Browsing by MeSH term "Chromatin"
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Publication 3D chromatin connectivity underlies replication origin efficiency in mouse embryonic stem cells.(Oxford University Press, 2022-11-28) Jodkowska, Karolina; Pancaldi, Vera; Rigau, Maria; Almeida, Ricardo; Fernández-Justel, José M; Graña-Castro, Osvaldo; Rodríguez-Acebes, Sara; Rubio-Camarillo, Miriam; Carrillo-de Santa Pau, Enrique; Pisano, David; Al-Shahrour, Fatima; Valencia, Alfonso; Gómez, María; Méndez, Juan; Ministerio de Ciencia y Tecnología (España); Fundação para a Ciência e Tecnologia (Portugal); Fundación La Caixa; Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF); Foundation for Polish ScienceIn mammalian cells, chromosomal replication starts at thousands of origins at which replisomes are assembled. Replicative stress triggers additional initiation events from 'dormant' origins whose genomic distribution and regulation are not well understood. In this study, we have analyzed origin activity in mouse embryonic stem cells in the absence or presence of mild replicative stress induced by aphidicolin, a DNA polymerase inhibitor, or by deregulation of origin licensing factor CDC6. In both cases, we observe that the majority of stress-responsive origins are also active in a small fraction of the cell population in a normal S phase, and stress increases their frequency of activation. In a search for the molecular determinants of origin efficiency, we compared the genetic and epigenetic features of origins displaying different levels of activation, and integrated their genomic positions in three-dimensional chromatin interaction networks derived from high-depth Hi-C and promoter-capture Hi-C data. We report that origin efficiency is directly proportional to the proximity to transcriptional start sites and to the number of contacts established between origin-containing chromatin fragments, supporting the organization of origins in higher-level DNA replication factories.Publication A Multisensory Network Drives Nuclear Mechanoadaptation.(Multidisciplinary Digital Publishing Institute (MDPI), 2022-03-04) Echarri, Asier; Instituto de Salud Carlos III; Ministerio de Ciencia e Innovación (España); Fundación ProCNIC; Ministerio de Ciencia e Innovación. Centro de Excelencia Severo Ochoa (España)Cells have adapted to mechanical forces early in evolution and have developed multiple mechanisms ensuring sensing of, and adaptation to, the diversity of forces operating outside and within organisms. The nucleus must necessarily adapt to all types of mechanical signals, as its functions are essential for virtually all cell processes, many of which are tuned by mechanical cues. To sense forces, the nucleus is physically connected with the cytoskeleton, which senses and transmits forces generated outside and inside the cell. The nuclear LINC complex bridges the cytoskeleton and the nuclear lamina to transmit mechanical information up to the chromatin. This system creates a force-sensing macromolecular complex that, however, is not sufficient to regulate all nuclear mechanoadaptation processes. Within the nucleus, additional mechanosensitive structures, including the nuclear envelope and the nuclear pore complex, function to regulate nuclear mechanoadaptation. Similarly, extra nuclear mechanosensitive systems based on plasma membrane dynamics, mechanotransduce information to the nucleus. Thus, the nucleus has the intrinsic structural components needed to receive and interpret mechanical inputs, but also rely on extra nuclear mechano-sensors that activate nuclear regulators in response to force. Thus, a network of mechanosensitive cell structures ensures that the nucleus has a tunable response to mechanical cues.Publication A short G1 phase imposes constitutive replication stress and fork remodelling in mouse embryonic stem cells(Nature Publishing Group, 2016-02-15) Ahuja, Akshay K; Jodkowska, Karolina; Teloni, Federico; Bizard, Anna H; Zellweger, Ralph; Herrador, Raquel; Ortega Jimenez, Sagrario; Hickson, Ian D; Altmeyer, Matthias; Mendez, Juan; Lopes, Massimo; Danish National Research Foundation; Ministerio de Economía y Competitividad (España); Unión Europea. Comisión Europea. European Research Council (ERC)Embryonic stem cells (ESCs) represent a transient biological state, where pluripotency is coupled with fast proliferation. ESCs display a constitutively active DNA damage response (DDR), but its molecular determinants have remained elusive. Here we show in cultured ESCs and mouse embryos that H2AX phosphorylation is dependent on Ataxia telangiectasia and Rad3 related (ATR) and is associated with chromatin loading of the ssDNA-binding proteins RPA and RAD51. Single-molecule analysis of replication intermediates reveals massive ssDNA gap accumulation, reduced fork speed and frequent fork reversal. All these marks of replication stress do not impair the mitotic process and are rapidly lost at differentiation onset. Delaying the G1/S transition in ESCs allows formation of 53BP1 nuclear bodies and suppresses ssDNA accumulation, fork slowing and reversal in the following S-phase. Genetic inactivation of fork slowing and reversal leads to chromosomal breakage in unperturbed ESCs. We propose that rapid cell cycle progression makes ESCs dependent on effective replication-coupled mechanisms to protect genome integrity.Publication Ammonia Affects Astroglial Proliferation in Culture(Public Library of Science (PLOS), 2015-09-30) Bodega, Guillermo; Segura-Collar, Berta; Ciordia, Sergio; Mena, María Del Carmen; López-Fernández, Luis Andrés; García, María Isabel; Trabado, Isabel; Suárez, Isabel; University of Alcalá (España)Primary cultures of rat astroglial cells were exposed to 1, 3 and 5 mM NH4Cl for up to 10 days. Dose- and time-dependent reductions in cell numbers were seen, plus an increase in the proportion of cells in the S phase. The DNA content was reduced in the treated cells, and BrdU incorporation diminished. However, neither ammonia nor ammonia plus glutamine had any effect on DNA polymerase activity. iTRAQ analysis showed that exposure to ammonia induced a significant reduction in histone and heterochromatin protein 1 expression. A reduction in cell viability was also noted. The ammonia-induced reduction of proliferative activity in these cultured astroglial cells seems to be due to a delay in the completion of the S phase provoked by the inhibition of chromatin protein synthesis.Publication ATM regulates ATR chromatin loading in response to DNA double-strand breaks.(Rockefeller University Press, 2006-02-20) Cuadrado, Myriam; Martinez-Pastor, Barbara; Murga, Matilde; Toledo, Luis I; Gutierrez-Martinez, Paula; Lopez, Eva; Fernandez-Capetillo, Oscar; Ministerio de Ciencia y Competitividad (España); Fundación La CaixaDNA double-strand breaks (DSBs) are among the most deleterious lesions that can challenge genomic integrity. Concomitant to the repair of the breaks, a rapid signaling cascade must be coordinated at the lesion site that leads to the activation of cell cycle checkpoints and/or apoptosis. In this context, ataxia telangiectasia mutated (ATM) and ATM and Rad-3-related (ATR) protein kinases are the earliest signaling molecules that are known to initiate the transduction cascade at damage sites. The current model places ATM and ATR in separate molecular routes that orchestrate distinct pathways of the checkpoint responses. Whereas ATM signals DSBs arising from ionizing radiation (IR) through a Chk2-dependent pathway, ATR is activated in a variety of replication-linked DSBs and leads to activation of the checkpoints in a Chk1 kinase-dependent manner. However, activation of the G2/M checkpoint in response to IR escapes this accepted paradigm because it is dependent on both ATM and ATR but independent of Chk2. Our data provides an explanation for this observation and places ATM activity upstream of ATR recruitment to IR-damaged chromatin. These data provide experimental evidence of an active cross talk between ATM and ATR signaling pathways in response to DNA damage.Publication BRCA1 Haploinsufficiency Is Masked by RNF168-Mediated Chromatin Ubiquitylation.(Cell Press, 2019-03-21) Zong, Dali; Adam, Salomé; Wang, Yifan; Sasanuma, Hiroyuki; Callén, Elsa; Murga, Matilde; Day, Amanda; Kruhlak, Michael J; Wong, Nancy; Munro, Meagan; Ray Chaudhuri, Arnab; Karim, Baktiar; Xia, Bing; Takeda, Shunichi; Johnson, Neil; Durocher, Daniel; Nussenzweig, André; Ministerio de Educación, Cultura y Deporte (España); United States Department of Health and Human Services; Lawrence Ellison Foundation; United States Department of Defense; Alex's Lemonade Stand Foundation; NIH - National Cancer Institute (NCI) (Estados Unidos); Rutgers Cancer InstituteBRCA1 functions at two distinct steps during homologous recombination (HR). Initially, it promotes DNA end resection, and subsequently it recruits the PALB2 and BRCA2 mediator complex, which stabilizes RAD51-DNA nucleoprotein filaments. Loss of 53BP1 rescues the HR defect in BRCA1-deficient cells by increasing resection, suggesting that BRCA1's downstream role in RAD51 loading is dispensable when 53BP1 is absent. Here we show that the E3 ubiquitin ligase RNF168, in addition to its canonical role in inhibiting end resection, acts in a redundant manner with BRCA1 to load PALB2 onto damaged DNA. Loss of RNF168 negates the synthetic rescue of BRCA1 deficiency by 53BP1 deletion, and it predisposes BRCA1 heterozygous mice to cancer. BRCA1+/-RNF168-/- cells lack RAD51 foci and are hypersensitive to PARP inhibitor, whereas forced targeting of PALB2 to DNA breaks in mutant cells circumvents BRCA1 haploinsufficiency. Inhibiting the chromatin ubiquitin pathway may, therefore, be a synthetic lethality strategy for BRCA1-deficient cancers.Publication Chromatin dynamics through mouse preimplantation development revealed by single molecule localisation microscopy.(The Company of Biologists, 2022-08-15) Portela, Marta; Jimenez-Carretero, Daniel; Labrador, Veronica; Andreu, Maria Jose; Arza, Elvira; Caiolfa, Valeria R; Manzanares, Miguel; Agencia Estatal de Investigación (España); Comunidad de Madrid (España); Ministerio de Ciencia e Innovación (España); Fundación Ramón Areces; Instituto de Salud Carlos III; Fundación ProCNIC; Ministerio de Ciencia e Innovación. Centro de Excelencia Severo Ochoa (España)Most studies addressing chromatin behaviour during preimplantation development are based on biochemical assays that lack spatial and cell-specific information, crucial during early development. Here, we describe the changes in chromatin taking place at the transition from totipotency to lineage specification, by using direct stochastical optical reconstruction microscopy (dSTORM) in whole-mount embryos during the first stages of mouse development. Through the study of two post-translational modifications of Histone 3 related to active and repressed chromatin, H3K4me3 and H3K9me3 respectively, we obtained a time-course of chromatin states, showing spatial differences between cell types, related to their differentiation state. This analysis adds a new layer of information to previous biochemical studies and provides novel insight to current models of chromatin organisation during the first stages of development.Publication Chromatin insulation dynamics in glioblastoma: challenges and future perspectives of precision oncology(BioMed Central (BMC), 2021-12) Sesé, Borja; Ensenyat-Mendez, Miquel; Iñiguez, Sandra; Llinàs-Arias, Pere; Marzese, Diego MGlioblastoma (GBM) is the most aggressive primary brain tumor, having a poor prognosis and a median overall survival of less than two years. Over the last decade, numerous findings regarding the distinct molecular and genetic profiles of GBM have led to the emergence of several therapeutic approaches. Unfortunately, none of them has proven to be effective against GBM progression and recurrence. Epigenetic mechanisms underlying GBM tumor biology, including histone modifications, DNA methylation, and chromatin architecture, have become an attractive target for novel drug discovery strategies. Alterations on chromatin insulator elements (IEs) might lead to aberrant chromatin remodeling via DNA loop formation, causing oncogene reactivation in several types of cancer, including GBM. Importantly, it is shown that mutations affecting the isocitrate dehydrogenase (IDH) 1 and 2 genes, one of the most frequent genetic alterations in gliomas, lead to genome-wide DNA hypermethylation and the consequent IE dysfunction. The relevance of IEs has also been observed in a small population of cancer stem cells known as glioma stem cells (GSCs), which are thought to participate in GBM tumor initiation and drug resistance. Recent studies revealed that epigenomic alterations, specifically chromatin insulation and DNA loop formation, play a crucial role in establishing and maintaining the GSC transcriptional program. This review focuses on the relevance of IEs in GBM biology and their implementation as a potential theranostic target to stratify GBM patients and develop novel therapeutic approaches. We will also discuss the state-of-the-art emerging technologies using big data analysis and how they will settle the bases on future diagnosis and treatment strategies in GBM patients.Publication Cohesin organizes chromatin loops at DNA replication factories.(Cold Spring Harbor Laboratory Press, 2010-12-15) Guillou, Emmanuelle; Ibarra, Arkaitz; Coulon, Vincent; Casado-Vela, Juan; Rico, Daniel; Casal, Ignacio; Schwob, Etienne; Losada, Ana; Mendez, Juan; Ministerio de Ciencia e Innovación (España); French National Cancer Institute; Fundación Caja Madrid; Fondation pour la recherche médicale (Francia); Basque Government (España)Genomic DNA is packed in chromatin fibers organized in higher-order structures within the interphase nucleus. One level of organization involves the formation of chromatin loops that may provide a favorable environment to processes such as DNA replication, transcription, and repair. However, little is known about the mechanistic basis of this structuration. Here we demonstrate that cohesin participates in the spatial organization of DNA replication factories in human cells. Cohesin is enriched at replication origins and interacts with prereplication complex proteins. Down-regulation of cohesin slows down S-phase progression by limiting the number of active origins and increasing the length of chromatin loops that correspond with replicon units. These results give a new dimension to the role of cohesin in the architectural organization of interphase chromatin, by showing its participation in DNA replication.Publication Contribution of variant subunits and associated factors to genome-wide distribution and dynamics of cohesin.(BioMed Central (BMC), 2022-11-24) Cuadrado, Ana; Giménez-Llorente, Daniel; De Koninck, Magali; Ruiz-Torres, Miguel; Kojic, Aleksandar; Rodríguez-Corsino, Miriam; Losada, Ana; European Union (EU); Ministerio de Ciencia y Competitividad (España)BACKGROUND The cohesin complex organizes the genome-forming dynamic chromatin loops that impact on all DNA-mediated processes. There are two different cohesin complexes in vertebrate somatic cells, carrying the STAG1 or STAG2 subunit, and two versions of the regulatory subunit PDS5, PDS5A and PDS5B. Mice deficient for any of the variant subunits are embryonic lethal, which indicates that they are not functionally redundant. However, their specific behavior at the molecular level is not fully understood. RESULTS The genome-wide distribution of cohesin provides important information with functional consequences. Here, we have characterized the distribution of cohesin subunits and regulators in mouse embryo fibroblasts (MEFs) either wild type or deficient for cohesin subunits and regulators by chromatin immunoprecipitation and deep sequencing. We identify non-CTCF cohesin-binding sites in addition to the commonly detected CTCF cohesin sites and show that cohesin-STAG2 is the preferred variant at these positions. Moreover, this complex has a more dynamic association with chromatin as judged by fluorescence recovery after photobleaching (FRAP), associates preferentially with WAPL and is more easily extracted from chromatin with salt than cohesin-STAG1. We observe that both PDS5A and PDS5B are exclusively located at cohesin-CTCF positions and that ablation of a single paralog has no noticeable consequences for cohesin distribution while double knocked out cells show decreased accumulation of cohesin at all its binding sites. With the exception of a fraction of cohesin positions in which we find binding of all regulators, including CTCF and WAPL, the presence of NIPBL and PDS5 is mutually exclusive, consistent with our immunoprecipitation analyses in mammalian cell extracts and previous results in yeast. CONCLUSION Our findings support the idea that non-CTCF cohesin-binding sites represent sites of cohesin loading or pausing and are preferentially occupied by the more dynamic cohesin-STAG2. PDS5 proteins redundantly contribute to arrest cohesin at CTCF sites, possibly by preventing binding of NIPBL, but are not essential for this arrest. These results add important insights towards understanding how cohesin regulates genome folding and the specific contributions of the different variants that coexist in the cell.Publication Cooperative Response to Endocardial Notch Reveals Interaction With Hippo Pathway.(Lippincott Williams & Wilkins (LWW), 2023-12-08) Luna-Zurita, Luis; Flores-Garza, Brenda Giselle; Grivas, Dimitrios; Siguero-Alvarez, Marcos; de la Pompa, Jose LuisBACKGROUND The endocardium is a crucial signaling center for cardiac valve development and maturation. Genetic analysis has identified several human endocardial genes whose inactivation leads to bicuspid aortic valve formation and calcific aortic valve disease, but knowledge is very limited about the role played in valve development and disease by noncoding endocardial regulatory regions and upstream factors. METHODS We manipulated Notch signaling in mouse embryonic endocardial cells by short-term and long-term coculture with OP9 stromal cells expressing Notch ligands and inhibition of Notch activity. We examined the transcriptional profile and chromatin accessibility landscape for each condition, integrated transcriptomic, transcription factor occupancy, chromatin accessibility, and proteomic datasets. We generated in vitro and in vivo models with CRISPR-Cas9-edited deletions of various noncoding regulatory elements and validated their regulatory potential. RESULTS We identified primary and secondary transcriptional responses to Notch ligands in the mouse embryonic endocardium, and a NOTCH-dependent transcriptional signature in valve development and disease. By defining the changes in the chromatin accessibility landscape and integrating with the landscape in developing mouse endocardium and adult human valves, we identify potential noncoding regulatory elements, validated selected candidates, propose interacting cofactors, and define the timeframe of their regulatory activity. Additionally, we found cooperative transcriptional repression with Hippo pathway by inhibiting nuclear Yap (Yes-associated protein) activity in the endocardium during cardiac valve development. CONCLUSIONS Sequential Notch-dependent transcriptional regulation in the embryonic endocardium involves multiple factors. Notch activates certain noncoding elements through these factors and simultaneously suppresses elements that could hinder cardiac valve development and homeostasis. Biorxviv: https://www.biorxiv.org/content/10.1101/2023.03.23.533882v1.full.Publication Different NIPBL requirements of cohesin-STAG1 and cohesin-STAG2.(Nature Publishing Group, 2023-03-10) Alonso-Gil, Dácil; Cuadrado, Ana; Giménez-Llorente, Daniel; Rodríguez-Corsino, Miriam; Losada, Ana; Ministerio de Ciencia e Innovación (España); Asociación Española Contra el CáncerCohesin organizes the genome through the formation of chromatin loops. NIPBL activates cohesin's ATPase and is essential for loop extrusion, but its requirement for cohesin loading is unclear. Here we have examined the effect of reducing NIPBL levels on the behavior of the two cohesin variants carrying STAG1 or STAG2 by combining a flow cytometry assay to measure chromatin-bound cohesin with analyses of its genome-wide distribution and genome contacts. We show that NIPBL depletion results in increased cohesin-STAG1 on chromatin that further accumulates at CTCF positions while cohesin-STAG2 diminishes genome-wide. Our data are consistent with a model in which NIPBL may not be required for chromatin association of cohesin but it is for loop extrusion, which in turn facilitates stabilization of cohesin-STAG2 at CTCF positions after being loaded elsewhere. In contrast, cohesin-STAG1 binds chromatin and becomes stabilized at CTCF sites even under low NIPBL levels, but genome folding is severely impaired.Publication Epigenomic Co-localization and Co-evolution Reveal a Key Role for 5hmC as a Communication Hub in the Chromatin Network of ESCs(Cell Press, 2016-02-09) Juan, David; Perner, Juliane; Carrillo de Santa Pau, Enrique; Marsili, Simone; Ochoa, David; Chung, Ho-Ryun; Vingron, Martin; Rico, Daniel; Valencia, Alfonso; Unión Europea. Comisión Europea. 7 Programa MarcoEpigenetic communication through histone and cytosine modifications is essential for gene regulation and cell identity. Here, we propose a framework that is based on a chromatin communication model to get insight on the function of epigenetic modifications in ESCs. The epigenetic communication network was inferred from genome-wide location data plus extensive manual annotation. Notably, we found that 5-hydroxymethylcytosine (5hmC) is the most-influential hub of this network, connecting DNA demethylation to nucleosome remodeling complexes and to key transcription factors of pluripotency. Moreover, an evolutionary analysis revealed a central role of 5hmC in the co-evolution of chromatin-related proteins. Further analysis of regions where 5hmC co-localizes with specific interactors shows that each interaction points to chromatin remodeling, stemness, differentiation, or metabolism. Our results highlight the importance of cytosine modifications in the epigenetic communication of ESCs.Publication Functional contribution of Pds5 to cohesin-mediated cohesion in human cells and Xenopus egg extracts.(The Company of Biologists, 2005-05-15) Losada, Ana; Yokochi, Tomoki; Hirano, Tatsuya; United States Department of Health and Human ServicesSister chromatid cohesion is essential for proper segregation of the genome in mitosis and meiosis. Central to this process is cohesin, a multi-protein complex conserved from yeast to human. Previous genetic studies in fungi have identified Pds5/BimD/Spo76 as an additional factor implicated in cohesion. Here we describe the biochemical and functional characterization of two Pds5-like proteins, Pds5A and Pds5B, from vertebrate cells. In HeLa cells, Pds5 proteins physically interact with cohesin and associate with chromatin in a cohesin-dependent manner. Depletion of the cohesin subunit Scc1 by RNA interference leads to the assembly of chromosomes with severe cohesion defects. A similar yet milder set of defects is observed in cells with reduced levels of Pds5A or Pds5B. In Xenopus egg extracts, mitotic chromosomes assembled in the absence of Pds5A and Pds5B display no discernible defects in arm cohesion, but centromeric cohesion is apparently loosened. Unexpectedly, these chromosomes retain an unusually high level of cohesin. Thus, Pds5 proteins seem to affect the stable maintenance of cohesin-mediated cohesion and its efficient dissolution during mitosis. We propose that Pds5 proteins play both positive and negative roles in sister chromatid cohesion, possibly by directly modulating the dynamic interaction of cohesin with chromatin. This idea would explain why cells lacking Pds5 function display rather complex and diverse phenotypes in different organisms.Publication H2AX is required for chromatin remodeling and inactivation of sex chromosomes in male mouse meiosis.(Cell Press, 2003-04) Fernandez-Capetillo, Oscar; Mahadevaiah, Shantha K; Celeste, Arkady; Romanienko, Peter J; Camerini-Otero, R Daniel; Bonner, William M; Manova, Katia; Burgoyne, Paul; Nussenzweig, André; capetilloDuring meiotic prophase in male mammals, the X and Y chromosomes condense to form a macrochromatin body, termed the sex, or XY, body, within which X- and Y-linked genes are transcriptionally repressed. The molecular basis and biological function of both sex body formation and meiotic sex chromosome inactivation (MSCI) are unknown. A phosphorylated form of H2AX, a histone H2A variant implicated in DNA repair, accumulates in the sex body in a manner independent of meiotic recombination-associated double-strand breaks. Here we show that the X and Y chromosomes of histone H2AX-deficient spermatocytes fail to condense to form a sex body, do not initiate MSCI, and exhibit severe defects in meiotic pairing. Moreover, other sex body proteins, including macroH2A1.2 and XMR, do not preferentially localize with the sex chromosomes in the absence of H2AX. Thus, H2AX is required for the chromatin remodeling and associated silencing in male meiosis.Publication Histone H2AX phosphorylation is dispensable for the initial recognition of DNA breaks.(Nature Publishing Group, 2003-07) Celeste, Arkady; Fernandez-Capetillo, Oscar; Kruhlak, Michael J; Pilch, Duane R; Staudt, David W; Lee, Alicia; Bonner, Robert F; Bonner, William M; Nussenzweig, AndréHistone H2AX is rapidly phosphorylated in the chromatin micro-environment surrounding a DNA double-strand break (DSB). Although H2AX deficiency is not detrimental to life, H2AX is required for the accumulation of numerous essential proteins into irradiation induced foci (IRIF). However, the relationship between IRIF formation, H2AX phosphorylation (gamma-H2AX) and the detection of DNA damage is unclear. Here, we show that the migration of repair and signalling proteins to DSBs is not abrogated in H2AX(-/-) cells, or in H2AX-deficient cells that have been reconstituted with H2AX mutants that eliminate phosphorylation. Despite their initial recruitment to DSBs, numerous factors, including Nbs1, 53BP1 and Brca1, subsequently fail to form IRIF. We propose that gamma-H2AX does not constitute the primary signal required for the redistribution of repair complexes to damaged chromatin, but may function to concentrate proteins in the vicinity of DNA lesions. The differential requirements for factor recruitment to DSBs and sequestration into IRIF may explain why essential regulatory pathways controlling the ability of cells to respond to DNA damage are not abolished in the absence of H2AX.Publication Human adipose tissue H3K4me3 histone mark in adipogenic, lipid metabolism and inflammatory genes is positively associated with BMI and HOMA-IR.(2019-04-08) Castellano-Castillo, Daniel; Denechaud, Pierre-Damien; Fajas, Lluis; Moreno-Indias, Isabel; Oliva-Olivera, Wilfredo; Tinahones, Francisco; Queipo-Ortuño, María Isabel; Cardona, FernandoAdipose tissue is considered an important metabolic tissue, in charge of energy storage as well as being able to act in systemic homeostasis and inflammation. Epigenetics involves a series of factors that are important for gene regulation or for chromatin structure, mostly DNA methylation and histone-tail modifications, which can be modified by environmental conditions (nutrition, lifestyle, smoking…). Since metabolic diseases like obesity and diabetes are closely related to lifestyle and nutrition, epigenetic deregulation could play an important role in the onset of these diseases and vice versa. However, little is known about histone marks in human adipose tissue. In a previous work, we developed a protocol for chromatin immunoprecipitation (ChIP) of frozen human adipose tissue. By using this method, this study investigates, for the first time, the H3K4 trimethylation (H3K4me3) mark (open chromatin) on the promoter of several factors involved in adipogenesis, lipid metabolism and inflammation in visceral adipose tissue (VAT) from human subjects with different degrees of body mass index (BMI) and metabolic disease. VAT was collected and frozen at -80°C. 100 mg VAT samples were fixed in 0.5% formaldehyde and homogenized. After sonication, the sheared chromatin was immune-precipitated with an anti-H3K4me3 antibody linked to magnetic beads and purified. H3K4me3 enrichment was analyzed by qPCR for LEP, LPL, SREBF2, SCD1, PPARG, IL6, TNF and E2F1 promoters. mRNA extraction on the same samples was performed to quantify gene expression of these genes. H3K4me3 was enriched at the promoter of E2F1, LPL, SREBF2, SCD1, PPARG and IL6 in lean normoglycemic compared to morbid obese subjects with prediabetes. Accordingly H3K4me3 mark enrichment at E2F1, LPL, SREBF2, SCD1, PPARG and IL6 promoters was positively correlated with the BMI and the HOMA-IR. Regression analysis showed a strong relationship between the BMI with H3K4me3 at the promoter of E2F1 and LPL, and with mRNA levels of LEP and SCD. In the case of HOMA-IR, the regression analysis showed associations with H3K4me3 enrichment at the promoter of SCD1 and IL6, and with the mRNA of LEP and SCD1. Moreover H3K4me3 at the E2F1 promoter was positively associated to E2F1 mRNA levels. H3K4me3 enrichment in the promoter of LEP, LPL, SREBF2, SCD1, PPARG, IL6, TNF and E2F1 is directly associated with increasing BMI and metabolic deterioration. The H3k4me3 mark could be regulating E3F1 mRNA levels in adipose tissue, while no associations between the promoter enrichment of this mark and mRNA levels existed for the other genes studied.Publication Human prefrontal cortex gene regulatory dynamics from gestation to adulthood at single-cell resolution.(Cell Press, 2022-11-10) Herring, Charles A; Simmons, Rebecca K; Freytag, Saskia; Poppe, Daniel; Moffet, Joel J D; Pflueger, Jahnvi; Buckberry, Sam; Vargas-Landin, Dulce B; Clément, Olivier; Echeverría, Enrique Goñi; Sutton, Gavin J; Alvarez-Franco, Alba; Hou, Rui; Pflueger, Christian; McDonald, Kerrie; Polo, Jose M; Forrest, Alistair R R; Nowak, Anna K; Voineagu, Irina; Martelotto, Luciano; Lister, Ryan; National Health and Medical Research Council (Australia); Australian Research CouncilHuman brain development is underpinned by cellular and molecular reconfigurations continuing into the third decade of life. To reveal cell dynamics orchestrating neural maturation, we profiled human prefrontal cortex gene expression and chromatin accessibility at single-cell resolution from gestation to adulthood. Integrative analyses define the dynamic trajectories of each cell type, revealing major gene expression reconfiguration at the prenatal-to-postnatal transition in all cell types followed by continuous reconfiguration into adulthood and identifying regulatory networks guiding cellular developmental programs, states, and functions. We uncover links between expression dynamics and developmental milestones, characterize the diverse timing of when cells acquire adult-like states, and identify molecular convergence from distinct developmental origins. We further reveal cellular dynamics and their regulators implicated in neurological disorders. Finally, using this reference, we benchmark cell identities and maturation states in organoid models. Together, this captures the dynamic regulatory landscape of human cortical development.Publication Integrating epigenomic data and 3D genomic structure with a new measure of chromatin assortativity(BioMed Central (BMC), 2016-07-08) Pancaldi, Vera; Carrillo-de-Santa-Pau, Enrique; Javierre, Biola Maria; Juan, David; Fraser, Peter; Spivakov, Mikhail; Valencia, Alfonso; Rico, Daniel; Unión Europea; Biotechnology and Biological Sciences Research Council (Reino Unido); Medical Research Council (Reino Unido); Instituto de Salud Carlos III; Unión Europea. Fondo Europeo de Desarrollo Regional (FEDER/ERDF)BACKGROUND: Network analysis is a powerful way of modeling chromatin interactions. Assortativity is a network property used in social sciences to identify factors affecting how people establish social ties. We propose a new approach, using chromatin assortativity, to integrate the epigenomic landscape of a specific cell type with its chromatin interaction network and thus investigate which proteins or chromatin marks mediate genomic contacts. RESULTS: We use high-resolution promoter capture Hi-C and Hi-Cap data as well as ChIA-PET data from mouse embryonic stem cells to investigate promoter-centered chromatin interaction networks and calculate the presence of specific epigenomic features in the chromatin fragments constituting the nodes of the network. We estimate the association of these features with the topology of four chromatin interaction networks and identify features localized in connected areas of the network. Polycomb group proteins and associated histone marks are the features with the highest chromatin assortativity in promoter-centered networks. We then ask which features distinguish contacts amongst promoters from contacts between promoters and other genomic elements. We observe higher chromatin assortativity of the actively elongating form of RNA polymerase 2 (RNAPII) compared with inactive forms only in interactions between promoters and other elements. CONCLUSIONS: Contacts among promoters and between promoters and other elements have different characteristic epigenomic features. We identify a possible role for the elongating form of RNAPII in mediating interactions among promoters, enhancers, and transcribed gene bodies. Our approach facilitates the study of multiple genome-wide epigenomic profiles, considering network topology and allowing the comparison of chromatin interaction networks.Publication NIPBL and cohesin: new take on a classic tale.(Elsevier, 2023-10) Alonso-Gil, Dácil; Losada, Ana; Ministerio de Ciencia e Innovación (España)Cohesin folds the genome in dynamic chromatin loops and holds the sister chromatids together. NIPBLScc2 is currently considered the cohesin loader, a role that may need reevaluation. NIPBL activates the cohesin ATPase, which is required for topological entrapment of sister DNAs and to fuel DNA loop extrusion, but is not required for chromatin association. Mechanistic dissection of these processes suggests that both NIPBL and the cohesin STAG subunit bind DNA. NIPBL also regulates conformational switches of the complex. Interactions of NIPBL with chromatin factors, including remodelers, replication proteins, and the transcriptional machinery, affect cohesin loading and distribution. Here, we discuss recent research addressing how NIPBL modulates cohesin activities and how its mutation causes a developmental disorder, Cornelia de Lange Syndrome (CdLS).