Mll1 is essential for the senescenceassociated secretory phenotype

Oncogene-induced senescence (OIS) and therapy-induced senescence (TIS), while tumor-suppressive, also promote procarcinogenic effects by activating the DNA damage response (DDR), which in turn induces inflammation. This inflammatory response prominently includes an array of cytokines known as the senescence-associated secretory phenotype (SASP). Previous observations link the transcription-associated methyltransferase and oncoprotein MLL1 to the DDR, leading us to investigate the role of MLL1 in SASP expression. Our findings reveal direct MLL1 epigenetic control over proproliferative cell cycle genes: MLL1 inhibition represses expression of proproliferative cell cycle regulators required for DNA replication and DDR activation, thus disabling SASP expression. Strikingly, however, these effects of MLL1 inhibition on SASP gene expression do not impair OIS and, furthermore, abolish the ability of the SASP to enhance cancer cell proliferation. More broadly, MLL1 inhibition also reduces “SASP-like” inflammatory gene expression from cancer cells in vitro and in vivo independently of senescence. Taken together, these data demonstrate that MLL1 inhibition may be a powerful and effective strategy for inducing cancerous growth arrest through the direct epigenetic regulation of proliferation-promoting genes and the avoidance of deleterious OIS- or TIS-related tumor secretomes, which can promote both drug resistance and tumor progression

Lamin B1 Depletion in Senescent Cells Triggers Large-Scale Changes in Gene Expression and the Chromatin Landscape

Senescence is a stable proliferation arrest, associated with an altered secretory pathway, thought to promote tumor suppression and tissue aging. While chromatin regulation and lamin B1 down-regulation have been implicated as senescence effectors, functional interactions between them are poorly understood. We compared genome-wide Lys4 trimethylation on histone H3 (H3K4me3) and H3K27me3 distributions between proliferating and senescent human cells and found dramatic differences in senescence, including large-scale domains of H3K4me3- and H3K27me3-enriched “mesas” and H3K27me3-depleted “canyons.” Mesas form at lamin B1-associated domains (LADs) in replicative senescence and oncogene-induced senescence and overlap DNA hypomethylation regions in cancer, suggesting that pre-malignant senescent chromatin changes foreshadow epigenetic cancer changes. Hutchinson-Gilford progeria syndrome fibroblasts (mutant lamin A) also show evidence of H3K4me3 mesas, suggesting a link between premature chromatin changes and accelerated cell senescence. Canyons mostly form between LADs and are enriched in genes and enhancers. H3K27me3 loss is correlated with up-regulation of key senescence genes, indicating a link between global chromatin changes and local gene expression regulation. Lamin B1 reduction in proliferating cells triggers senescence and formation of mesas and canyons. Our data illustrate profound chromatin reorganization during senescence and suggest that lamin B1 down-regulation in senescence is a key trigger of global and local chromatin changes that impact gene expression, aging, and cancer

Lysosome-mediated processing of chromatin in senescence

Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C–negative, but strongly γ-H2AX–positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression

H4K44 Acetylation Facilitates Chromatin Accessibility during Meiosis

Meiotic recombination hotspots are associated with histone post-translational modifications and open chromatin. However, it remains unclear how histone modifications and chromatin structure regulate meiotic recombination. Here, we identify acetylation of histone H4 at Lys44 (H4K44ac) occurring on the nucleosomal lateral surface. We show that H4K44 is acetylated at pre-meiosis and meiosis and displays genome-wide enrichment at recombination hotspots in meiosis. Acetylation at H4K44 is required for normal meiotic recombination, normal levels of double-strand breaks (DSBs) during meiosis, and optimal sporulation. Non-modifiable H4K44R results in increased nucleosomal occupancy around DSB hotspots. Our results indicate that H4K44ac functions to facilitate chromatin accessibility favorable for normal DSB formation and meiotic recombination

Systematic genetic and proteomic screens during gametogenesis identify H2BK34 methylation as an evolutionary conserved meiotic mark

International audienceBackground: Gametes are highly differentiated cells specialized to carry and protect the parental genetic information. During male germ cell maturation, histone proteins undergo distinct changes that result in a highly compacted chromatin organization. Technical difficulties exclude comprehensive analysis of precise histone mutations during mammalian spermatogenesis. The model organism Saccharomyces cerevisiae possesses a differentiation pathway termed sporulation which exhibits striking similarities to mammalian spermatogenesis. This study took advantage of this yeast pathway to first perform systematic mutational and proteomics screens on histones, revealing amino acid residues which are essential for the formation of spores. Methods: A systematic mutational screen has been performed on the histones H2A and H2B, generating ~ 250 mutants using two genetic backgrounds and assessing their ability to form spores. In addition, histones were purified at key stages of sporulation and post-translational modifications analyzed by mass spectrometry. Results: The mutation of 75 H2A H2B residues affected sporulation, many of which were localized to the nucleosome lateral surface. The use of different genetic backgrounds confirmed the importance of many of the residues, as 48% of yeast histone mutants exhibited impaired formation of spores in both genetic backgrounds. Extensive proteomic analysis identified 67 unique post-translational modifications during sporulation, 27 of which were previously unre-ported in yeast. Furthermore, 33 modifications are located on residues that were found to be essential for efficient sporulation in our genetic mutation screens. The quantitative analysis of these modifications revealed a massive deacetylation of all core histones during the pre-meiotic phase and a close interplay between H4 acetylation and methylation during yeast sporulation. Methylation of H2BK37 was also identified as a new histone marker of meiosis and the mouse paralog, H2BK34, was also enriched for methylation during meiosis in the testes, establishing conservation during mammalian spermatogenesis. Conclusion: Our results demonstrate that a combination of genetic and proteomic approaches applied to yeast sporulation can reveal new aspects of chromatin signaling pathways during mammalian spermatogenesis. © The Author(s) 2020. This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article' s Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article

Co-depletion of NIPBL and WAPL balance cohesin activity to correct gene misexpression.

The relationship between cohesin-mediated chromatin looping and gene expression remains unclear. NIPBL and WAPL are two opposing regulators of cohesin activity; depletion of either is associated with changes in both chromatin folding and transcription across a wide range of cell types. However, a direct comparison of their individual and combined effects on gene expression in the same cell type is lacking. We find that NIPBL or WAPL depletion in human HCT116 cells each alter the expression of ~2,000 genes, with only ~30% of the genes shared between the conditions. We find that clusters of differentially expressed genes within the same topologically associated domain (TAD) show coordinated misexpression, suggesting some genomic domains are especially sensitive to both more or less cohesin. Finally, co-depletion of NIPBL and WAPL restores the majority of gene misexpression as compared to either knockdown alone. A similar set of NIPBL-sensitive genes are rescued following CTCF co-depletion. Together, this indicates that altered transcription due to reduced cohesin activity can be functionally offset by removal of either its negative regulator (WAPL) or the physical barriers (CTCF) that restrict loop-extrusion events

Additional information related to Fig 2.

(A) Top 5 GO Biological Processes scored by adjusted p-value for NIPBL DEGs and their significance. (B) Top 5 GO Biological Processes scored by adjusted p-value for WAPL DEGs and their significance. (C) Top 5 GO Biological Processes scored by adjusted p-value for RAD21 DEGs and their significance. (D) The log2(fold change) of shared DEGs across NIPBL and WAPL knockdown conditions. (E) Percentage of up, down, NIPBL, WAPL, or nonDEGs with a TSS within 5kb of a RAD21 ChIP-Seq peak co-occupied by CTCF. Fisher’s exact test, **** p (TIF)</p

Cohesin-sensitive genes are clustered and coordinated within TADs.

(A) The observed average percentage of NIPBL DEGs per TAD compared to a null distribution (expected). Permutations generated by shuffling the DEG and nonDEG designations across genes 1,000 times. Analysis limited to TADs with at least one expressed gene. Exact test, p = 0. (B) The observed average percentage of WAPL DEGs per TAD compared to a null distribution (expected). Permutations generated by shuffling the DEG and nonDEG designations across genes 1,000 times. Analysis limited to TADs with at least one expressed gene. Exact test, p = 0. (C) The average coordination of NIPBL DEGs compared to a null distribution generated by shuffling the fold change amongst the DEGs 1,000 times. Analysis limited to TADs with at least two expressed genes. Exact test, p = 0. (D) The average coordination of WAPL DEGs compared to a null distribution generated by shuffling the fold change amongst the DEGs 1,000 times. Analysis limited to TADs with at least two expressed genes. Exact test, p = 0. (E) Representative TAD with 100% DEG coordination on chr5 which contains six downregulated NIPBL DEGs. Black lines represent TADs, cyan boxes represent loops. (F) Representative TAD with 100% DEG coordination on chr17 which contains nine upregulated NIPBL DEGs. Black lines represent TADs, cyan boxes represent loops.</p