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Protein arginine deimination leading to the non-coded amino acid citrulline remains a key question in the field of post-translational modifications ever since its discovery by Rogers and Simmonds in 1958. Citrullination is catalyzed by a family of enzymes called peptidyl arginine deiminases (PADIs). Initially, increased citrullination was associated with autoimmune diseases, including rheumatoid arthritis and multiple sclerosis, as well as other neurological disorders and multiple types of cancer. During the last decade, research efforts have focused on how citrullination contributes to disease pathogenesis by modulating epigenetic events, pluripotency, immunity and transcriptional regulation. However, our knowledge regarding the functional implications of citrullination remains quite limited, so we still do not completely understand its role in physiological and pathological conditions. Here, we review the recently discovered functions of PADI2-mediated citrullination of the C-terminal domain of RNA polymerase II in transcriptional regulation in breast cancer cells and the proposed mechanisms to reshape the transcription regulatory network that promotes cancer progression. ; This work was supported by Spanish MEC (SAF2016–75006), the Catalan Government (2017–2019 SGR 747_MBeato), and the European Research Council Synergy Grant "4DGenome" (609989). Also, we acknowledge the support of the Spanish Ministry of Economy and Competitiveness, "Centro de Excelencia Severo Ochoa" and the CERCA Programme/Generalitat de Catalunya"

Initial studies showed that ligand-activated hormone receptors act by binding to the proximal promoters of individual target genes. Genome-wide studies have now revealed that regulation of transcription by steroid hormones mainly depends on binding of the receptors to distal regulatory elements. Those distal elements, either enhancers or silencers, act on the regulation of target genes by chromatin looping to the gene promoters. In the nucleus, this level of chromatin folding is integrated within dynamic higher orders of genome structures, which are organized in a non-random fashion. Terminally differentiated cells exhibit a tissue-specific three-dimensional (3D) organization of the genome that favors or restrains the activity of transcription factors and modulates the function of steroid hormone receptors, which are transiently activated upon hormone exposure. Conversely, integration of the hormones signal may require modifications of the 3D organization to allow appropriate transcriptional outcomes. In this review, we summarize the main levels of organization of the genome, review how they can modulate the response to steroids in a cell specific manner and discuss the role of receptors in shaping and rewiring the structure in response to hormone. Taking into account the dynamics of 3D genome organization will contribute to a better understanding of the pleiotropic effects of steroid hormones in normal and cancer cells. ; We thank all members of the Chromatin and Gene Expression group (CRG, Barcelona) and the members of the 4DGenome project (CRG and CNAG-CRG, Barcelona) for helpful discussions. Research in the Beato's laboratory receives funding from the European Research Council under the European Union's Seventh Framework Programme (FP7/2007-2013)/ERC Synergy grant agreement 609989 (4DGenome). The content of this manuscript reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. We also acknowledge support of the Spanish Ministry of Economy and Competitiveness, "Centro de Excelencia Severo Ochoa 2013-2017" and Plan Nacional (SAF2016-75006-P), as well as support of the CERCA Programme/Generalitat de Catalunya.

At least six histone H1 variants exist in somatic mammalian cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition, H1 seems to be actively involved in the regulation of gene expression. However, it is not well known whether the different variants have distinct roles or if they regulate specific promoters. We have explored this by inducible shRNA-mediated knock-down of each of the H1 variants in a human breast cancer cell line. Rapid inhibition of each H1 variant was not compensated for by changes of expression of other variants. Microarray experiments have shown a different subset of genes to be altered in each H1 knock-down. Interestingly, H1.2 depletion caused specific effects such as a cell cycle G1-phase arrest, the repressed expression of a number of cell cycle genes, and decreased global nucleosome spacing. On its side, H1.4 depletion caused cell death in T47D cells, providing the first evidence of the essential role of an H1 variant for survival in a human cell type. Thus, specific phenotypes are observed in breast cancer cells depleted of individual histone H1 variants, supporting the theory that distinct roles exist for the linker histone variants. © 2008 Sancho et al. ; This work was supported by grants from the Departament d'Universitats, Recerca i Societat de la Informacio (DURSI), and Ministerio de Ciencia y Tecnología (MCYT) and FEDER (SAF2002-03320). AJ was recipient of a 'Ramón y Cajal' appointment from the MCYT; MS was recipient of a FPI predoctoral fellowship from the Ministerio de Educación; ED was recipient of a Leonardo da Vinci-Unipharma short-term fellowship from the European Union ; Peer Reviewed

BACKGROUND: Recent advances in the field of high-throughput genomics have rendered possible the performance of genome-scale studies to define the nucleosomal landscapes of eukaryote genomes. Such analyses are aimed towards providing a better understanding of the process of nucleosome positioning, for which several models have been suggested. Nevertheless, questions regarding the sequence constraints of nucleosomal DNA and how they may have been shaped through evolution remain open. In this paper, we analyze in detail different experimental nucleosome datasets with the aim of providing a hypothesis for the emergence of nucleosome-forming sequences. RESULTS: We compared the complete sets of nucleosome positions for the budding yeast (Saccharomyces cerevisiae) as defined in the output of two independent experiments with the use of two different experimental techniques. We found that < 10% of the experimentally defined nucleosome positions were consistently positioned in both datasets. This subset of well-positioned nucleosomes, when compared with the bulk, was shown to have particular properties at both sequence and structural levels. Consistently positioned nucleosomes were also shown to occur preferentially in pairs of dinucleosomes, and to be surprisingly less conserved compared with their adjacent nucleosome-free linkers. CONCLUSION: Our findings may be combined into a hypothesis for the emergence of a weak nucleosome-positioning code. According to this hypothesis, consistent nucleosomes may be partly guided by nearby nucleosome-free regions through statistical positioning. Once established, a set of well-positioned consistent nucleosomes may impose secondary constraints that further shape the structure of the underlying DNA. We were able to capture these constraints through the application of a recently introduced structural property that is related to the symmetry of DNA curvature. Furthermore, we found that both consistently positioned nucleosomes and their adjacent nucleosome-free regions show an increased tendency towards conservation of this structural feature. ; This work was funded by grants from the Spanish Ministry of Education and Science (Plan Nacional BIO2006-03380) and Consolider project CSD2006-0004 as well as the European Union International Project HEROIC (018883).

Chromatin is known to undergo extensive remodeling during nuclear reprogramming. However, the factors and mechanisms involved in this remodeling are still poorly understood and current experimental approaches to study it are not best suited for molecular and genetic analyses. Here we report on the use of Drosophila preblastodermic embryo extracts (DREX) in chromatin remodeling experiments. Our results show that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming, such as rapid binding of the germline specific linker histone dBigH1 variant to somatic chromatin, heterochromatin reorganization, changes in the epigenetic state of chromatin, and nuclear lamin disassembly. These results raise the possibility of using the powerful tools of Drosophila genetics for the analysis of chromatin changes associated with this essential process. ; This work was supported by grants from MINECO (BFU2015-65082-P), the Generalitat de Catalunya (SGR2014-204), and the European Community FEDER program. This work was carried out within the framework of the "Centre de Referència en Biotecnologia" of the Generalitat de Catalunya. EŠ acknowledges receipt of a NEWFELPRO Fellowship of the Croatian Government and Ministry of Science and Education within EU FP7 Programme framework. ; Peer reviewed

Chromatin is known to undergo extensive remodeling during nuclear reprogramming. However, the factors and mechanisms involved in this remodeling are still poorly understood and current experimental approaches to study it are not best suited for molecular and genetic analyses. Here we report on the use of Drosophila preblastodermic embryo extracts (DREX) in chromatin remodeling experiments. Our results show that incubation of somatic nuclei in DREX induces changes in chromatin organization similar to those associated with nuclear reprogramming, such as rapid binding of the germline specific linker histone dBigH1 variant to somatic chromatin, heterochromatin reorganization, changes in the epigenetic state of chromatin, and nuclear lamin disassembly. These results raise the possibility of using the powerful tools of Drosophila genetics for the analysis of chromatin changes associated with this essential process. ; This work was supported by grants from MINECO (BFU2015-65082-P), the Generalitat de Catalunya (SGR2014-204), and the European Community FEDER program. This work was carried out within the framework of the "Centre de Referència en Biotecnologia" of the Generalitat de Catalunya. EŠ acknowledges receipt of a NEWFELPRO Fellowship of the Croatian Government and Ministry of Science and Education within EU FP7 Programme framework.

Breast cancer prognosis and response to endocrine therapy strongly depends on the expression of the estrogen and progesterone receptors (ER and PR, respectively). Although much is known about ERα gene (ESR1) regulation after hormonal stimulation, how it is regulated in hormone-free condition is not fully understood. We used ER-/PR-positive breast cancer cells to investigate the role of PR in ESR1 regulation in the absence of hormones. We show that PR binds to the low-methylated ESR1 promoter and maintains both gene expression and DNA methylation of the ESR1 locus in hormone-deprived breast cancer cells. Depletion of PR reduces ESR1 expression, with a concomitant increase in gene promoter methylation. The high amount of methylation in the ESR1 promoter of PR-depleted cells persists after the stable re-expression of PR and inhibits PR binding to this genomic region. As a consequence, the rescue of PR expression in PR-depleted cells is insufficient to restore ESR1 expression. Consistently, DNA methylation impedes PR binding to consensus progesterone responsive elements. These findings contribute to understanding the complex crosstalk between PR and ER and suggest that the analysis of ESR1 promoter methylation in breast cancer cells can help to design more appropriate targeted therapies for breast cancer patients. ; We received funding from the Spanish Ministry of Economy and Competitiveness, Plan Nacional Project SAF 2013-42497-P; Centro de Excelencia Severo Ochoa 2013–2017; the Centre de Recerca de Catalunya (CERCA) Programme/Generalitat de Catalunya; G.V. has received funding from the Spanish Ministry of Economy and Competitiveness, "Juan de la Cierva Incorporation" fellowship (Ref. IJCI-2014-20723), the European Union Seventh Framework Programme (FP7/2007-2013) under Grant Agreement Number 299429 and the European Molecular Biology Organization (EMBO long-term fellowship ALTF 1106-2011, cofunded with the European Commission EMBOCOFUND2010, GA-2010-267146).

While the importance of gene enhancers in transcriptional regulation is well established, the mechanisms and the protein factors that determine enhancers activity have only recently begun to be unravelled. Recent studies have shown that progesterone receptor (PR) binds regions that display typical features of gene enhancers. Here, we show by ChIP-seq experiments that the chromatin remodeler CHD8 mostly binds promoters under proliferation conditions. However, upon progestin stimulation, CHD8 re-localizes to PR enhancers also enriched in p300 and H3K4me1. Consistently, CHD8 depletion severely impairs progestin-dependent gene regulation. CHD8 binding is PR-dependent but independent of the pioneering factor FOXA1. The SWI/SNF chromatin-remodelling complex is required for PR-dependent gene activation. Interestingly, we show that CHD8 interacts with the SWI/SNF complex and that depletion of BRG1 and BRM, the ATPases of SWI/SNF complex, impairs CHD8 recruitment. We also show that CHD8 is not required for H3K27 acetylation, but contributes to increase accessibility of the enhancer to DNaseI. Furthermore, CHD8 was required for RNAPII recruiting to the enhancers and for transcription of enhancer-derived RNAs (eRNAs). Taken together our data demonstrate that CHD8 is involved in late stages of PR enhancers activation. ; This work was supported by Spanish Ministerio de Ciencia e Innovación (BFU2011-23442, CSD2006-00049 to JCR and BMC 2003-02902 and 2010-15313; CSD2006-00049, to MB and an FPU fellowship to EVC); Junta de Andalucía (P06-CVI- 4844) and Fundación Ramón Areces to JCR, and by the Catalan government (AGAUR) to MB. ASR was recipient of a JAE grant from CSIC. ; Peer Reviewed

Nuclear architecture is decisive for the assembly of transcriptional responses. However, how chromosome organization is dynamically modulated to permit rapid and transient transcriptional changes in response to environmental challenges remains unclear. Here we show that hyperosmotic stress disrupts different levels of chromosome organization, ranging from A/B compartment changes to reduction in the number and insulation of topologically associating domains (TADs). Concomitantly, transcription is greatly affected, TAD borders weaken, and RNA Polymerase II runs off from hundreds of transcription end sites. Stress alters the binding profiles of architectural proteins, which explains the disappearance of local chromatin organization. These processes are dynamic, and cells rapidly reconstitute their default chromatin conformation after stress removal, uncovering an intrinsic organization. Transcription is not required for local chromatin reorganization, while compartment recovery is partially transcription-dependent. Thus, nuclear organization in mammalian cells can be rapidly modulated by environmental changes in a reversible manner. ; The study was supported by grants from the Spanish Ministry of Economy and Competitiveness (BFU2015-64437-P and FEDER, BFU2014-52125-REDT, and BFU2014-51672-REDC to F.P.; BFU2017-85152-P and FEDER to E.d.N.), the Catalan Government (2017 SGR 799), the Fundación Botín, and the Banco Santander through its Santander Universities Global Division to F.P. and the Unidad de Excelencia Maria de Maeztu, MDM-2014-0370. F.P. is recipient of an ICREA Acadèmia (Generalitat de Catalunya). M.B. received the support of the European Research Council (ERC Synergy Grant 4D Genome 609989) and CERCA.

While protein ADP-ribosylation was reported to regulate differentiation and dedifferentiation, it has so far not been studied during transdifferentiation. Here, we found that MyoD-induced transdifferentiation of fibroblasts to myoblasts promotes the expression of the ADP-ribosyltransferase ARTD1. Comprehensive analysis of the genome architecture by Hi-C and RNA-seq analysis during transdifferentiation indicated that ARTD1 locally contributed to A/B compartmentalization and coregulated a subset of MyoD target genes that were however not sufficient to alter transdifferentiation. Surprisingly, the expression of ARTD1 was accompanied by the continuous synthesis of nuclear ADP ribosylation that was neither dependent on the cell cycle nor induced by DNA damage. Conversely to the H2O2-induced ADP-ribosylation, the MyoD-dependent ADP-ribosylation was not associated to chromatin but rather localized to the nucleoplasm. Together, these data describe a MyoD-induced nucleoplasmic ADP-ribosylation that is observed particularly during transdifferentiation and thus potentially expands the plethora of cellular processes associated with ADP-ribosylation. ; Research in Beato's lab is supported by funds from the European Research Council under the European Union's Seventh Framework Program (FP7/2007-2013)/ERC Synergy grant agreement 609989 (4DGenome), from the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013-2017' and Plan Nacional (SAF2016-75006-P), as well as support of the CERCA Program/Generalitat de Catalunya. L.B was supported by the Forschungskredit 2019 of the University of Zurich and K.G by a grant of the Oncosuisse (Nr. KFS-3740-08-2015-R). ADP-ribosylation research in the laboratory of MOH is funded by the Kanton of Zurich and the Swiss National Science Foundation, Switzerland (grant 31003A_176177).

In fission yeast cells, Cds1 is the effector kinase of the DNA replication checkpoint. We previously showed that when the DNA replication checkpoint is activated, the repressor Yox1 is phosphorylated and inactivated by Cds1, resulting in activation of MluI-binding factor (MBF)-dependent transcription. This is essential to reinitiate DNA synthesis and for correct G1-to-S transition. Here we show that Cdc10, which is an essential part of the MBF core, is the target of the DNA damage checkpoint. When fission yeast cells are treated with DNA-damaging agents, Chk1 is activated and phosphorylates Cdc10 at its carboxy-terminal domain. This modification is responsible for the repression of MBF-dependent transcription through induced release of MBF from chromatin. This inactivation of MBF is important for survival of cells challenged with DNA-damaging agents. Thus Yox1 and Cdc10 couple normal cell cycle regulation in unperturbed conditions and the DNA replication and DNA damage checkpoints into a single transcriptional complex. ; The experimental work was supported by grants from the Spanish government (BMC 2003-02902 and 2010-15313; CSD2006-00049), the European Union (IP HEROIC), and the Catalan government (AGAUR). L.G. was a recipient of a fellowship from the International PhD program of LaCaixa; G.P.V. was a recipient of a fellowship from the Ramón y Cajal program.

How repetitive elements, epigenetic modifications, and architectural proteins interact ensuring proper genome expression remains poorly understood. Here, we report regulatory mechanisms unveiling a central role of Alu elements (AEs) and RNA polymerase III transcription factor C (TFIIIC) in structurally and functionally modulating the genome via chromatin looping and histone acetylation. Upon serum deprivation, a subset of AEs pre-marked by the activity-dependent neuroprotector homeobox Protein (ADNP) and located near cell-cycle genes recruits TFIIIC, which alters their chromatin accessibility by direct acetylation of histone H3 lysine-18 (H3K18). This facilitates the contacts of AEs with distant CTCF sites near promoter of other cell-cycle genes, which also become hyperacetylated at H3K18. These changes ensure basal transcription of cell-cycle genes and are critical for their re-activation upon serum re-exposure. Our study reveals how direct manipulation of the epigenetic state of AEs by a general transcription factor regulates 3D genome folding and expression. ; This work was supported by the Spanish Ministry of Economy and Competitiveness Centro de Excelencia Severo Ochoa 2013–2017 SEV-2012-0208 (to CRG) and BFU2016-76141-P (to S.d.l.L.); ACER (to CRG); Italian Association for Cancer Research (AIRC, grant IG16877 to G.D.); the Cancer Research UK Programme Foundation (CR-UK C47547/A21536 to A.V.); a Wellcome Trust Investigator Award (200818/Z/16/Z to A.V.); and the European Research Council (ERC) under the European Union's Seventh Framework Programme (FP7/2007–2013/ERC Synergy grant agreement 609989-4DGenome to M.B.). We acknowledge the support of the Spanish Ministry of Science, Innovation, and Universities to the EMBL partnership, the CERCA Programme/Generalitat de Catalunya , and the Centro de Excelencia Severo Ochoa. The ERC provided funding for the open access charge. The proteomics analyses were performed at the CRG/UPF Proteomics Unit (part of the of Proteored, PRB3; supported by grant PT17/0019 [ISCIII and ERDF]). This work was also supported by the Ligue Contre le Cancer (to M.T.)

In breast cancer cells, some topologically associating domains (TADs) behave as hormonal gene regulation units, within which gene transcription is coordinately regulated in response to steroid hormones. Here we further describe that responsive TADs contain 20- to 100-kb-long clusters of intermingled estrogen receptor (ESR1) and progesterone receptor (PGR) binding sites, hereafter called hormone-control regions (HCRs). In T47D cells, we identified more than 200 HCRs, which are frequently bound by unliganded ESR1 and PGR. These HCRs establish steady long-distance inter-TAD interactions between them and organize characteristic looping structures with promoters in their TADs even in the absence of hormones in ESR1+-PGR+ cells. This organization is dependent on the expression of the receptors and is further dynamically modulated in response to steroid hormones. HCRs function as platforms that integrate different signals, resulting in some cases in opposite transcriptional responses to estrogens or progestins. Altogether, these results suggest that steroid hormone receptors act not only as hormone-regulated sequence-specific transcription factors but also as local and global genome organizers. ; We received funding from the European Research Council under the European Union's Seventh Framework Program (FP7/2007–2013)/ERC Synergy grant agreement 609989 (4DGenome). The content of this manuscript reflects only the author's views and the Union is not liable for any use that may be made of the information contained therein. We acknowledge support of the Spanish Ministry of Economy and Competitiveness, 'Centro de Excelencia Severo Ochoa 2013–2017' and Plan Nacional (SAF2016-75006-P), as well as support of the CERCA Programme/Generalitat de Catalunya.