Nicolas Descostes

Argonaute proteins couple chromatin silencing to alternative splicing

Argonaute proteins play a major part in transcriptional gene silencing in many organisms, but their role in the nucleus of somatic mammalian cells remains elusive. Here, we have immunopurified human Argonaute-1 and Argonaute-2 (AGO1 and AGO2) chromatin-embedded proteins and found them associated with chromatin modifiers and, notably, with splicing factors. Using the CD44 gene as a model, we show that AGO1 and AGO2 facilitate spliceosome recruitment and modulate RNA polymerase II elongation rate, thereby affecting alternative splicing. Proper AGO1 and AGO2 recruitment to CD44 transcribed regions required the endonuclease Dicer and the chromobox protein HP1γ, and resulted in increased histone H3 lysine 9 methylation on variant exons. Our data thus uncover a new model for the regulation of alternative splicing, in which Argonaute proteins couple RNA polymerase II elongation to chromatin modification.

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.

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

Site-specific methylation and acetylation of lysine residues in the C-terminal domain (CTD) of RNA polymerase II

Dynamic modification of heptad-repeats with the consensus sequence Tyr1-Ser2-Pro3-Thr4-Ser5-Pro6-Ser7 of RNA polymerase II (RNAPII) C-terminal domain (CTD) regulates transcription-coupled processes. Mass spectrometry analysis revealed that K7-residues in non-consensus repeats of human RNAPII are modified by acetylation, or mono-, di-, and tri-methylation. K7ac, K7me2, and K7me3 were found exclusively associated with phosphorylated CTD peptides, while K7me1 occurred also in non-phosphorylated CTD. The monoclonal antibody 1F5 recognizes K7me1/2 residues in CTD and reacts with RNAPIIA. Treatment of cellular extracts with phosphatase or of cells with the kinase inhibitor flavopiridol unmasked the K7me1/2 epitope in RNAPII0, consistent with the association of K7me1/2 marks with phosphorylated CTD peptides. Genome-wide profiling revealed high levels of K7me1/2 marks at the transcriptional start site of genes for sense and antisense transcribing RNAPII. The new K7 modifications further expand the mammalian CTD code to allow regulation of differential gene expression.

ARS2 is a general suppressor of pervasive transcription

Abstract Termination of transcription is important for establishing gene punctuation marks. It is also critical for suppressing many of the pervasive transcription events occurring throughout eukaryotic genomes and coupling their RNA products to efficient decay. In human cells, the ARS2 protein has been implicated in such function as its depletion causes transcriptional read-through of selected gene terminators and because it physically interacts with the ribonucleolytic nuclear RNA exosome. Here, we study the role of ARS2 on transcription and RNA metabolism genome wide. We show that ARS2 depletion negatively impacts levels of promoter-proximal RNA polymerase II at protein-coding (pc) genes. Moreover, our results reveal a general role of ARS2 in transcription termination-coupled RNA turnover at short transcription units like snRNA-, replication-dependent histone-, promoter upstream transcript- and enhancer RNA-loci. Depletion of the ARS2 interaction partner ZC3H18 mimics the ARS2 depletion, although to a milder extent, whereas depletion of the exosome core subunit RRP40 only impacts RNA abundance post-transcriptionally. Interestingly, ARS2 is also involved in transcription termination events within first introns of pc genes. Our work therefore establishes ARS2 as a general suppressor of pervasive transcription with the potential to regulate pc gene expression.

Pasha: a versatile R package for piling chromatin HTS data

UNLABELLED We describe an R package designed for processing aligned reads from chromatin-oriented high-throughput sequencing experiments. Pasha (preprocessing of aligned sequences from HTS analyses) allows easy manipulation of aligned reads from short-read sequencing technologies (ChIP-seq, FAIRE-seq, MNase-Seq, …) and offers innovative approaches such as ChIP-seq reads elongation, nucleosome midpoint piling strategy for positioning analyses, or the ability to subset paired-end reads by groups of insert size that can contain biologically relevant information. AVAILABILITY AND IMPLEMENTATION Pasha is a multi-platform R package, available on CRAN repositories under GPL-3 license (https://cran.r-project.org/web/packages/Pasha/). CONTACTS rfenouil@gmail.com or jean-christophe.andrau@igmm.cnrs.fr SUPPLEMENTARY INFORMATION Supplementary data are available at Bioinformatics online.

Precise re-deposition of nucleosomes on repressive chromatin domains sustain epigenetic inheritance during DNA replication

Summary Chromatin domains and their associated structures must be faithfully inherited through cellular division to maintain cellular identity. Yet, accessing the localized strategies preserving chromatin domain inheritance, specifically the transfer of parental, pre-existing nucleosomes with their associated post-translational modifications (PTMs) during DNA replication is challenging in living cells. We devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histone-containing nucleosomes at single desired loci in mouse embryonic stem cells such that their fate after DNA replication could be followed. Strikingly, repressed chromatin domains are preserved through the local re-deposition of parental nucleosomes. In contrast, nucleosomes decorating active chromatin domains do not exhibit such preservation. Notably, altering cell fate leads to an adjustment in the positional inheritance of parental nucleosomes that reflects the corresponding changes in chromatin structure. These findings point to important mechanisms that contribute to parental nucleosome segregation to preserve cellular identity.Whether chromatin domains display localized strategies to transfer pre-existing nucleosomal (H3-H4)2 core histones and their post-translational modifications (PTMs) during DNA replication remains unknown, largely due to the limitations of direct and precise methods to follow the fate of parental nucleosomes behind the replication fork. Here, we devised an inducible, proximity-dependent labeling system to irreversibly mark replication-dependent H3.1 and H3.2 histones at desired loci in mouse embryonic stem cells such that their position before and after replication could be determined at high resolution. We found both local and non-local re-deposition of parental histones during DNA replication, with a ‘repressed’ chromatin state being locally preserved and an ‘active’ chromatin domain lacking such inheritance. One Sentence Summary A method that permanently labels histones at chosen loci revealed that nucleosomes from repressed (but not active) chromatin domains are re-deposited locally after DNA replication.

ChIPSeqSpike: A R/Bioconductor package for ChIP-Seq data scaling according to spike-in control

Motivation Chromatin Immuno-Precipitation followed by Sequencing (ChlP-Seq) is used to determine the binding sites of any protein of interest. ChIP-Seq data suffer from being more qualitative than quantitative. The recent use of Spike-in controls along with the standard protocol tackled this problem. However, no dedicated tool is available for a robust evaluation of this new ChIP-seq approach Results We developed ChIPSeqSpike, an R/Bioconductor package that enables ChIP-Seq spike-in normalization, assessment and analysis. Ready to use scaled bigwig files and scaling factors values are obtained as output. ChIPSeqSpike also provides tools for ChIP-Seq spike-in assessment and analysis through a versatile collection of graphical functions. Availability The package is implemented in R (as of version 3.4) and is available from Bioconductor at the URL: https://www.bioconductor.org/packages/3.7/bioc/html/ChIPSeqSpike.html, where installation and usage instructions can be found. Contact nicolas.descostes@nyumc.org

Multiple modes of PRC2 inhibition elicit global chromatin alterations in H3K27M pediatric glioma

A methionine substitution at lysine 27 on histone H3 variants (H3K27M) characterizes ~80% of diffuse intrinsic pontine gliomas (DIPG) and inhibits PRC2 in a dominant negative fashion. Yet, the mechanisms for this inhibition and abnormal epigenomic landscape have not been resolved. Using quantitative proteomics, we discovered that robust PRC2 inhibition requires levels of H3K27M greatly exceeding those of PRC2, seen in DIPG. While PRC2 inhibition requires interaction with H3K27M, we found this interaction on chromatin is transient with PRC2 largely being released from H3K27M. Unexpectedly, inhibition persisted even after PRC2 dissociated from H3K27M-chromatin suggesting a lasting impact on PRC2. Furthermore, allosterically activated PRC2 is particularly sensitive to K27M leading to a failure to spread H3K27me3 at distinct foci. In turn, levels of Polycomb antagonists such as H3K36me2 are elevated suggesting a more global, downstream effect on the epigenome. Together, these findings reveal the conditions required for H3K27M-mediated PRC2 inhibition and reconcile seemingly paradoxical effects of H3K27M on PRC2 recruitment and activity.