SETD7 regulates the differentiation of human embryonic stem cells

The successful use of specialized cells in regenerative medicine requires an optimization in the differentiation protocols that are currently used. Understanding the molecular events that take place during the differentiation of human pluripotent cells is essential for the improvement of these protocols and the generation of high quality differentiated cells. In an effort to understand the molecular mechanisms that govern differentiation we identify the methyltransferase SETD7 as highly induced during the differentiation of human embryonic stem cells and differentially expressed between induced pluripotent cells and somatic cells. Knock-down of SETD7 causes differentiation defects in human embryonic stem cell including delay in both the silencing of pluripotency-related genes and the induction of differentiation genes. We show that SETD7 methylates linker histone H1 in vitro causing conformational changes in H1. These effects correlate with a decrease in the recruitment of H1 to the pluripotency genes OCT4 and NANOG during differentiation in the SETD7 knockdown that might affect the proper silencing of these genes during differentiation.M.J.B. was partially supported by the Ramón y Cajal program of MEC (RYC-2007-01510). B.S. was a recipient of a predoctoral fellowship from MEC (BES-2008-009567). C.M. was supported by PT13/0001/0041 PRB2-ISCIII-SGEFI- FEDER-PE I+D+i 2013-2016. J.C. was partially supported by Fundación CELLEX. This work was partially supported by grant RD12/0019/0034 TERCEL-RETICS-ISCIII-MINECO-FEDER, grant SAF2009-08588 from MICINN to M.J.B and grant BFU2014-52237 to A.J.Peer Reviewe

Lysine 27 dimethylation of Drosophila linker histone dH1 contributes to heterochromatin organization independently of H3K9 methylation

Post-translational modifications (PTMs) of core histones are important epigenetic determinants that correlate with functional chromatin states. However, despite multiple linker histone H1s PTMs have been identified, little is known about their genomic distribution and contribution to the epigenetic regulation of chromatin. Here, we address this question in Drosophila that encodes a single somatic linker histone, dH1. We previously reported that dH1 is dimethylated at K27 (dH1K27me2). Here, we show that dH1K27me2 is a major PTM of Drosophila heterochromatin. At mitosis, dH1K27me2 accumulates at pericentromeric heterochromatin, while, in interphase, it is also detected at intercalary heterochromatin. ChIPseq experiments show that >98% of dH1K27me2 enriched regions map to heterochromatic repetitive DNA elements, including transposable elements, simple DNA repeats and satellite DNAs. Moreover, expression of a mutated dH1K27A form, which impairs dH1K27me2, alters heterochromatin organization, upregulates expression of heterochromatic transposable elements and results in the accumulation of RNA:DNA hybrids (R-loops) in heterochromatin, without affecting H3K9 methylation and HP1a binding. The pattern of dH1K27me2 is H3K9 methylation independent, as it is equally detected in flies carrying a H3K9R mutation, and is not affected by depletion of Su(var)3–9, HP1a or Su(var)4–20. Altogether these results suggest that dH1K27me2 contributes to heterochromatin organization independently of H3K9 methylation.MICIN/AEI 10.13039/501100011033 [BFU2015-65082-P and PGC2018-094538-B-100]; ‘FEDER, una manera de hacer Europa’; Generalitat de Catalunya [SGR2014-204, SGR2017-475]; this work was carried out within the framework of the ‘Centre de Referencia en Biotecnologia’ of ` the Generalitat de Catalunya. Funding for open access charge: MINECO [PGC2018-094538-B-100]. Conflict of interest statement. None declared

Histone H1 depletion triggers an interferon response in cancer cells via activation of heterochromatic repeats

Histone H1 has seven variants in human somatic cells and contributes to chromatin compaction and transcriptional regulation. Knock-down (KD) of each H1 variant in breast cancer cells results in altered gene expression and proliferation differently in a variant specific manner with H1.2 and H1.4 KDs being most deleterious. Here we show combined depletion of H1.2 and H1.4 has a strong deleterious effect resulting in a strong interferon (IFN) response, as evidenced by an up-regulation of many IFN-stimulated genes (ISGs) not seen in individual nor in other combinations of H1 variant KDs. Although H1 participates to repress ISG promoters, IFN activation upon H1.2 and H1.4 KD is mainly generated through the activation of the IFN response by cytosolic nucleic acid receptors and IFN synthesis, and without changes in histone modifications at induced ISG promoters. H1.2 and H1.4 co-KD also promotes the appearance of accessibility sites genome wide and, particularly, at satellites and other repeats. The IFN response may be triggered by the expression of noncoding RNA generated from heterochromatic repeats or endogenous retroviruses upon H1 KD. In conclusion, redundant H1-mediated silencing of heterochromatin is important to maintain cell homeostasis and to avoid an unspecific IFN response

Histone H1 depletion triggers an interferon response in cancer cells via activation of heterochromatic repeats

Trabajo presentado en la VIII Jornada de Cromatina i Epigenètica, organizada por la Secció de Biologia Molecular de la Societat Catalana de Biologia (SCB) y celebrada el 16 de marzo de 2018Seven linker histone H1 variants exist in human somatic cells with distinct prevalence depending on the cell type and along differentiation. H1 bind to linker DNA contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. It is not well known whether the different variants have specific roles. We have shown that H1 variants are not distributed uniformly along the genome and there are differences between variants, H1.2 being the one showing the most specific pattern. We have explored functions of H1 variants by inducible shRNA-mediated knock-down of each of the variants. Knock-down of each H1 variant alters expression of a different, reduced subset of genes. Combined depletion of H1.2 and H1.4 has a strong deleterious effect in the cancer cells examined, and induces a strong interferon (IFN) response with up-regulation of many IFN-stimulated genes (ISGs). Although H1 participates to repress ISG promoters, its activation upon H1 KD is mainly generated by the expression of noncoding RNA generated from heterochromatic repeats including satellites. In conclusion, redundant H1-mediated silencing of heterochromatin is important to maintain genome stability and to avoid an unspecific growth-inhibiting IFN response.N

The GAGA protein of Drosophila is phosphorylated by CK2

The GAGA factor of Drosophila is a sequence-specific DNA-binding protein that contributes to multiple processes from the regulation of gene expression to the structural organisation of heterochromatin and chromatin remodelling. GAGA is known to interact with various other proteins (tramtrack, pipsqueak, batman and dSAP18) and protein complexes (PRC1, NURF and FACT). GAGA functions are likely regulated at the level of post-translational modifications. Little is known, however, about its actual pattern of modification. It was proposed that GAGA can be O-glycosylated. Here, we report that GAGA519 isoform is a phosphoprotein that is phosphorylated by CK2 at the region of the DNA-binding domain. Our results indicate that phosphorylation occurs at S388 and, to a lesser extent, at S378. These two residues are located in a region of the DNA-binding domain that makes no direct contact with DNA, being dispensable for sequence-specific recognition. Phosphorylation at these sites does not abolish DNA binding but reduces the affinity of the interaction. These results are discussed in the context of the various functions and interactions that GAGA supports. © 2005 Elsevier Ltd. All rights reserved.This work was financed by grants from the Ministerio de Ciencia y Tecnología (BMC2003-243; BMC2003-616; BIO2002-2301) and the CIRIT (2001SGR00344; 2001SGR00047). X.A. acknowledges receipt of a doctoral fellowship from the CIRIT. This work was carried out within the framework of the “Centre de Referència en Biotecnologia” of the Generalitat de CatalunyaPeer Reviewe

Combined bottom-up and top-down mass spectrometry analyses of the pattern of post-translational modifications of Drosophila melanogaster linker histone H1

Linker histone H1 is a major chromatin component that binds internucleosomal DNA and mediates the folding of nucleosomes into a higher-order structure, namely the 30-nm chromatin fiber. Multiple post-translational modifications (PTMs) of core histones H2A, H2B, H3 and H4 have been identified and their important contribution to the regulation of chromatin structure and function is firmly established. In contrast, little is known about histone H1 modifications and their function. Here we address this question in Drosophila melanogaster, which, in contrast to most eukaryotic species, contains a single histone H1 variant, dH1. For this purpose, we combined bottom-up and top-down mass-spectrometry strategies. Our results indicated that dH1 is extensively modified by phosphorylation, methylation, acetylation and ubiquitination, with most PTMs falling in the N-terminal domain. Interestingly, several dH1 N-terminal modifications have also been reported in specific human and/or mouse H1 variants, suggesting that they have conserved functions. In this regard, we also provide evidence for the contribution of one of such conserved PTMs, dimethylation of K27, to heterochromatin organization during mitosis. Furthermore, our results also identified multiple dH1 isoforms carrying several phosphorylations and/or methylations, illustrating the high structural heterogeneity of dH1. In particular, we identified several non-CDK sites at the N-terminal domain that appear to be hierarchically phosphorylated. This study provides the most comprehensive PTM characterization of any histone H1 variant to date. © 2012 Elsevier B.V.This work was supported by grants from MICINN (CSD2006-49, BIO2008-00799 and BFU2009-07111), the CSIC (200420E391) and the “Generalitat de Catalunya” (SGR2009-1023 and SGR2009-1005). This work was carried out within the framework of the “Xarxa de Referència en Biotecnologia” of the “Generalitat de Catalunya”.Peer Reviewe

Acetylation of GAGA factor modulates its interaction with DNA

GAGA is a Drosophila transcription factor that shows a high degree of post-translational modification. Here, we show that GAGA factor is acetylated in vivo. Lysine residues K325 and K373 on basic regions BR1 and BR3 of the DNA binding domain, respectively, are shown to be acetylated by PCAF. While BR1 is strictly required to stabilize DNA binding, BR3 is dispensable. However, acetylation of both lysine residues, either alone or in combination, weakens the binding to DNA. Despite the high degree of conservation of K325 and K373 in flies, their mutation to glutamine does not affect DNA binding. Molecular dynamics simulations, using acetylated K325 and a K325Q mutant of GAGA DNA binding domain in complex with DNA, are fully consistent with these results and provide a thermodynamic explanation for this observation. We propose that while K325 and K373 are not essential for DNA binding they have been largely conserved for regulatory purposes, thus highlighting a key regulatory system for GAGA factor in flies. © 2010 American Chemical Society.Este trabajo fue financiado por el Ministerio de Ciencia e Innovación del Gobierno de España (concesiones BFU2006-09761 y BFU2007-64395/BMC para Jordi Benués), y fue llevado a cabo dentro del marco del “Centre de Referència en Biotecnologia” de la Generalitat de Catalunya.Peer Reviewe

The SETD7 knock-down affects the cell cycle profile of differentiating cells.

<p>(A) Cell cycle profile of undifferentiated and after 15 days of <i>in vitro</i> differentiation of ES[4] transduced with a non target shRNA (shSCR) and a shRNA that targets SETD7 (shSETD7). (B) Mean of the percentage of cells in each phase of the cell cycle in three independent differentiation experiments. *Differences in the percentage of cells in S-phase between shSETD7 and shSCR in differentiated cells was found significant at a p-value<0.05.</p

Residues modified by SETD7 in H1.0 and H1.4 found by mass spectrometry.

<p>Residues modified by SETD7 in H1.0 and H1.4 found by mass spectrometry.</p