Frederic Koch

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.

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.

Splicing enhances recruitment of methyltransferase HYPB/Setd2 and methylation of histone H3 Lys36

Several lines of recent evidence support a role for chromatin in splicing regulation. Here, we show that splicing can also contribute to histone modification, which implies bidirectional communication between epigenetic mechanisms and RNA processing. Genome-wide analysis of histone methylation in human cell lines and mouse primary T cells reveals that intron-containing genes are preferentially marked with histone H3 Lys36 trimethylation (H3K36me3) relative to intronless genes. In intron-containing genes, H3K36me3 marking is proportional to transcriptional activity, whereas in intronless genes, H3K36me3 is always detected at much lower levels. Furthermore, splicing inhibition impairs recruitment of H3K36 methyltransferase HYPB (also known as Setd2) and reduces H3K36me3, whereas splicing activation has the opposite effect. Moreover, the increase of H3K36me3 correlates with the length of the first intron, consistent with the view that splicing enhances H3 methylation. We propose that splicing is mechanistically coupled to recruitment of HYPB/Setd2 to elongating RNA polymerase II.

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.

Threonine‐4 of mammalian RNA polymerase II CTD is targeted by Polo‐like kinase 3 and required for transcriptional elongation

Eukaryotic RNA polymerase II (Pol II) has evolved an array of heptad repeats with the consensus sequence Tyr1‐Ser2‐Pro3‐Thr4‐Ser5‐Pro6‐Ser7 at the carboxy‐terminal domain (CTD) of the large subunit (Rpb1). Differential phosphorylation of Ser2, Ser5, and Ser7 in the 5′ and 3′ regions of genes coordinates the binding of transcription and RNA processing factors to the initiating and elongating polymerase complexes. Here, we report phosphorylation of Thr4 by Polo‐like kinase 3 in mammalian cells. ChIPseq analyses indicate an increase of Thr4‐P levels in the 3′ region of genes occurring subsequently to an increase of Ser2‐P levels. A Thr4/Ala mutant of Pol II displays a lethal phenotype. This mutant reveals a global defect in RNA elongation, while initiation is largely unaffected. Since Thr4 replacement mutants are viable in yeast we conclude that this amino acid has evolved an essential function(s) in the CTD of Pol II for gene transcription in mammalian cells.

The Ccr4–Not Deadenylase Subunits CNOT7 and CNOT8 Have Overlapping Roles and Modulate Cell Proliferation

Accurate gene expression requires the precise control of mRNA levels, which are determined by the relative rates of nuclear (pre-)mRNA synthesis and processing, and cytoplasmic mRNA turnover. A key step in mRNA degradation is the removal of the poly(A) tail, which involves several deadenylases including components of the Ccr4-Not complex. Here, we focused on the role of the human paralogues CNOT7 (hCaf1/Caf1a) and CNOT8 (hPop2/Caf1b/Calif), which possess deadenylase activity mediated by DEDD nuclease domains. We show that efficient proliferation requires both subunits, although combined knockdown of CNOT7 and CNOT8 further reduces cell proliferation indicating partial redundancy between these proteins. Interestingly, the function of CNOT7 in cell proliferation partly depends on its catalytic activity. On the other hand, the interaction between CNOT7 and BTG2, a member of the antiproliferative BTG/Tob family involved in transcription and mRNA decay appears less important for proliferation of MCF7 cells, suggesting that CNOT7 does not function solely in conjunction with BTG2. Further analysis of gene expression profiles of CNOT7 and/or CNOT8 knockdown cells underscores the partial redundancy between these subunits and suggests that regulation of several genes, including repression of the antiproliferative genes MSMB and PMP22, by the Ccr4-Not complex contributes to cell proliferation.

Genome-wide RNA polymerase II: not genes only!

RNA polymerase (Pol) II transcriptional regulation is an essential process for guiding eukaryotic gene expression. Early in vitro studies deciphered the essential steps for transcription, including recruitment, initiation, elongation and termination. Based on these findings, the idea emerged that Pol II should essentially be located on promoters or genic regions of transcribed genes. The development of in vivo localization protocols has enabled the investigation of genome-wide Pol II occupancy. Recent studies from yeast to human show that Pol II can be poised at the transcription start site or can be located outside of gene-coding regions, sometimes dependent on the growth or differentiation stage. These recent results regarding Pol II genomic location and transcription challenge our classical views of transcriptional regulation.

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.

Tyrosine phosphorylation of RNA Polymerase II CTD is associated with antisense promoter transcription and active enhancers in mammalian cells

In mammals, the carboxy-terminal domain (CTD) of RNA polymerase (Pol) II consists of 52 conserved heptapeptide repeats containing the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7. Post-translational modifications of the CTD coordinate the transcription cycle and various steps of mRNA maturation. Here we describe Tyr1 phosphorylation (Tyr1P) as a hallmark of promoter (5′ associated) Pol II in mammalian cells, in contrast to what was described in yeast. Tyr1P is predominantly found in antisense orientation at promoters but is also specifically enriched at active enhancers. Mutation of Tyr1 to phenylalanine (Y1F) prevents the formation of the hyper-phosphorylated Pol IIO form, induces degradation of Pol II to the truncated Pol IIB form, and results in a lethal phenotype. Our results suggest that Tyr1P has evolved specialized and essential functions in higher eukaryotes associated with antisense promoter and enhancer transcription, and Pol II stability. DOI: http://dx.doi.org/10.7554/eLife.02105.001

CoCAS: A ChIP-on-chip Analysis Suite

Motivation: High-density tiling microarrays are increasingly used in combination with ChIP assays to study transcriptional regulation. To ease the analysis of the large amounts of data generated by this approach, we have developed ChIP-on-chip Analysis Suite (CoCAS), a standalone software suite which implements optimized ChIP-on-chip data normalization, improved peak detection, as well as quality control reports. Our software allows dye swap, replicate correlation and connects easily with genome browsers and other peak detection algorithms. CoCAS can readily be used on the latest generation of Agilent high-density arrays. Also, the implemented peak detection methods are suitable for other datasets, including ChIP-Seq output. Availability: The software is available for download along with a sample dataset at http://www.ciml.univ-mrs.fr/software/ferrier.htm. Contact: ferrier@ciml.univ-mrs.fr; andrau@ciml.univ-mrs.fr; spicuglia@ciml.univ-mrs.fr Supplementary information: Supplementary data are available at Bioinformatics online.