and silence a diverse set of TEs in germ cells. In mice, piRNA-guided transposon repression correlates with establishment of CpG DNA methylation on their sequences, yet the mechanism and the spectrum of genomic targets of piRNA silencing are unknown. Here we show that in addition to DNA methylation, the piRNA pathway is required to maintain a high level of the repressive H3K9me3 histone modification on long interspersed nuclear elements (LINEs) in germ cells. piRNA-dependent chromatin repression targets exclusively full-length elements of actively transposing LINE families, demonstrating the remarkable ability of the piRNA pathway to recognize active elements among the large number of genomic transposon fragments.

In mammals, extracellular miRNAs circulate in biofluids as stable entities that are secreted by normal and diseased tissues, and can enter cells and regulate gene expression. Drosophila melanogaster is a proven system for the study human diseases. They have an open circulatory system in which hemolymph (HL) circulates in direct contact with all internal organs, in a manner analogous to vertebrate blood plasma. Here we show using deep sequencing that Drosophila HL contains RNase resistant, circulating miRNAs (HL-miRNAs). Limited subsets of body tissue miRNAs (BT-miRNAs) accumulated in HL, suggesting they may be specifically released from cells or particularly stable in HL. Alternatively, they might arise from specific cells such as hemocytes, in intimate contact with HL. Young and old flies accumulated unique populations HL-miRNAs, suggesting their accumulation is responsive to the physiological status of the fly. These HL-miRNAs may function in flies similarly to the miRNAs circulating in mammalian biofluids. The discovery of these HL-miRNAs will provide a new venue for health and disease-related research in Drosophila.

In mammals, extracellular miRNAs circulate in biofluids as stable entities that are secreted by normal and diseased tissues, and can enter cells and regulate gene expression. Drosophila melanogaster is a proven system for the study human diseases. They have an open circulatory system in which hemolymph (HL) circulates in direct contact with all internal organs, in a manner analogous to vertebrate blood plasma. Here we show using deep sequencing that Drosophila HL contains RNase resistant, circulating miRNAs (HL-miRNAs). Limited subsets of body tissue miRNAs (BT-miRNAs) accumulated in HL, suggesting they may be specifically released from cells or particularly stable in HL. Alternatively, they might arise from specific cells such as hemocytes, in intimate contact with HL. Young and old flies accumulated unique populations HL-miRNAs, suggesting their accumulation is responsive to the physiological status of the fly. These HL-miRNAs may function in flies similarly to the miRNAs circulating in mammalian biofluids. The discovery of these HL-miRNAs will provide a new venue for health and disease-related research in Drosophila.

The generation of naïve T lymphocytes is critical for immune function yet the mechanisms governing their maturation remain incompletely understood. We have identified a mouse mutant, bloto, that harbors a hypomorphic mutation in the zinc finger protein Zfp335. Mutant blt/blt mice exhibit a naïve T cell deficiency due to an intrinsic developmental defect that begins to manifest in the thymus and continues into the periphery, affecting T cells that have recently undergone thymic egress. Zfp335 binds to promoter regions via a consensus motif, and its target genes are enriched in categories related to protein metabolism, mitochondrial function and transcriptional regulation. Restoring the expression of one target, Ankle2, partially rescues T cell maturation. Our findings identify Zfp335 as a transcription factor and essential regulator of late-stage intrathymic and post-thymic T cell maturation.

The prevailing theory for the molecular basis of evolution involves genetic mutations that ultimately generate the heritable phenotypic variation on which natural selection acts. However, epigenetic transgenerational inheritance of phenotypic variation may also play an important role in evolutionary change. A growing number of studies have demonstrated the presence of epigenetic inheritance in a variety of different organisms that can persist for hundreds of generations. The possibility that epigenetic changes could accumulate over macroevolutionary time has been considered, but not yet seldom been tested empirically. The current study was designed to compare epigenetic changes among several closely related species of Darwin’s finches, a well-known example of adaptive radiation. Erythrocyte DNA was obtained from five species of sympatric Darwin’s finches that vary in phylogenetic relatedness. Genome wide alterations in genetic mutations using copy number variation (CNV) were compared to epigenetic alterations associated with differential DNA methylation regions (epimutations). Epimutations were more common than genetic CNV mutations among the five species; furthermore, the number of epimutations increased monotonically with phylogenetic distance. Interestingly, the number of genetic CNV mutations did not consistently increase with phylogenetic distance. The number, chromosomal locations, regional clustering, and lack of overlap of epimutations and genetic mutations suggests that epigenetic changes are distinct and that they correlate with the evolutionary history of Darwin’s finches. The potential functional significance of the epimutations was explored by comparing their locations on the genome to the location of evolutionarily important genes and cellular pathways in birds. Specific epimutations were associated with genes related to the bone morphogenic protein (BMP), toll receptor, and melanogenesis signaling pathways. Species- specific epimutations were significantly over-represented in these pathways. Since environmental factors are known to rapidly alter heritable changes in the epigenome, it is possible that epigenetic changes have played a contributing role in the molecular basis of the evolution of Darwin’s finches.

The liver x receptor (LXR) and peroxisome proliferator-activated receptor gamma (PPARG) nuclear receptor transcription factors (TFs) are master regulators of energy homeostasis that impact tumor cell metabolism and proliferation. To better define the underlying molecular processes governing the genetic control of cellular growth in response to extracellular metabolic signals, we performed a comprehensive, genome-wide characterization of the temporal regulatory cascades mediated by LXR and PPARG signaling in HT29 colorectal cancer cells. For this analysis we applied a multi-tiered approach that incorporated cellular phenotypic assays, gene expression profiles, chromatin state dynamics and nuclear receptor binding patterns. By integrating these orthogonal datasets over time, we uncovered the regulatory architecture of LXR and PPARG activity. Both sets of nuclear receptors inhibited cell proliferation and increased cellular oxidative stress, validating results from independent studies. Despite this metabolic reprogramming, the gene regulatory network programs initiated by these nuclear receptors were widely distinct. PPARG generated a rapid and short-term response while maintaining a gene activator role. By contrast, LXR signaling was prolonged, with initial, predominantly activating functions altering to repressive gene regulatory activities at late time points through RNA polymerase II (RNAP2) promoter pausing. This exhaustive dataset illustrates the complexity of genome function and structure by elucidating how common phenotypic outcomes are genetically encoded through diverse transcriptional programs. Our results further provide a detailed, molecular framework that can be applied to better understanding how extracellular nutrients impact cancer cell physiology and highlight the importance of study designs that incorporate complementary genomic features in a dynamic manner.

Substantial effort is currently devoted to identifying cancer-associated alterations using genomics. Here, we show that standard blood collection procedures rapidly change the transcriptional and post-transcriptional landscapes of hematopoietic cells, resulting in biased activation of specific biological pathways, up-regulation of pseudogenes, antisense RNAs, and unannotated coding isoforms, and RNA surveillance inhibition. Affected genes include common mutational targets and thousands of other genes participating in processes such as chromatin modification, RNA splicing, T and B cell activation, and NF-κB signaling. The majority of published leukemic transcriptomes exhibit signals of this incubation-induced dysregulation, explaining up to 40% of differences in gene expression and alternative splicing between leukemias and reference normal transcriptomes. The effects of sample processing are particularly evident in pan-cancer analyses. We provide biomarkers that detect prolonged incubation of individual samples, and show that keeping blood on ice markedly reduces changes to the transcriptome. In addition to highlighting the potentially confounding effects of technical artifacts in cancer genomics data, our study emphasizes the need to survey the diversity of normal as well as neoplastic cells when characterizing tumors. This study is complemented by GSE61410: transcriptomic profiling of bone marrow cells from healthy individuals.

This SuperSeries is composed of the SubSeries listed below.

We report the effect on genome-wide gene expression after deletion of an enhancer region downstream of Sox2 in F1 ES cells. The Sox2 transcription factor must be robustly transcribed in embryonic stem (ES) cells to maintain pluripotency.  Reporter assays reveal novel enhancers, including two enhancers over 100 kb downstream (SRR107 and SRR111) which, through the formation of chromatin loops, localise to the Sox2 promoter in ES cells.  Using CRISPR/Cas9 we deleted a region containing these two enhancers, which we term the Sox2 control region (SCR). This deletion revealed that the SCR is required for Sox2 transcription in ES cells.  Furthermore, homozygous deletion of this distal Sox2 control region (SCR) caused significant reduction in Sox2 mRNA and protein levels, loss of ES cell colony morphology, genome-wide changes in gene expression and impaired neuroectodermal formation upon spontaneous differentiation to embryoid bodies. Together these data identify a distal control region essential for Sox2 transcription in ES cells.

Aberrant activation of the Hedgehog (Hh) signaling pathway is implicated in the pathogenesis of many cancers, including medulloblastoma and basal cell carcinoma (BCC). In this study, using neonatally irradiated Ptch1+/- mice as a model of Hh-dependent tumors, we investigated the in vivo effects of MK-4101, a novel SMO antagonist, for treatment of medulloblastoma and BCC. Results clearly demonstrate a robust antitumor activity of MK-4101, achieved through the inhibition of proliferation and induction of extensive apoptosis in tumor cells. Of note, beside antitumor activity on transplanted tumors, MK-4101 was highly efficacious against primary medulloblastoma and BCC developing in the cerebellum and skin of Ptch1+/- mice. By identifying the changes induced by MK-4101 in gene expression profiles in tumors, we also elucidate the mechanism of action of this novel, orally administrable compound. MK-4101 targets the Hh pathway in the tumor cells, showing the maximum inhibitory effect on Gli1 activity. MK-4101 also induced deregulation of cell cycle and block of DNA replication in tumors. Members of the IGF and Wnt signaling pathways , were among the most highly deregulated genes by MK-4101, suggesting that the interplay among Hh, IGF and Wnt is crucial in Hh-dependent tumorigenesis. Altogether, the results of this preclinical study support a therapeutic opportunity for MK-4101 in the treatment of Hh-driven cancers, also providing useful information for combination therapy with drugs targeting pathways cooperating with Hh oncogenic activity.

The Malaria Host-Pathogen Interaction Center (MaHPIC) is a transdisciplinary malaria systems biology research program initially supported by an NIH/NIAID contract (# HHSN272201200031C, 2012-2017; see http://www.systemsbiology.emory.edu). The MaHPIC continues with ongoing support from the Defense Advanced Research Project Agency (DARPA) and others. The MaHPIC generates many data types (e.g., clinical, hematological, parasitological, metabolomics, functional genomics, lipidomics, proteomics, immune response, telemetry) and mathematical models, to iteratively test and develop hypotheses related to the complex host-parasite dynamics in the course of malaria in non-human primates (NHPs), and metabolomics data via collaborations with investigators conducting clinical studies in malaria endemic countries, with the overarching goal of better understanding human disease, pathogenesis, and immunity. Curation and maintenance of all data and metadata are the responsibility of the MaHPIC:  Mary Galinski [email protected] (MaHPIC Program Director), Jessica Kissinger [email protected] (MaHPIC Co-Program Director), and Alberto Moreno [email protected] (MaHPIC Co-Program Director).

The largest and most diverse class of eukaryotic transcription factors contain Cys2-His2 zinc fingers (C2H2-ZFs), each of which typically binds a DNA nucleotide triplet within a larger binding site. Frequent recombination and diversification of their DNA-contacting residues suggests that these zinc fingers play a prevalent role in adaptive evolution. Very little is known about the function and evolution of the vast majority of C2H2-ZFs, including whether they even bind DNA. We determined in vivo binding sites of 39 human C2H2-ZF proteins, and correlated them with potential functions for these proteins.

Entamoeba histolytica is a protozoan parasite responsible for amebiasis, a disease which is characterized by acute inflammation of the colon. Adaptation of the parasite to toxic level of nitric oxide (NO) produced by phagocytes may be essential for the establishment of chronic amebiasis and for the survival of parasite within the host. To obtain insight into the mechanism of adaptation to NO, E. histolytica trophozoites were selected in vitro by stepwise exposures to increasing amounts of NO donor S-Nitrosoglutathione (GSNO) up to a concentration of 110 µM. These NO adapted trophozoites (NAT) were more resistance to acute exposure of GSNO (350μM), to activated macrophages and have a better capability to invade porcine colon explants compared to wild-type trophozoites. The transcriptome of NAT was investigated by RNA-sequencing (RNA-seq) and it showed only a weak overlapping with the transcriptome of trophozoites exposed to acute nitrosative stress (TEANS).

Background: Clinical characteristics of ovarian clear cell adenocarcinoma (CCC) include 1) higher incidence among Japanese, 2) association with endometriosis, 3) poor prognosis in advanced stage, 4) higher incidence of thrombosis as complication. We applied high resolution comparative genomic hybridization (CGH) array to screen somatic copy number alterations (SCNAs) associated with these clinical characteristics. Methods: We conducted a prospective cohort study. DNA obtained from tumors was assayed by array comparative genomic hybridization using Agilent Whole Human Genome 244K.

We report gene expression profiling in the fission yeast Schizosaccharomyces pombe. We performed high-throughput sequencing of RNA isolated from wild-type, ago1d, mmi1d and dcr1d strains.

Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7kb lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high mRNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a suite of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25 nucleotide “TINCR box” motif which is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyze TINCR binding capacity to ~9,400 human recombinant proteins revealed direct binding of TINCR RNA to the Staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay (SMD), however, lacked differentiation impacts. Instead, the TINCR/STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through inducible lncRNA binding to differentiation mRNAs to ensure their expression.

Several of the thousands of human long non-coding RNAs (lncRNAs) have been functionally characterized; however, potential roles for lncRNAs in somatic tissue differentiation remain poorly understood. Here we show that a 3.7kb lncRNA, terminal differentiation-induced ncRNA (TINCR), controls human epidermal differentiation by a post-transcriptional mechanism. TINCR is required for high mRNA abundance of key differentiation genes, many of which are mutated in human skin diseases, including FLG, LOR, ALOXE3, ALOX12B, ABCA12, CASP14 and ELOVL3. TINCR-deficient epidermis lacked terminal differentiation ultrastructure, including keratohyalin granules and intact lamellar bodies. Genome-scale RNA interactome analysis revealed that TINCR interacts with a suite of differentiation mRNAs. TINCR-mRNA interaction occurs through a 25 nucleotide “TINCR box” motif which is strongly enriched in interacting mRNAs and required for TINCR binding. A high-throughput screen to analyze TINCR binding capacity to ~9,400 human recombinant proteins revealed direct binding of TINCR RNA to the Staufen1 (STAU1) protein. STAU1-deficient tissue recapitulated the impaired differentiation seen with TINCR depletion. Loss of UPF1 and UPF2, both of which are required for STAU1-mediated RNA decay (SMD), however, lacked differentiation impacts. Instead, the TINCR/STAU1 complex seems to mediate stabilization of differentiation mRNAs, such as KRT80. These data identify TINCR as a key lncRNA required for somatic tissue differentiation, which occurs through inducible lncRNA binding to differentiation mRNAs to ensure their expression.

This SuperSeries is composed of the SubSeries listed below.

The subcellular localization and translation of messenger RNA (mRNA) supports functional differentiation between cellular compartments. In neuronal dendrites, local translation of mRNA provides a rapid and specific mechanism for synaptic plasticity and memory formation, and might be involved in the pathophysiology of certain brain disorders. Despite the importance of dendritic mRNA translation, little is known about which mRNAs can be translated in dendrites in vivo and when their translation occurs. Here we collect ribosome-bound mRNA from the dendrites of CA1 pyramidal neurons in the adult mouse hippocampus. We find that dendritic mRNA rapidly associates with ribosomes following a novel experience consisting of a contextual fear conditioning trial. High throughput RNA sequencing followed by machine learning classification reveals an unexpected breadth of ribosome-bound dendritic mRNAs, including mRNAs expected to be entirely somatic. Our findings are in agreement with a mechanism of synaptic plasticity that engages the acute local translation of functionally diverse dendritic mRNAs.

Bile acids play multiple roles in vertebrate metabolism by facilitating lipid absorption in the intestine and acting as a signaling molecule in lipid and carbohydrate metabolism. Bile acids are also the main route to excrete excess cholesterol out of the body. Alpha-methyl-Coa racemase (Amacr) is one of the enzymes needed to produce bile acids from cholesterol. The mouse model lacking Amacr can produce only minor (less than 10%) amounts of bile acids, but still they are symptomless in normal laboratory conditions.

In this study, we generated a zebrafish model of DBA with RPL5 morphants and implemented high-throughput RNA-seq and miRNA-seq to identify key genes, lncRNAs, and miRNAs during zebrafish development and hematopoiesis. We found that RPL5 is required for both primitive and definitive hematopoiesis processes that are partially mediated by the P53 pathway. Several genes such as cirh1a, noc2l, tars, and nol6 and miRNAs such as dre-miR-10a*, dre-miR-722, dre-miR-737, and dre-miR-142a-3p were significantly deregulated, and these changes may play a crucial role in hematopoiesis, ribosome biogenesis and development process. We also characterized the lncRNome in zebrafish with RPL5 deficiency. By constructing a comprehensive regulatory network, we identified central node genes in the network connected to the P53 pathway, almost all of which were targeted by the significantly deregulated miRNAs listed above. Our results therefore establish a regulatory network for critical genes and miRNAs involved in the RPL5-deficient zebrafish model and provide a comprehensive basis for the molecular pathogenesis of RPL5-mediated DBA and other ribosomopathies.

Overexpression of the transcription factor SRY-related box 2 (SOX2) is characteristic of Glioblastoma (GBM), yet its regulatory network in vivo is poorly understood. Using a human orthotopic tumor model of GBM for ChIP-Seq analysis, we mapped the SOX2 cistrome. Integrative analysis of SOX2 cistrome and GBM transcriptome data identified two transcription factors, oligodendrocyte lineage transcription factor 2 (OLIG2) and zinc finger E-box binding homeobox 1 (ZEB1) as critical SOX2 targets. Their expression is strongly correlated with SOX2 expression in clinical GBM specimens, and all three proteins are frequently co-expressed in primary Glioblastoma cells, even in the setting of EGFR intratumoral heterogeneity. Sox2, Olig2, and Zeb1 expression is activated by the oncogene EGFRvIII in a murine glioma model, and we demonstrate that co-expression of these transcription factors transforms tumor suppressor deficient astrocytes in the absence of an upstream oncogene and that the resulting tumors exhibit the major histopathological features of GBM.

In this study, we generated a zebrafish model of DBA with RPL5 morphants and implemented high-throughput RNA-seq and miRNA-seq to identify key genes, lncRNAs, and miRNAs during zebrafish development and hematopoiesis. We found that RPL5 is required for both primitive and definitive hematopoiesis processes that are partially mediated by the P53 pathway. Several genes such as cirh1a, noc2l, tars, and nol6 and miRNAs such as dre-miR-10a*, dre-miR-722, dre-miR-737, and dre-miR-142a-3p were significantly deregulated, and these changes may play a crucial role in hematopoiesis, ribosome biogenesis and development process. We also characterized the lncRNome in zebrafish with RPL5 deficiency. By constructing a comprehensive regulatory network, we identified central node genes in the network connected to the P53 pathway, almost all of which were targeted by the significantly deregulated miRNAs listed above. Our results therefore establish a regulatory network for critical genes and miRNAs involved in the RPL5-deficient zebrafish model and provide a comprehensive basis for the molecular pathogenesis of RPL5-mediated DBA and other ribosomopathies.

NCoR and SMRT are two paralogous vertebrate proteins that function as corepressors with unliganded nuclear receptors. Although C. elegans has a large number of nuclear receptors, orthologues of the corepressors NCoR and SMRT have not unambiguously been identified in Drosophila or C. elegans. Here, we identify GEI-8 as the closest homologue of NCoR and SMRT in C. elegans and demonstrate that GEI-8 is expressed as at least two isoforms throughout development in multiple tissues, including neurons, muscle and intestinal cells. We demonstrate that a homozygous deletion within the gei-8 coding region, which is predicted to encode a truncated protein lacking the predicted NR domain, results in severe mutant phenotypes with developmental defects, slow movement and growth, arrested gonadogenesis and defects in cholinergic neurotransmission. Whole genome expression analysis by microarrays identified sets of de-regulated genes consistent with both the observed mutant phenotypes and a role of GEI-8 in regulating transcription. Interestingly, the upregulated transcripts included a predicted mitochondrial sulfide:quinine reductase encoded by Y9C9A.16. This locus also contains non-coding, 21-U RNAs of the piRNA. Inhibition of the expression of the region coding for 21-U RNAs leads to irregular gonadogenesis in the homozygous gei-8 mutants, but not in an otherwise wild-type background, suggesting that GEI-8 may function in concert with the 21-U RNAs to regulate gonadogenesis. Our results confirm that GEI-8 is the orthologue of the vertebrate NCoR/SMRT corepressors and demonstrate important roles for this putative transcriptional corepressor in development and neuronal function.