Active Transcription Start Sites Research Articles

GENE-58. ACTIVE TRANSCRIPTION START SITES OF MENINGIOMA SAMPLES ASSOCIATED WITH RISK OF RECURRENCE

Abstract Meningiomas are mostly benign brain tumors but have a substantial risk of recurrence, sometimes to more aggressive subtypes. Recently, a DNA methylation signature in meningioma was described as able to stratify patients by recurrence risk (favorable and unfavorable). It is well recognized that epigenetic deregulation at distinct genomic elements can affect changes in gene expression and contribute to cancer initiation and progression. Our goal for this study is to define genes that are actively expressed or repressed by both DNA methylation and chromatin histone modification (defined by H3K4me3). For this pilot study, we selected two favorable (grades I and II) and two unfavorable (grades II and III) meningioma primary tumor samples (N=4) and mapped H3K4me3 genome-wide and whole-genome DNA methylation, in an attempt to identify active transcription start sites at known promoters. After data alignment, preprocessing and peak calling, we identified 29,514 consensus peaks for H3K4me3. The differential binding analysis resulted in 5,752 H3K4me3 regions that distinguish favorable from unfavorable meningioma, mostly gain of peaks in the unfavorable group. We identified 1,505 peaks overlapping with known promoters, 51% associated with gain of peaks in the unfavorable group. Promoter-associated chromatin changes coincided with hypomethylation in 23 unique genes in the unfavorable group. Genes such as MET, PTEN, and the long non-coding RNA RP11-60L3.1 were identified as potential regulators of meningioma recurrence. Our preliminary results describe the identification of distinct genome-wide changes in chromatin associated with meningioma patient with high risk for recurrence. Identification of candidate genes will provide knowledge of the role of epigenomics in the development of malignant meningioma and of opportunities for targeted therapy.

Identification and dynamic quantification of regulatory elements using total RNA.

The spatial and temporal regulation of transcription initiation is pivotal for controlling gene expression. Here, we introduce capped-small RNA-seq (csRNA-seq), which uses total RNA as starting material to detect transcription start sites (TSSs) of both stable and unstable RNAs at single-nucleotide resolution. csRNA-seq is highly sensitive to acute changes in transcription and identifies an order of magnitude more regulated transcripts than does RNA-seq. Interrogating tissues from species across the eukaryotic kingdoms identified unstable transcripts resembling enhancer RNAs, pri-miRNAs, antisense transcripts, and promoter upstream transcripts in multicellular animals, plants, and fungi spanning 1.6 billion years of evolution. Integration of epigenomic data from these organisms revealed that histone H3 trimethylation (H3K4me3) was largely confined to TSSs of stable transcripts, whereas H3K27ac marked nucleosomes downstream from all active TSSs, suggesting an ancient role for posttranslational histone modifications in transcription. Our findings show that total RNA is sufficient to identify transcribed regulatory elements and capture the dynamics of initiated stable and unstable transcripts at single-nucleotide resolution in eukaryotes.

Functional annotation of the cattle genome through systematic discovery and characterization of chromatin states and butyrate-induced variations

BackgroundThe functional annotation of genomes, including chromatin accessibility and modifications, is important for understanding and effectively utilizing the increased amount of genome sequences reported. However, while such annotation has been well explored in a diverse set of tissues and cell types in human and model organisms, relatively little data are available for livestock genomes, hindering our understanding of complex trait variation, domestication, and adaptive evolution. Here, we present the first complete global landscape of regulatory elements in cattle and explore the dynamics of chromatin states in rumen epithelial cells induced by the rumen developmental regulator—butyrate.ResultsWe established the first global map of regulatory elements (15 chromatin states) and defined their coordinated activities in cattle, through genome-wide profiling for six histone modifications, RNA polymerase II, CTCF-binding sites, DNA accessibility, DNA methylation, and transcriptome in rumen epithelial primary cells (REPC), rumen tissues, and Madin-Darby bovine kidney epithelial cells (MDBK). We demonstrated that each chromatin state exhibited specific enrichment for sequence ontology, transcription, methylation, trait-associated variants, gene expression-associated variants, selection signatures, and evolutionarily conserved elements, implying distinct biological functions. After butyrate treatments, we observed that the weak enhancers and flanking active transcriptional start sites (TSS) were the most dynamic chromatin states, occurred concomitantly with significant alterations in gene expression and DNA methylation, which was significantly associated with heifer conception rate and stature economic traits.ConclusionOur results demonstrate the crucial role of functional genome annotation for understanding genome regulation, complex trait variation, and adaptive evolution in livestock. Using butyrate to induce the dynamics of the epigenomic landscape, we were able to establish the correlation among nutritional elements, chromatin states, gene activities, and phenotypic outcomes.

I-104 Distinct chromosomal positions of intact HIV-1 proviruses

A small reservoir of chromosomally-integrated, genome-intact but transcriptionally-silent HIV-1 proviruses persists lifelong despite suppressive antiretroviral therapy, and makes HIV-1 infection an incurable disease. Yet, very little is known about the positions of such intact proviruses in the human genome, or the mechanisms that contribute to maintaining their transcriptional latency. Here, we used a novel experimental approach involving multiple displacement amplification of individual HIV-1 proviral species, followed by near full-length HIV-1 next-generation sequencing and corresponding chromosomal integration site analysis to selectively map the chromosomal positions of intact HIV-1 proviruses in 3 HIV-1-infected individuals undergoing long-term antiretroviral therapy. We observed that in comparison to defective proviral sequences, intact HIV-1 proviruses were enriched for non-genic chromosomal positions and more frequently displayed an opposite orientation relative to host genes. In addition, intact HIV-1 proviruses were preferentially integrated either in relative proximity or in increased distance to active transcriptional start sites and to accessible chromatin regions. These studies strongly suggest selection of intact proviruses with features of deeper viral latency during prolonged antiretroviral therapy, and may be important for developing targeted strategies to purge the genome-intact viral reservoir.

CpG binding protein (CFP1) occupies open chromatin regions of active genes, including enhancers and non-CpG islands

BackgroundThe mechanism by which protein complexes interact to regulate the deposition of post-translational modifications of histones remains poorly understood. This is particularly important at regulatory regions, such as CpG islands (CGIs), which are known to recruit Trithorax (TrxG) and Polycomb group proteins. The CxxC zinc finger protein 1 (CFP1, also known as CGBP) is a subunit of the TrxG SET1 protein complex, a major catalyst of trimethylation of H3K4 (H3K4me3).ResultsHere, we used ChIP followed by high-throughput sequencing (ChIP-seq) to analyse genomic occupancy of CFP1 in two human haematopoietic cell types. We demonstrate that CFP1 occupies CGIs associated with active transcription start sites (TSSs), and is mutually exclusive with H3K27 trimethylation (H3K27me3), a marker of polycomb repressive complex 2. Strikingly, rather than being restricted to active CGI TSSs, CFP1 also occupies a substantial fraction of active non-CGI TSSs and enhancers of transcribed genes. However, relative to other TrxG subunits, CFP1 was specialised to TSSs. Finally, we found enrichment of CpG-containing DNA motifs in CFP1 peaks at CGI promoters.ConclusionsWe found that CFP1 is not solely recruited to CpG islands as it was originally defined, but also other regions including non-CpG island promoters and enhancers.

Methylome-wide association findings for major depressive disorder overlap in blood and brain and replicate in independent brain samples.

We present the first large-scale methylome-wide association studies (MWAS) for major depressive disorder (MDD) to identify sites of potential importance for MDD etiology. Using a sequencing-based approach that provides near-complete coverage of all 28 million common CpGs in the human genome, we assay methylation in MDD cases and controls from both blood (N=1,132) and postmortem brain tissues (N=61 samples from Brodmann Area 10, BA10). The MWAS for blood identified several loci with P 1.91×10−8-4.39×10−8 and a resampling approach showed that the cumulative association was significant (P=4.03×10−10) with the signal coming from the top 25,000 MWAS markers. Furthermore, a permutation based analysis showed significant overlap (P=5.4×10−3) between the MWAS findings in blood and brain (BA10). This overlap was significantly enriched for a number of features including being in eQTLs in blood and frontal cortex, CpG islands and shores, and exons. The overlapping sites were also enriched for active chromatin states in brain including genic enhancers and active transcription start sites. Furthermore, three loci located in GABBR2, RUFY3 and in an intergenic region on chromosome 2 replicated with the same direction of effect in the second brain tissue (BA25, N=60) from the same individuals and in two independent brain collections (BA10, N=81 and 64). GABBR2 inhibits neuronal activity through G protein-coupled second-messenger systems and RUFY3 is implicated in the establishment of neuronal polarity and axon elongation. In conclusion, we identified and replicated methylated loci associated with MDD that are involved in biological functions of likely importance to MDD etiology.

TOPMed Whole Genome Sequence Combined with Pancreas-Specific Annotation for Rare Variants Tests of Type 2 Diabetes Risk

Genetic studies have shown that common variants associated with type 2 diabetes are enriched in pancreatic-specific regulatory regions. Most whole genome sequence (WGS) studies have used general annotation of regulatory regions not specific to relevant tissues. In contrast, we used functional annotation from pancreatic islets to define rare variant groupings for aggregate tests in coding and non-coding regions in a pilot analysis on chr 10 with 13,200 individuals across six NHLBI TOPMed studies: 2,601 individuals with type 2 diabetes and 10,599 normoglycemic controls with <30X WGS coverage. We identified genes expressed in pancreas and nearby regions showing islet-specific regulatory activity, including open chromatin and chromatin states associated with promoters and enhancers. Variants with minor allele frequency < 1% were aggregated by gene, including the region 5 kbp upstream and downstream of transcribed regions. We only included variants annotated as: "splice acceptor", "splice donor", "splice region", "missense", or "stop gained" within exons expressed in islets, while outside of those exons we included only those variants within “active transcription start sites” and “active enhancer” predicted chromatin states. We performed SKAT association tests, adjusting the association with type 2 diabetes for age, sex, study, and a genetic relatedness matrix to accounted for genetic similarities within families and between populations. Overall, 219 genes (median of 161 variants per gene-region) expressed in islets were included in the analysis: ATP5C1, AVPI1, and YME1L1 reached nominal significance (P<0.01). Chr 10 pilot analyses provide a framework for tailoring rare variant analyses in WGS data through the use of disease-specific functional annotation. WGS analysis in larger samples and additional functional data generated from collaborators in the NIDDK’s AMP-T2D Consortium will extend the genomic spectrum associated with type 2 diabetes risk. Disclosure A. Manning: None. T. Majarian: None. P.S. de Vries: None. J. Wessel: Employee; Spouse/Partner; Eli Lilly and Company. J.B. Meigs: None.

Benefit from decline: the primary transcriptome of Alteromonas macleodii str. Te101 during Trichodesmium demise

Interactions between co-existing microorganisms deeply affect the physiology of the involved organisms and, ultimately, the function of the ecosystem as a whole. Copiotrophic Alteromonas are marine gammaproteobacteria that thrive during the late stages of phytoplankton blooms in the marine environment and in laboratory co-cultures with cyanobacteria such as Trichodesmium. The response of this heterotroph to the sometimes rapid and transient changes in nutrient supply when the phototroph crashes is not well understood. Here, we isolated and sequenced the strain Alteromonas macleodii str. Te101 from a laboratory culture of Trichodesmium erythraeum IMS101, yielding a chromosome of 4.63 Mb and a single plasmid of 237 kb. Increasing salinities to ≥43 ppt inhibited the growth of Trichodesmium but stimulated growth of the associated Alteromonas. We characterized the transcriptomic responses of both microorganisms and identified the complement of active transcriptional start sites in Alteromonas at single-nucleotide resolution. In replicate cultures, a similar set of genes became activated in Alteromonas when growth rates of Trichodesmium declined and mortality was high. The parallel activation of fliA, rpoS and of flagellar assembly and growth-related genes indicated that Alteromonas might have increased cell motility, growth, and multiple biosynthetic activities. Genes with the highest expression in the data set were three small RNAs (Aln1a-c) that were identified as analogs of the small RNAs CsrB-C in E. coli or RsmX-Z in pathogenic bacteria. Together with the carbon storage protein A (CsrA) homolog Te101_05290, these RNAs likely control the expression of numerous genes in responding to changes in the environment.

ATIM-24. SYSTEMATIC IDENTIFICATION OF TOCA 511 RETROVIRUS INTEGRATION SITES AND MUTATION PROFILES IN TUMOR AND BLOOD SAMPLES FROM PATIENTS UNDERGOING INVESTIGATIONAL TREATMENT FOR RECURRENT HIGH GRADE GLIOMA CORRELATES WITH FAVORABLE CLINICAL SAFETY PROFILE

Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector (RRV) based on a gamma retrovirus encoding a cytosine deaminase transgene that allows infected cells to selectively convert the antifungal drug 5-fluorocytosine into the antineoplastic drug 5-fluorouracil. Toca 511 can be delivered by multiple routes, and is designed to selectively infect and spread in tumor cells, and through multiple mechanisms elicit an anti-tumor immune response. Toca 511 and Toca FC have been administered to >125 recurrent high grade glioma patients in three phase I studies (NCT01156584, NCT01470794, NCT01985256) and, based on evidence for safety and drug activity from these trials, a phase 2/3 trial (Toca 5) is in progress (NCT02414165). In addition, we investigated potential off-target (blood) viral infection, and whether such off-target events have potential toxicity. We also investigated the stability profile of Toca 511 genomes after infection of tumor and off-target tissue. To date, approximately 25% of patients show quantifiable viral DNA (LOQ 250 copies/mL) in blood. In all cases, quantifiable virus RNA and DNA signal was cleared from blood. We systematically mapped Toca 511 integration sites from patient samples with detectable virus and sequenced, at high depth, integrated Toca 511 genomes, and in some cases RNA genomes, from these samples. As reported for gamma retroviruses, Toca 511 integration profiles display a preference for integration near active transcription start sites. There was no evidence for clonal expansion of Toca 511 integrated sites or cells. Also there was no preferential insertion of sites nearby oncogenes. Toca 511 sequences display an array of mutations in the tumor and blood, often transitions consistent with cytidine deaminase (APOBEC) activity. The data provide molecular correlates to the previously reported clinical safety profile of Toca 511 and Toca FC treatment and provide support for the activity of Toca 511 and Toca FC treatment in humans.

MdRNA-Seq analysis of marine microbial communities from the northern Red Sea.

Metatranscriptomic differential RNA-Seq (mdRNA-Seq) identifies the suite of active transcriptional start sites at single-nucleotide resolution through enrichment of primary transcript 5′ ends. Here we analyzed the microbial community at 45 m depth at Station A in the northern Gulf of Aqaba, Red Sea, during 500 m deep mixing in February 2012 using mdRNA-Seq and a parallel classical RNA-Seq approach. We identified promoters active in situ for five different pico-planktonic genera (the SAR11 clade of Alphaproteobacteria, Synechococcus of Cyanobacteria, Euryarchaeota, Thaumarchaeota, and Micromonas as an example for picoeukaryotic algae), showing the applicability of this approach to highly diverse microbial communities. 16S rDNA quantification revealed that 24% of the analyzed community were group II marine Euryarchaeota in which we identified a highly abundant non-coding RNA, Tan1, and detected very high expression of genes encoding intrinsically disordered proteins, as well as enzymes for the synthesis of specific B vitamins, extracellular peptidases, carbohydrate-active enzymes, and transport systems. These results highlight previously unknown functions of Euryarchaeota with community-wide relevance. The complementation of metatranscriptomic studies with mdRNA-Seq provides substantial additional information regarding transcriptional start sites, promoter activities, and the identification of non-coding RNAs.

Base-pair-resolution genome-wide mapping of active RNA polymerases using precision nuclear run-on (PRO-seq).

We provide a protocol for precision nuclear run-on sequencing (PRO-seq) and its variant, PRO-cap, which map the location of active RNA polymerases (PRO-seq) or transcription start sites (TSSs) (PRO-cap) genome-wide at high resolution. The density of RNA polymerases at a particular genomic locus directly reflects the level of nascent transcription at that region. Nuclei are isolated from cells and, under nuclear run-on conditions, transcriptionally engaged RNA polymerases incorporate one or, at most, a few biotin-labeled nucleotide triphosphates (biotin-NTPs) into the 3' end of nascent RNA. The biotin-labeled nascent RNA is used to prepare sequencing libraries, which are sequenced from the 3' end to provide high-resolution positional information for the RNA polymerases. PRO-seq provides much higher sensitivity than ChIP-seq, and it generates a much larger fraction of usable sequence reads than ChIP-seq or NET-seq (native elongating transcript sequencing). Similarly to NET-seq, PRO-seq maps the RNA polymerase at up to base-pair resolution with strand specificity, but unlike NET-seq it does not require immunoprecipitation. With the protocol provided here, PRO-seq (or PRO-cap) libraries for high-throughput sequencing can be generated in 4-5 working days. The method has been applied to human, mouse, Drosophila melanogaster and Caenorhabditis elegans cells and, with slight modifications, to yeast.

403. Toca Retroviral Replicating Vector in Tumor and Blood from Clinical Trial Subjects with Recurrent High Grade Glioma

Toca 511 (vocimagene amiretrorepvec) is a retroviral replicating vector (RRV) based on an amphotropic murine gammaretrovirus, that encodes a cytosine deaminase transgene that allows infected cells to selectively convert the antifungal drug 5-fluorocytosine (5-FC) into the antineoplastic drug 5-fluorouracil (5-FU). Toca 511 can be delivered by multiple routes, selectively infects and spreads in tumor cells and through multiple mechanisms can elicit an anti-tumor immune response. Thus, clinically, treatment with Toca 511 and extended-release 5-FC (Toca FC) is expected to selectively destroy tumor cells within the body, while leaving healthy cells unharmed. Toca 511 and Toca FC have been administered to >120 high grade glioma subjects in three phase I studies (NCT01156584, NCT01470794, NCT01985256) and, based on results from these trials, a phase 2/3 trial (Toca 5) has recently started recruitment (NCT02414165). Although Toca 511 plus Toca FC has been well tolerated, we investigated potential off-target viral infection and integration, and whether such off-target events have potential toxicity; we also investigated the stability profile of the Toca 511 genome after infection of tumor targets and off-target tissue (blood). To date, few subjects (approximately 10-25%) show quantifiable viral signal (LOQ approximately 4000 copies/mL) in blood after initial clearance, following Toca 511 injection into the brain tumor or tumor bed. In all cases, quantifiable virus RNA and DNA signal was cleared from blood. In order to better understand interactions among Toca 511 and subjects’ tumor and blood, we systematically mapped Toca 511 integration sites from subject samples with detectable virus and also sequenced, at high depth, integrated Toca 511 genomes, and in some cases RNA genomes, from these samples. Toca 511 integration profiles display a preference for integration near active transcription start sites, as has been seen generally for gammaretroviruses. There was no evidence for clonal expansion of Toca 511 integrated sites/cells or preferential retrieval of sites nearby oncogenes. Toca 511 sequences display an array of mutations in the tumor and blood, including transitions consistent with cytosine deaminase activity. The data provide a molecular correlate to the clinical safety profile of Toca 511 and Toca FC treatment as well as molecular characterization of RRV after infection, replication and therapeutic use in humans.

Identification of Conserved and Potentially Regulatory Small RNAs in Heterocystous Cyanobacteria.

Small RNAs (sRNAs) are a growing class of non-protein-coding transcripts that participate in the regulation of virtually every aspect of bacterial physiology. Heterocystous cyanobacteria are a group of photosynthetic organisms that exhibit multicellular behavior and developmental alternatives involving specific transcriptomes exclusive of a given physiological condition or even a cell type. In the context of our ongoing effort to understand developmental decisions in these organisms we have undertaken an approach to the global identification of sRNAs. Using differential RNA-Seq we have previously identified transcriptional start sites for the model heterocystous cyanobacterium Nostoc sp. PCC 7120. Here we combine this dataset with a prediction of Rho-independent transcriptional terminators and an analysis of phylogenetic conservation of potential sRNAs among 89 available cyanobacterial genomes. In contrast to predictive genome-wide approaches, the use of an experimental dataset comprising all active transcriptional start sites (differential RNA-Seq) facilitates the identification of bona fide sRNAs. The output of our approach is a dataset of predicted potential sRNAs in Nostoc sp. PCC 7120, with different degrees of phylogenetic conservation across the 89 cyanobacterial genomes analyzed. Previously described sRNAs appear among the predicted sRNAs, demonstrating the performance of the algorithm. In addition, new predicted sRNAs are now identified that can be involved in regulation of different aspects of cyanobacterial physiology, including adaptation to nitrogen stress, the condition that triggers differentiation of heterocysts (specialized nitrogen-fixing cells). Transcription of several predicted sRNAs that appear exclusively in the genomes of heterocystous cyanobacteria is experimentally verified by Northern blot. Cell-specific transcription of one of these sRNAs, NsiR8 (nitrogen stress-induced RNA 8), in developing heterocysts is also demonstrated.

Whole-genome sequencing reveals activation-induced cytidine deaminase signatures during indolent chronic lymphocytic leukaemia evolution.

Patients with chromosome 13q deletion or normal cytogenetics represent the majority of chronic lymphocytic leukaemia (CLL) cases, yet have relatively few driver mutations. To better understand their genomic landscape, here we perform whole-genome sequencing on a cohort of patients enriched with these cytogenetic characteristics. Mutations in known CLL drivers are seen in only 33% of this cohort, and associated with normal cytogenetics and unmutated IGHV. The most commonly mutated gene in our cohort, IGLL5, shows a mutational pattern suggestive of activation-induced cytidine deaminase (AID) activity. Unsupervised analysis of mutational signatures demonstrates the activities of canonical AID (c-AID), leading to clustered mutations near active transcriptional start sites; non-canonical AID (nc-AID), leading to genome-wide non-clustered mutations, and an ageing signature responsible for most mutations. Using mutation clonality to infer time of onset, we find that while ageing and c-AID activities are ongoing, nc-AID-associated mutations likely occur earlier in tumour evolution.

Identification of TNF-α-responsive promoters and enhancers in the intestinal epithelial cell model Caco-2.

The Caco-2 cell line is one of the most important in vitro models for enterocytes, and is used to study drug absorption and disease, including inflammatory bowel disease and cancer. In order to use the model optimally, it is necessary to map its functional entities. In this study, we have generated genome-wide maps of active transcription start sites (TSSs), and active enhancers in Caco-2 cells with or without tumour necrosis factor (TNF)-α stimulation to mimic an inflammatory state. We found 520 promoters that significantly changed their usage level upon TNF-α stimulation; of these, 52% are not annotated. A subset of these has the potential to confer change in protein function due to protein domain exclusion. Moreover, we locate 890 transcribed enhancer candidates, where ∼50% are changing in usage after TNF-α stimulation. These enhancers share motif enrichments with similarly responding gene promoters. As a case example, we characterize an enhancer regulating the laminin-5 γ2-chain (LAMC2) gene by nuclear factor (NF)-κB binding. This report is the first to present comprehensive TSS and enhancer maps over Caco-2 cells, and highlights many novel inflammation-specific promoters and enhancers.

Characteristic bimodal profiles of RNA polymerase II at thousands of active mammalian promoters.

BackgroundIn mammals, ChIP-seq studies of RNA polymerase II (PolII) occupancy have been performed to reveal how recruitment, initiation and pausing of PolII may control transcription rates, but the focus is rarely on obtaining finely resolved profiles that can portray the progression of PolII through sequential promoter states.ResultsHere, we analyze PolII binding profiles from high-coverage ChIP-seq on promoters of actively transcribed genes in mouse and humans. We show that the enrichment of PolII near transcription start sites exhibits a stereotypical bimodal structure, with one peak near active transcription start sites and a second peak 110 base pairs downstream from the first. Using an empirical model that reliably quantifies the spatial PolII signal, gene by gene, we show that the first PolII peak allows for refined positioning of transcription start sites, which is corroborated by mRNA sequencing. This bimodal signature is found both in mouse and humans. Analysis of the pausing-related factors NELF and DSIF suggests that the downstream peak reflects widespread pausing at the +1 nucleosome barrier. Several features of the bimodal pattern are correlated with sequence features such as CpG content and TATA boxes, as well as the histone mark H3K4me3.ConclusionsWe thus show how high coverage DNA sequencing experiments can reveal as-yet unnoticed bimodal spatial features of PolII accumulation that are frequent at individual mammalian genes and reminiscent of transcription initiation and pausing. The initiation-pausing hypothesis is corroborated by evidence from run-on sequencing and immunoprecipitation in other cell types and species.

Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1γ in reprogramming to pluripotency

Reprogramming of somatic cells into iPSCs involves a dramatic reorganization of chromatin. To identify posttranslational histone modifications that change in global abundance during this process, we have applied a quantitative mass-spectrometry-based approach. We found that iPSCs, compared to both the starting fibroblasts and a late reprogramming intermediate (pre-iPSCs), are enriched for histone modifications associated with active chromatin, and depleted for marks of transcriptional elongation and a subset of repressive modifications including H3K9me2/me3. Dissecting the contribution of H3K9methylation to reprogramming, we show that the H3K9methyltransferases Ehmt1, Ehmt2, and Setdb1 regulate global H3K9me2/me3 levels and that their depletion increases iPSC formation from both fibroblasts and pre-iPSCs. Similarly, inhibition of heterochromatin-protein-1γ (Cbx3), a protein known to recognize H3K9methylation, enhances reprogramming. Genome-wide location analysis revealed that Cbx3 predominantly binds active genes in both pre-iPSCs and pluripotent cells but with a strikingly different distribution: in pre-iPSCs, but not in ESCs, Cbx3 associates with active transcriptional start sites, suggesting a developmentally-regulated role for Cbx3 in transcriptional activation. Despite largely non-overlapping functions and the association of Cbx3 with active transcription, the H3K9methyltransferases and Cbx3 both inhibit reprogramming by repressing the pluripotency factor Nanog. Together, our findings demonstrate that Cbx3 and H3K9methylation restrict late reprogramming events, and suggest that a dramatic change in global chromatin character is an epigenetic roadblock for reprogramming.

Genome-wide profiles of H2AX and γ-H2AX differentiate endogenous and exogenous DNA damage hotspots in human cells

Phosphorylation of the histone variant H2AX forms γ-H2AX that marks DNA double-strand break (DSB). Here, we generated the sequencing-based maps of H2AX and γ-H2AX positioning in resting and proliferating cells before and after ionizing irradiation. Genome-wide locations of possible endogenous and exogenous DSBs were identified based on γ-H2AX distribution in dividing cancer cells without irradiation and that in resting cells upon irradiation, respectively. γ-H2AX-enriched regions of endogenous origin in replicating cells included sub-telomeres and active transcription start sites, apparently reflecting replication- and transcription-mediated stress during rapid cell division. Surprisingly, H2AX itself, prior to phosphorylation, was specifically located at these endogenous hotspots. This phenomenon was only observed in dividing cancer cells but not in resting cells. Endogenous H2AX was concentrated on the transcription start site of actively transcribed genes but was irrelevant to pausing of RNA polymerase II (pol II), which precisely coincided with γ-H2AX of endogenous origin. γ-H2AX enrichment upon irradiation also coincided with actively transcribed regions, but unlike endogenous γ-H2AX, it extended into the gene body and was not specifically concentrated on the pausing site of pol II. Sub-telomeres were less responsive to external DNA damage than to endogenous stress. Our findings provide insight into DNA repair programs of cancer and may have implications for cancer therapy.

Induction of Myelin Protein Zero by Early Growth Response 2 through Upstream and Intragenic Elements

The Mpz (myelin protein zero) gene codes for the principal component of myelin in the peripheral nervous system, and mutations in this gene cause human peripheral myelinopathies. Expression of the Mpz gene is controlled by two major transactivators that coordinate Schwann cell development: Egr2/Krox20 and Sox10. Our in vivo ChIP-chip analysis in myelinating peripheral nerve identified major sites of Egr2 interaction within the first intron of the Mpz gene and approximately 5 kb upstream of the transcription start site. In addition, the sites of Egr2 binding display many of the hallmarks associated with enhancer elements. Interestingly, the upstream Egr2 binding sites lie proximal to the divergently transcribed succinate dehydrogenase C gene, but Sdhc expression was not affected by the massive induction of Mpz mediated by Egr2. Mpz induction was greatly enhanced in the presence of the Egr2 binding sites, and removal of them markedly diminished transgenic expression of a construct derived from the Mpz locus. Sox10 was also found to be associated with the upstream region, and its binding was required for Egr2-mediated activation in this distal regulatory region. Our findings highlight that peripheral nerve-specific expression of Mpz is primarily regulated by both upstream and intron-associated regulatory elements. Overall, these results provide a locus-wide analysis of the role and activity of Egr2 in regulation of the Mpz gene within its native chromosomal context.

Epigenetics of human T cells during the G0→G1 transition

We investigated functional epigenetic changes that occur in primary human T lymphocytes during entry into the cell cycle and mapped these at the single-nucleosome level by ChIP-chip on tiling arrays for chromosomes 1 and 6. We show that nucleosome loss and flanking active histone marks define active transcriptional start sites (TSSs). Moreover, these signatures are already set at many inducible genes in quiescent cells prior to cell stimulation. In contrast, there is a dearth of the inactive histone mark H3K9me3 at the TSS, and under-representation of H3K9me2 and H3K9me3 defines the body of active genes. At the DNA level, cytosine methylation (meC) is enriched for nucleosomes that remain at the TSS, whereas in general there is a dearth of meC at TSSs. Furthermore, a drop in meC also marks 3' transcription termination, and a peak of meC occurs at stop codons. This mimics the 3' nucleosomal distribution in yeast, which we show does not occur in human T cells.