Dynamics of broad H3K4me3 domains uncover an epigenetic switch between cell identity and cancer-related genes.

Broad domains of H3K4 methylation have been associated with consistent expression of tissue-specific, cell identity, and tumor suppressor genes. Here, we identified broad domain-associated genes in healthy human thymic T cell populations and a collection of T cell acute lymphoblastic leukemia (T-ALL) primary samples and cell lines. We found that broad domains are highly dynamic throughout T cell differentiation, and their varying breadth allows the distinction between normal and neoplastic cells. Although broad domains preferentially associate with cell identity and tumor suppressor genes in normal thymocytes, they flag key oncogenes in T-ALL samples. Moreover, the expression of broad domain-associated genes, both coding and noncoding, is frequently deregulated in T-ALL. Using two distinct leukemic models, we showed that the ectopic expression of T-ALL oncogenic transcription factor preferentially impacts the expression of broad domain-associated genes in preleukemic cells. Finally, an H3K4me3 demethylase inhibitor differentially targets T-ALL cell lines depending on the extent and number of broad domains. Our results show that the regulation of broad H3K4me3 domains is associated with leukemogenesis, and suggest that the presence of these structures might be used for epigenetic prioritization of cancer-relevant genes, including long noncoding RNAs.

Low level CpG island promoter methylation predicts a poor outcome in adult T-cell acute lymphoblastic leukemia.

Cancer cells undergo massive alterations in their DNA methylation patterns which result in aberrant gene expression and malignant phenotypes. Abnormal DNA methylation is a prognostic marker in several malignancies, but its potential prognostic significance in adult T-cell acute lymphoblastic leukemia (T-ALL) is poorly defined. Here, we performed methylated DNA immunoprecipitation to obtain a comprehensive genome-wide analysis of promoter methylation in adult T-ALL (n=24) compared to normal thymi (n=3). We identified a CpG hypermethylator phenotype that distinguishes two T-ALL subgroups and further validated it in an independent series of 17 T-lymphoblastic lymphoma. Next, we identified a methylation classifier based on nine promoters which accurately predict the methylation phenotype. This classifier was applied to an independent series of 168 primary adult T-ALL treated accordingly to the GRAALL03/05 trial using methylation-specific multiplex ligation-dependent probe amplification. Importantly hypomethylation correlated with specific oncogenic subtypes of T-ALL and identified patients associated with a poor clinical outcome. This methylation-specific multiplex ligation-dependent probe amplification based methylation profiling could be useful for therapeutic stratification of adult T-ALL in routine practice. The GRAALL-2003 and -2005 studies were registered at http://www.clinicaltrials.gov as #NCT00222027 and #NCT00327678, respectively.

Dynamic recruitment of Ets1 to both nucleosome-occupied and -depleted enhancer regions mediates a transcriptional program switch during early T-cell differentiation.

Ets1 is a sequence-specific transcription factor that plays an important role during hematopoiesis, and is essential for the transition of CD4(-)/CD8(-) double negative (DN) to CD4(+)/CD8(+) double positive (DP) thymocytes. Using genome-wide and functional approaches, we investigated the binding properties, transcriptional role and chromatin environment of Ets1 during this transition. We found that while Ets1 binding at distal sites was associated with active genes at both DN and DP stages, its enhancer activity was attained at the DP stage, as reflected by levels of the core transcriptional hallmarks H3K4me1/3, RNA Polymerase II and eRNA. This dual, stage-specific ability reflected a switch from non-T hematopoietic toward T-cell specific gene expression programs during the DN-to-DP transition, as indicated by transcriptome analyses of Ets1(-/-) thymic cells. Coincidentally, Ets1 associates more specifically with Runx1 in DN and with TCF1 in DP cells. We also provide evidence that Ets1 predominantly binds distal nucleosome-occupied regions in DN and nucleosome-depleted regions in DP. Finally and importantly, we demonstrate that Ets1 induces chromatin remodeling by displacing H3K4me1-marked nucleosomes. Our results thus provide an original model whereby the ability of a transcription factor to bind nucleosomal DNA changes during differentiation with consequences on its cognate enhancer activity.

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.

Transcription-dependent generation of a specialized chromatin structure at the TCRß locus.

V(D)J recombination assembles Ag receptor genes during lymphocyte development. Enhancers at AR loci are known to control V(D)J recombination at associated alleles, in part by increasing chromatin accessibility of the locus, to allow the recombination machinery to gain access to its chromosomal substrates. However, whether there is a specific mechanism to induce chromatin accessibility at AR loci is still unclear. In this article, we highlight a specialized epigenetic marking characterized by high and extended H3K4me3 levels throughout the Dß-Jß-Cß gene segments. We show that extended H3K4 trimethylation at the Tcrb locus depends on RNA polymerase II (Pol II)-mediated transcription. Furthermore, we found that the genomic regions encompassing the two DJCß clusters are highly enriched for Ser(5)-phosphorylated Pol II and short-RNA transcripts, two hallmarks of transcription initiation and early transcription. Of interest, these features are shared with few other tissue-specific genes. We propose that the entire DJCß regions behave as transcription "initiation" platforms, therefore linking a specialized mechanism of Pol II transcription with extended H3K4 trimethylation and highly accessible Dß and Jß gene segments.

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.

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 enhancer's 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.

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.

Epigenetic control of Tcrb gene rearrangement.

V(D)J recombination assembles antigen receptor genes from germline V, D and J segments during lymphocyte development. In αßT-cells, this leads to the subsequent expression of T-cell receptor (TCR) ß and α chains. Generally, V(D)J recombination is closely controlled at various levels, including cell-type and cell-stage specificities, order of locus/gene segment recombination, and allele usage to mediate allelic exclusion. Many of these controls rely on the modulation of gene accessibility to the recombination machinery, involving not only biochemical changes in chromatin arrangement and structural modifications of chromosomal organization and positioning, but also the refined composition of the recombinase targets, the so-called recombination signal sequences. Here, we summarize current knowledge regarding the regulation of V(D)J recombination at the Tcrb gene locus, certainly one for which these various levels of control and regulatory components have been most extensively investigated.

Epigenetic aspects of lymphocyte antigen receptor gene rearrangement or 'when stochasticity completes randomness'.

To perform their specific functional role, B and T lymphocytes, cells of the adaptive immune system of jawed vertebrates, need to express one (and, preferably, only one) form of antigen receptor, i.e. the immunoglobulin or T-cell receptor (TCR), respectively. This end goal depends initially on a series of DNA cis-rearrangement events between randomly chosen units from separate clusters of V, D (at some immunoglobulin and TCR loci) and J gene segments, a biomolecular process collectively referred to as V(D)J recombination. V(D)J recombination takes place in immature T and B cells and relies on the so-called RAG nuclease, a site-specific DNA cleavage apparatus that corresponds to the lymphoid-specific moiety of the VDJ recombinase. At the genome level, this recombinase's mission presents substantial biochemical challenges. These relate to the huge distance between (some of) the gene segments that it eventually rearranges and the need to achieve cell-lineage-restricted and developmentally ordered routines with at times, mono-allelic versus bi-allelic discrimination. The entire process must be completed without any recombination errors, instigators of chromosome instability, translocation and, potentially, tumorigenesis. As expected, such a precisely choreographed and yet potentially risky process demands sophisticated controls; epigenetics demonstrates what is possible when calling upon its many facets. In this vignette, we will recall the evidence that almost from the start appeared to link the two topics, V(D)J recombination and epigenetics, before reviewing the latest advances in our knowledge of this joint venture.

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.

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.

TCR beta allelic exclusion in dynamical models of V(D)J recombination based on allele independence.

Allelic exclusion represents a major aspect of TCRbeta gene assembly by V(D)J recombination in developing T lymphocytes. Despite recent progress, its comprehension remains problematic when confronted with experimental data. Existing models fall short in terms of incorporating into a unique distribution all the cell subsets emerging from the TCRbeta assembly process. To revise this issue, we propose dynamical, continuous-time Markov chain-based modeling whereby essential steps in the biological procedure (D-J and V-DJ rearrangements and feedback inhibition) evolve independently on the two TCRbeta alleles in every single cell while displaying random modes of initiation and duration. By selecting parameters via fitting procedures, we demonstrate the capacity of the model to offer accurate fractions of all distinct TCRbeta genotypes observed in studies using developing and mature T cells from wild-type or mutant mice. Selected parameters in turn afford relative duration for each given step, hence updating TCRbeta recombination distinctive timings. Overall, our dynamical modeling integrating allele independence and noise in recombination and feedback-inhibition events illustrates how the combination of these ingredients alone may enforce allelic exclusion at the TCRbeta locus.

Thymocytes trigger self-antigen-controlling pathways in immature medullary thymic epithelial stages.

Interactions of developing T cells with Aire+ medullary thymic epithelial cells expressing high levels of MHCII molecules (mTEChi) are critical for the induction of central tolerance in the thymus. In turn, thymocytes regulate the cellularity of Aire+ mTEChi. However, it remains unknown whether thymocytes control the precursors of Aire+ mTEChi that are contained in mTEClo cells or other mTEClo subsets that have recently been delineated by single-cell transcriptomic analyses. Here, using three distinct transgenic mouse models, in which antigen presentation between mTECs and CD4+ thymocytes is perturbed, we show by high-throughput RNA-seq that self-reactive CD4+ thymocytes induce key transcriptional regulators in mTEClo and control the composition of mTEClo subsets, including Aire+ mTEChi precursors, post-Aire and tuft-like mTECs. Furthermore, these interactions upregulate the expression of tissue-restricted self-antigens, cytokines, chemokines, and adhesion molecules important for T-cell development. This gene activation program induced in mTEClo is combined with a global increase of the active H3K4me3 histone mark. Finally, we demonstrate that these self-reactive interactions between CD4+ thymocytes and mTECs critically prevent multiorgan autoimmunity. Our genome-wide study thus reveals that self-reactive CD4+ thymocytes control multiple unsuspected facets from immature stages of mTECs, which determines their heterogeneity.

Experimental evidence for long-distance electrodynamic intermolecular forces.

Both classical and quantum electrodynamics predict the existence of dipole-dipole long-range electrodynamic intermolecular forces; however, these have never been hitherto experimentally observed. The discovery of completely new and unanticipated forces acting between biomolecules could have considerable impact on our understanding of the dynamics and functioning of the molecular machines at work in living organisms. Here, using two independent experiments, on the basis of different physical effects detected by fluorescence correlation spectroscopy and terahertz spectroscopy, respectively, we demonstrate experimentally the activation of resonant electrodynamic intermolecular forces. This is an unprecedented experimental proof of principle of a physical phenomenon that, having been observed for biomacromolecules and with long-range action (up to 1000 Å), could be of importance for biology. In addition to thermal fluctuations that drive molecular motion randomly, these resonant (and thus selective) electrodynamic forces may contribute to molecular encounters in the crowded cellular space.

Duality of enhancer functioning mode revealed in a reduced TCR beta gene enhancer knockin mouse model.

The TCRbeta gene enhancer (Ebeta) commands TCRbeta gene expression through the lifespan of T lymphocytes. Genetic and molecular studies have implied that in early thymocytes, Ebeta directs chromatin opening over the Dbeta-Jbeta-Cbeta domains and triggers initial Dbeta-Jbeta recombination. In mature T cells, Ebeta is required for expression of the assembled TCRbeta gene. Whether these separate activities rely on distinct Ebeta regulatory sequences and involve differing modes of activation is unclear. Using gene targeting in mouse embryonic stem cells, we replaced Ebeta by a conserved core fragment (Ebeta169). We found that Ebeta169-carrying alleles were capable of sustaining beta gene expression and the development of mature T cells in homozygous knockin mice. Surprisingly, these procedures and underlying molecular transactions were affected to a wide range of degrees depending on the developmental stage. Early thymocytes barely achieved Dbeta-Jbeta germline transcription and recombination. In contrast, T cells displayed substantial though heterogeneous levels of VDJ-rearranged TCRbeta gene expression. Our results have implications regarding enhancer function in cells of the adaptive immune system and, potentially, TCRbeta gene recombination and allelic exclusion.

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. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

In vivo reinsertion of excised episomes by the V(D)J recombinase: a potential threat to genomic stability.

It has long been thought that signal joints, the byproducts of V(D)J recombination, are not involved in the dynamics of the rearrangement process. Evidence has now started to accumulate that this is not the case, and that signal joints play unsuspected roles in events that might compromise genomic integrity. Here we show both ex vivo and in vivo that the episomal circles excised during the normal process of receptor gene rearrangement may be reintegrated into the genome through trans-V(D)J recombination occurring between the episomal signal joint and an immunoglobulin/T-cell receptor target. We further demonstrate that cryptic recombination sites involved in T-cell acute lymphoblastic leukemia-associated chromosomal translocations constitute hotspots of insertion. Eventually, the identification of two in vivo cases associating episomal reintegration and chromosomal translocation suggests that reintegration events are linked to genomic instability. Altogether, our data suggest that V(D)J-mediated reintegration of episomal circles, an event likely eluding classical cytogenetic screenings, might represent an additional potent source of genomic instability and lymphoid cancer.

Regulation of V(D)J recombination.

Adaptive immunity is intimately linked to the expression of antigen-specific immunoglobulin and T cell receptor genes and their recombination assembly from germline V, D and J gene segments. This developmentally regulated process relies on the activity of the Rag1-Rag2 recombinase, on accessibility of target gene segments and on monoallelic gene activation. Recent studies have revealed new mechanisms that, along with recombinase activity and locus accessibility, are likely to contribute to the control of V(D)J recombination, including target-site bias by the recombinase, RNA processing and chromosome positioning.

Molecular genetics at the T-cell receptor beta locus: insights into the regulation of V(D)J recombination.

The V(D)J recombination machinery assembles antigen receptor genes from germline V, D and J segments duringlymphocyte development. In alphabetaT cells, this leads to the production of the T-cell receptor (TCR) alpha and beta chains. Notably, V(D)J recombination at the Tcrb locus is tightly controlled at various levels, including cell-type and stage specificities, intralocus ordering and allelic exclusion. Although many of these controls are partly mediated at the level of genomic accessibility to the V(D)J recombinase, recent studies have uncovered novel mechanisms that are also likely to contribute to the developmental regulation of Tcrb gene rearrangement events. In this chapter, we summarize our current knowledge and highlight unanswered questions regarding the regulation of V(D)J recombination at the Tcrb locus, placing emphasis on mouse transgenesis and gene-targeting approaches.