Two distinct repressive mechanisms for histone 3 lysine 4 methylation through promoting 3'-end antisense transcription

International audienceHistone H3 di- and trimethylation on lysine 4 are major chromatin marks that correlate with active transcription. The influence of these modifications on transcription itself is, however, poorly understood. We have investigated the roles of H3K4 methylation in Saccharomyces cerevisiae by determining genome-wide expression-profiles of mutants in the Set1 complex, COMPASS, that lays down these marks. Loss of H3K4 trimethylation has virtually no effect on steady-state or dynamically-changing mRNA levels. Combined loss of H3K4 tri- and dimethylation results in steady-state mRNA upregulation and delays in the repression kinetics of specific groups of genes. COMPASS-repressed genes have distinct H3K4 methylation patterns, with enrichment of H3K4me3 at the 3'-end, indicating that repression is coupled to 3'-end antisense transcription. Further analyses reveal that repression is mediated by H3K4me3-dependent 3'-end antisense transcription in two ways. For a small group of genes including PHO84, repression is mediated by a previously reported trans-effect that requires the antisense transcript itself. For the majority of COMPASS-repressed genes, however, it is the process of 3'-end antisense transcription itself that is the important factor for repression. Strand-specific qPCR analyses of various mutants indicate that this more prevalent mechanism of COMPASS-mediated repression requires H3K4me3-dependent 3'-end antisense transcription to lay down H3K4me2, which seems to serve as the actual repressive mark. Removal of the 3'-end antisense promoter also results in derepression of sense transcription and renders sense transcription insensitive to the additional loss of SET1. The derepression observed in COMPASS mutants is mimicked by reduction of global histone H3 and H4 levels, suggesting that the H3K4me2 repressive effect is linked to establishment of a repressive chromatin structure. These results indicate that in S. cerevisiae, the non-redundant role of H3K4 methylation by Set1 is repression, achieved through promotion of 3'-end antisense transcription to achieve specific rather than global effects through two distinct mechanisms

Ubiquitylation of the COMPASS component Swd2 links H2B ubiquitylation to H3K4 trimethylation.

International audienceMono-ubiquitylation of histone H2B correlates with transcriptional activation and is required for di- and trimethylation at Lys 4 on the histone H3 tail (H3K4) by the SET1/COMPASS methyltransferase complex through a poorly characterized trans-tail pathway. Here we show that mono-ubiquitylation of histone H2B promotes ubiquitylation at Lys 68 and Lys 69 of Swd2, the essential component of SET1/COMPASS in Saccharomyces cerevisiae. We found that Rad6/Bre1 ubiquitylation enzymes responsible for H2B ubiquitylation also participate directly in Swd2 modification. Preventing Swd2 or H2B ubiquitylation did not affect Set1 stability, interaction of Swd2 with Set1 or the ability of Swd2 to interact with chromatin. However, we found that mutation of Lys 68 and Lys 69 of Swd2 markedly reduced trimethylation, and to a lesser extent dimethylation, of H3K4 at the 5'-end of transcribing genes without affecting monomethylation. This effect results from the ability of Swd2 ubiquitylation to control recruitment of Spp1, a COMPASS subunit necessary for trimethylation. Our results further indicate that Swd2 is a major H3-binding component of COMPASS. Swd2 thus represents a key factor that mediates crosstalk between H2B ubiquitylation and H3K4 trimethylation on chromatin

Interferon controls SUMO availability via the Lin28 and let-7 axis to impede virus replication

International audienceSmall ubiquitin-related modifier (SUMO) protein conjugation onto target proteins regulates multiple cellular functions, including defence against pathogens, stemness and senescence. SUMO1 peptides are limiting in quantity and are thus mainly conjugated to high-affinity targets. Conjugation of SUMO2/3 paralogues is primarily stress inducible and may initiate target degradation. Here we demonstrate that the expression of SUMO1/2/3 is dramatically enhanced by interferons through an miRNA-based mechanism involving the Lin28/let-7 axis, a master regulator of stemness. Normal haematopoietic progenitors indeed display much higher SUMO contents than their differentiated progeny. Critically, SUMOs contribute to the antiviral effects of interferons against HSV1 or HIV. Promyelocytic leukemia (PML) nuclear bodies are interferon-induced domains, which facilitate sumoylation of a subset of targets. Our findings thus identify an integrated interferon-responsive PML/SUMO pathway that impedes viral replication by enhancing SUMO conjugation and possibly also modifying the repertoire of targets. Interferon-enhanced post-translational modifications may be essential for senescence or stem cell self-renewal, and initiate SUMO-dependent proteolysis

H2B Ubiquitylation Controls the Formation of Export-Competent mRNP.

International audienceHistone H2B ubiquitylation is a transcription-dependent modification that not only regulates nucleosome dynamics but also controls the trimethylation of histone H3 on lysine 4 by promoting ubiquitylation of Swd2, a component of both the histone methyltransferase COMPASS complex and the cleavage and polyadenylation factor(CPF). We show that preventing either H2B ubiquitylation or H2B-dependent modification of Swd2 results in nuclear accumulation of poly(A) RNA due to a defect in the integrity and stability of APT, a subcomplex of the CPF. Ubiquitin-regulated APT complex dynamics is required for the correct recruitment of the mRNA export receptor Mex67 to nuclear mRNPs. While H2B ubiquitylation controls the recruitment of the different Mex67 adaptors to mRNPs, the effect of Swd2 ubiquitylation is restricted to Yra1 and Nab2, which, in turn, controls poly(A) tail length. Modification of H2B thus participates in the crosstalk between cotranscriptional events and assembly of mRNPs linking nuclear processing and mRNA export

Transcription and methylation analyses of preleukemic promyelocytes indicate a dual role for PML/RARA in leukemia initiation

International audienceAcute promyelocytic leukemia is an aggressive malignancy characterized by the accumulation of promyelocytes in the bone marrow. PML/RARA is the primary abnormality implicated in this pathology, but the mechanisms by which this chimeric fusion protein initiates disease are incompletely understood. Identifying PML/RARA targets in vivo is critical for comprehending the road to pathogenesis. Utilizing a novel sorting strategy, we isolated highly purified promyelocyte populations from normal and young preleukemic animals, carried out microarray and methylation profiling analyses, and compared the results from the two groups of animals. Surprisingly, in the absence of secondary lesions, PML/RARA had an overall limited impact on both the transcriptome and methylome. Of interest, we did identify down-regulation of secondary and tertiary granule genes as the first step engaging the myeloid maturation block. Although initially not sufficient to arrest terminal granulopoiesis in vivo, such alterations set the stage for the later, complete differentiation block seen in leukemia. Further, gene set enrichment analysis revealed that PML/RARA promyelocytes exhibit a subtle increase in expression of cell cycle genes, and we show that this leads to both increased proliferation of these cells and expansion of the promyelocyte compartment. Importantly, this proliferation signature was absent from the poorly leukemogenic p50/RARA fusion model, implying a critical role for PML in the altered cell-cycle kinetics and ability to initiate leukemia. Thus, our findings challenge the predominant model in the field and we propose that PML/RARA initiates leukemia by subtly shifting cell fate decisions within the promyelocyte compartment

COMPASS-repressed genes have aberrant H3K4 methylation patterns, indicative of 3′-end antisense transcription.

<p>(A) Heatmaps of enrichment of H3K4me2(left) and H3K4me3(right) over H3 in the gene body and flanking regions of the 69 COMPASS repressed genes, based on <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002952#pgen.1002952-Kirmizis1" target="_blank">[53]</a>. The enrichments are rescaled for each individual gene with blue and white corresponding to the highest and lowest enrichment, respectively. <i>PHO84</i> is marked by P. (B) The average enrichment of H3K4me1 (blue), H3K4me2 (red) and H3K4me3 (grey) over H3, for the set of 47 COMPASS-repressed genes that show at least a two-fold enrichment of H3K4me2 or H3K4me3 somewhere across the gene or flanking region <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002952#pgen.1002952-Kirmizis1" target="_blank">[53]</a>.</p

COMPASS repression is mediated through 3′-end antisense transcriptional gene silencing.

<p>(A) Hierarchical clustering of the 69 COMPASS-repressed genes in the <i>rrp6</i>Δ, <i>set1</i>Δ and the <i>set1</i>Δ <i>rrp6</i>Δ strains. <i>PHO84</i> is marked by P and <i>AMS1</i>, <i>YGR110W</i>, <i>ARG1</i>, <i>SPR3</i> and <i>OYE3</i> are marked by 1 to 5, respectively. The black bar marks the subset of genes where the two mutations are epistatic. Three quarters of these genes are related to phosphate metabolism. (B) Sense and antisense RNA levels analyzed by qPCR in indicated backgrounds. The schematic representation of the follow-up genes shows the relative positions of the primers used for strand-specific reverse transcription reactions in arrows, while the black box indicates the location of the DNA fragment produced during the qPCR. Error bars reflect standard deviations of an average signal obtained from at least two independent experiments. The significance of the difference in expression changes observed between the mutant cells and the corresponding background strain (rrp6<i>Δ</i> in the case of <i>spp1Δrrp6Δ</i> and <i>set1Δrrp6Δ</i>, <i>wt for the others</i>), was evaluated using Student's <i>t</i>-test (^*<i>P</i> 0.01–0.05; ^**<i>P</i> 0.001–0.01; ^***<i>P</i><0.001).</p

Loss of H3K4 di- and trimethylation results in upregulation of a subset of genes.

<p>(A) Commassie-stained gel of purified histones from the indicated strains (top) and western blots with antibodies directed against H3 carboxy-terminus and the different H3K4 methylation states (bottom). (B) Hierarchical clustering of all genes with significantly changed mRNA expression (p-value less than 0.01 and fold-change versus wild-type more than 1.7) in at least two COMPASS mutants. Fold-change of mRNA expression in mutant versus wild-type is indicated by the colour bar as log₂ values. Number of genes below each heatmap correspond to the genes called significant in each mutant. (C) Genes depicted in the same order as in B for the H3K4R point mutant and <i>bre1</i>Δ.</p