Salvatore Spicuglia

H3K4 tri‐methylation provides an epigenetic signature of active enhancers

Combinations of post‐translational histone modifications shape the chromatin landscape during cell development in eukaryotes. However, little is known about the modifications exactly delineating functionally engaged regulatory elements. For example, although histone H3 lysine 4 mono‐methylation (H3K4me1) indicates the presence of transcriptional gene enhancers, it does not provide clearcut information about their actual position and stage‐specific activity. Histone marks were, therefore, studied here at genomic loci differentially expressed in early stages of T‐lymphocyte development. The concomitant presence of the three H3K4 methylation states (H3K4me1/2/3) was found to clearly reflect the activity of bona fide T‐cell gene enhancers. Globally, gain or loss of H3K4me2/3 at distal genomic regions correlated with, respectively, the induction or the repression of associated genes during T‐cell development. In the Tcrb gene enhancer, the H3K4me3‐to‐H3K4me1 ratio decreases with the enhancers strength. Lastly, enhancer association of RNA‐polymerase II (Pol II) correlated with the presence of H3K4me3 and Pol II accumulation resulted in local increase of H3K4me3. Our results suggest the existence of functional links between Pol II occupancy, H3K4me3 enrichment and enhancer activity.

BLUEPRINT to decode the epigenetic signature written in blood

volume 30 number 3 march 2012 nature biotechnology To the Editor: Last October, scientists gathered in Amsterdam to celebrate the start of BLUEPRINT (http://www.blueprintepigenome.eu/), an EU-funded consortium that will generate epigenomic maps of at least 100 different blood cell types. With this initiative, Europe has pledged a substantial contribution to the ultimate goal of the International Human Epigenome Consortium (IHEC) to map 1,000 human epigenomes. Here, we provide a brief background to the scientific questions that prompted the formation of BLUEPRINT, summarize the overall goals of BLUEPRINT and detail the specific areas in which the consortium will focus its initial efforts and resources. In mammals, nucleated cells share the same genome but have different epigenomes depending on the cell type and many other factors, resulting in an astounding diversity in phenotypic plasticity with respect to morphology and function. This diversity is defined by cell-specific patterns of gene expression, which are controlled through regulatory sites in the genome to which transcription factors bind. In eukaryotes, access to these sites is orchestrated via chromatin, the complex of DNA, RNA and proteins that constitutes the functional platform of the genome. In contrast with DNA, chromatin is not static but highly dynamic, particularly through modifications of histones at nucleosomes and cytosines at the DNA level that together define the epigenome, the epigenetic state of the cell. Advances in new genomics technologies, particularly next-generation sequencing, allow the epigenome to be studied in a holistic fashion, leading to a better understanding of chromatin function and functional annotation of the genome. Yet little is known about how epigenetic characteristics vary between different cell types, in health and disease or among individuals. This lack of a quantitative framework for the dynamics of the epigenome and its determinants is a major hurdle for the translation of epigenetic observations into regulatory models, the identification of associations between epigenotypes and diseases, and the subsequent development of new classes of compounds for disease prevention and treatment. The task, however, is daunting as each of the several hundred cell types in the human body is expected to show specific epigenomic features that are further expected to respond to environmental inputs in time and space. The research community has realized these limitations and the need for concerted action. The IHEC was founded to coordinate large-scale international efforts toward the goal of a comprehensive human epigenome reference atlas (http://www.ihec-epigenomes. org/). The IHEC will coordinate epigenomic mapping and characterization worldwide to avoid redundant research efforts, implement high data quality standards, coordinate data storage, management and analysis, and provide free access to the epigenomes produced. The maps generated under the umbrella of the IHEC contain detailed information on DNA methylation, histone modification, nucleosome occupancy, and corresponding coding and noncoding RNA expression in different normal and diseased cell types. This will allow integration of different layers of epigenetic information for a wide variety of distinct cell types and thus provide a resource for both basic and applied research. BLUEPRINT aims to bridge the gap in our current knowledge between individual components of the epigenome and their functional dynamics through state-of-the-art analysis in a defined set of primarily human hematopoietic cells from healthy and diseased individuals. Mammalian blood formation or hematopoiesis is one of the best-studied systems of stem cell biology. Blood formation can be viewed as a hierarchical process, and classically, differentiation is defined to occur along the myeloid and lymphoid lineages. The identity of cellular intermediates and the geometry of branch points are still under intense investigation and therefore provide a paradigm for delineation of fundamental principles of cell fate determination and regulation of proliferation and lifespan, which differ considerably between different types of blood cells. BLUEPRINT will generate reference epigenomes of at least 50 specific blood cell types and their malignant counterparts and aim to provide high-quality reference epigenomes of primary cells from >60 individuals with detailed genetic and, where appropriate, medical records. To account for and quantify the impact of DNA sequence variation on epigenome differences, BLUEPRINT will work whenever possible on samples of known genetic variation, including samples from the Cambridge BioResource (Cambridge, UK), the International Cancer Genome Consortium and the British Diabetic Twin Study for disease-discordant monozygotic twin samples. The Wellcome Trust Sanger Institute (Hinxton, UK) will also provide full genomic sequencing for up to 100 samples. BLUEPRINT will harness existing proven technologies to generate reference epigenomes, including RNA-Seq for transcriptome analysis, bisulfite sequencing for methylome analysis, DNaseI-Seq for analysis of hypersensitive sites and ChIPSeq for analysis of at least six histone marks. Moreover, BLUEPRINT aims to develop new technologies to enhance high-throughput epigenome mapping, particularly when using few cells. BLUEPRINT is initially focusing on four main areas. One main goal of the project is to comprehensively analyze diverse epigenomic maps and make them available as an integrated BLUEPRINT-IHEC resource to the scientific community. Integration is envisioned for related projects within species (e.g., the 1000 Genomes Project) and between species (e.g., modENCODE) to better understand functional aspects (e.g., shared pathways) and the evolution of cell lineage development. Analysis of the BLUEPRINT data is expected to catalyze a better understanding of the relationship between epigenetic and genomic information and will form the basis for generation of new methods (e.g., epigenetic imputation) for prediction of epigenetic states from epigenomic profiles. Such prediction methods will facilitate a move toward a more quantitative knowledge and modeling of epigenetic mechanisms. As a result, such models could in the future assist in ‘reverse engineering’ of regulatory networks to repair or restore epigenetic codes that have been perturbed by disease. A second goal of BLUEPRINT is to systematically link epigenetic variation with phenotypic plasticity in health and disease. This will be attempted in three ways. First, genetic and epigenetic varation in two blood cell types from 100 healthy individuals will be analyzed. These measurements will be combined with whole-genome and transcriptome sequencing to dissect the interplay between common DNA sequence BLUEPRINT to decode the epigenetic signature written in blood CORRESPONDENCE

Transcription initiation platforms and GTF recruitment at tissue-specific enhancers and promoters

Recent work has shown that RNA polymerase (Pol) II can be recruited to and transcribe distal regulatory regions. Here we analyzed transcription initiation and elongation through genome-wide localization of Pol II, general transcription factors (GTFs) and active chromatin in developing T cells. We show that Pol II and GTFs are recruited to known T cell–specific enhancers. We extend this observation to many new putative enhancers, a majority of which can be transcribed with or without polyadenylation. Importantly, we also identify genomic features called transcriptional initiation platforms (TIPs) that are characterized by large areas of Pol II and GTF recruitment at promoters, intergenic and intragenic regions. TIPs show variable widths (0.4–10 kb) and correlate with high CpG content and increased tissue specificity at promoters. Finally, we also report differential recruitment of TFIID and other GTFs at promoters and enhancers. Overall, we propose that TIPs represent important new regulatory hallmarks of the genome.

CpG islands and GC content dictate nucleosome depletion in a transcription-independent manner at mammalian promoters

One clear hallmark of mammalian promoters is the presence of CpG islands (CGIs) at more than two-thirds of genes, whereas TATA boxes are only present at a minority of promoters. Using genome-wide approaches, we show that GC content and CGIs are major promoter elements in mammalian cells, able to govern open chromatin conformation and support paused transcription. First, we define three classes of promoters with distinct transcriptional directionality and pausing properties that correlate with their GC content. We further analyze the direct influence of GC content on nucleosome positioning and depletion and show that CpG content and CGI width correlate with nucleosome depletion both in vivo and in vitro. We also show that transcription is not essential for nucleosome exclusion but influences both a weak +1 and a well-positioned nucleosome at CGI borders. Altogether our data support the idea that CGIs have become an essential feature of promoter structure defining novel regulatory properties in mammals.

Promoter Activation by Enhancer-Dependent and -Independent Loading of Activator and Coactivator Complexes

Activation of the pDbeta1 promoter at the TCRbeta locus requires a functional distal enhancer, Ebeta. Here, we have analyzed the mechanism of promoter activation in thymocytes from mice containing or lacking Ebeta. We found that pDbeta1 shows a complex profile of transcription factor and chromatin remodeling complex occupancy even at Ebeta(-) alleles. The presence of Ebeta, however, results in a few specific changes in factor occupancy at the promoter. These differences correlate with localized alterations in histone modifications and in the recruitment of the basal transcriptional machinery. In addition, Ebeta is also bound by CBP and Pol II, suggesting a mechanism for delivery of a holoenzyme complex to the pDbeta1 promoter. These results illustrate a specialized, long-range function of an enhancer in the hierarchical events that regulate assembly of a cell type-specific promoter.

A unique H3K4me2 profile marks tissue-specific gene regulation

Characterization of the epigenetic landscape fundamentally contributes toward deciphering the regulatory mechanisms that govern gene expression. However, despite an increasing flow of newly generated data, no clear pattern of chromatin modifications has so far been linked to specific modes of transcriptional regulation. Here, we used high-throughput genomic data from CD4(+) T lymphocytes to provide a comprehensive analysis of histone H3 lysine 4 dimethylation (H3K4me2) enrichment in genomic regions surrounding transcriptional start sites (TSSs). We discovered that a subgroup of genes linked to T cell functions displayed high levels of H3K4me2 within their gene body, in sharp contrast to the TSS-centered profile typical of housekeeping genes. Analysis of additional chromatin modifications and DNase I hypersensitive sites (DHSS) revealed a combinatorial chromatin signature characteristic of this subgroup. We propose that this epigenetic feature reflects the activity of an as yet unrecognized, intragenic cis-regulatory platform dedicated to refining tissue-specificity in gene expression.

Integrative analysis of public ChIP-seq experiments reveals a complex multi-cell regulatory landscape

The large collections of ChIP-seq data rapidly accumulating in public data warehouses provide genome-wide binding site maps for hundreds of transcription factors (TFs). However, the extent of the regulatory occupancy space in the human genome has not yet been fully apprehended by integrating public ChIP-seq data sets and combining it with ENCODE TFs map. To enable genome-wide identification of regulatory elements we have collected, analysed and retained 395 available ChIP-seq data sets merged with ENCODE peaks covering a total of 237 TFs. This enhanced repertoire complements and refines current genome-wide occupancy maps by increasing the human genome regulatory search space by 14% compared to ENCODE alone, and also increases the complexity of the regulatory dictionary. As a direct application we used this unified binding repertoire to annotate variant enhancer loci (VELs) from H3K4me1 mark in two cancer cell lines (MCF-7, CRC) and observed enrichments of specific TFs involved in biological key functions to cancer development and proliferation. Those enrichments of TFs within VELs provide a direct annotation of non-coding regions detected in cancer genomes. Finally, full access to this catalogue is available online together with the TFs enrichment analysis tool (http://tagc.univ-mrs.fr/remap/).

Uncleaved TFIIA Is a Substrate for Taspase 1 and Active in Transcription

ABSTRACT In higher eukaryotes, the large subunit of the general transcription factor TFIIA is encoded by the single TFIIAαβ gene and posttranslationally cleaved into α and β subunits. The molecular mechanisms and biological significance of this proteolytic process have remained obscure. Here, we show that TFIIA is a substrate of taspase 1 as reported for the trithorax group mixed-lineage leukemia protein. We demonstrate that recombinant taspase 1 cleaves TFIIA in vitro. Transfected taspase 1 enhances cleavage of TFIIA, and RNA interference knockdown of endogenous taspase 1 diminishes cleavage of TFIIA in vivo. In taspase 1−/− MEF cells, only uncleaved TFIIA is detected. In Xenopus laevis embryos, knockdown of TFIIA results in phenotype and expression defects. Both defects can be rescued by expression of an uncleavable TFIIA mutant. Our study shows that uncleaved TFIIA is transcriptionally active and that cleavage of TFIIA does not serve to render TFIIA competent for transcription. We propose that cleavage fine tunes the transcription regulation of a subset of genes during differentiation and development.

Divergent transcription is associated with promoters of transcriptional regulators

BackgroundDivergent transcription is a wide-spread phenomenon in mammals. For instance, short bidirectional transcripts are a hallmark of active promoters, while longer transcripts can be detected antisense from active genes in conditions where the RNA degradation machinery is inhibited. Moreover, many described long non-coding RNAs (lncRNAs) are transcribed antisense from coding gene promoters. However, the general significance of divergent lncRNA/mRNA gene pair transcription is still poorly understood. Here, we used strand-specific RNA-seq with high sequencing depth to thoroughly identify antisense transcripts from coding gene promoters in primary mouse tissues.ResultsWe found that a substantial fraction of coding-gene promoters sustain divergent transcription of long non-coding RNA (lncRNA)/mRNA gene pairs. Strikingly, upstream antisense transcription is significantly associated with genes related to transcriptional regulation and development. Their promoters share several characteristics with those of transcriptional developmental genes, including very large CpG islands, high degree of conservation and epigenetic regulation in ES cells. In-depth analysis revealed a unique GC skew profile at these promoter regions, while the associated coding genes were found to have large first exons, two genomic features that might enforce bidirectional transcription. Finally, genes associated with antisense transcription harbor specific H3K79me2 epigenetic marking and RNA polymerase II enrichment profiles linked to an intensified rate of early transcriptional elongation.ConclusionsWe concluded that promoters of a class of transcription regulators are characterized by a specialized transcriptional control mechanism, which is directly coupled to relaxed bidirectional transcription.

Definition of a T-cell receptor beta gene core enhancer of V(D)J recombination by transgenic mapping.

ABSTRACT V(D)J recombination in differentiating lymphocytes is a highly regulated process in terms of both cell lineage and the stage of cell development. Transgenic and knockout mouse studies have demonstrated that transcriptional enhancers from antigen receptor genes play an important role in this regulation by activatingcis-recombination events. A striking example is the T-cell receptor β-chain (TCRβ) gene enhancer (Eβ), which in the mouse consists of at least seven nuclear factor binding motifs (βE1 to βE7). Here, using a well-characterized transgenic recombination substrate approach, we define the sequences within Eβ required for recombination enhancer activity. The Eβ core is comprised of a limited set of motifs (βE3 and βE4) and an additional previously uncharacterized 20-bp sequence 3′ of the βE4 motif. This core element confers cell lineage- and stage-specific recombination within the transgenic substrates, although it cannot bypass the suppressive effects resulting from transgene integration in heterochromatic centromeres. Strikingly, the core enhancer is heavily occupied by nuclear factors in immature thymocytes, as shown by in vivo footprinting analyses. A larger enhancer fragment including the βE1 through βE4 motifs but not the 3′ sequences, although active in inducing germ line transcription within the transgenic array, did not retain the Eβ recombinational activity. Our results emphasize the multifunctionality of the TCRβ enhancer and shed some light on the molecular mechanisms by which transcriptional enhancers and associated nuclear factors may impact on cis recombination, gene expression, and lymphoid cell differentiation.