Dynamics and dispensability of variant-specific histone H1 Lys-26/Ser-27 and Thr-165 post-translational modifications

Jean-Michel Terme et al.In mammals, the linker histone H1, involved in DNA packaging into chromatin, is represented by a family of variants. H1 tails undergo post-translational modifications (PTMs) that can be detected by mass spectrometry. We developed antibodies to analyze several of these as yet unexplored PTMs including the combination of H1.4 K26 acetylation or trimethylation and S27 phosphorylation. H1.2-T165 phosphorylation was detected at S and G2/M phases of the cell cycle and was dispensable for chromatin binding and cell proliferation; while the H1.4-K26 residue was essential for proper cell cycle progression. We conclude that histone H1 PTMs are dynamic over the cell cycle and that the recognition of modified lysines may be affected by phosphorylation of adjacent residues. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.This work was supported by the Spanish Ministry of Science and Innovation (MICINN) and European Regional Development Fund (Grant BFU2011-23057 to A.J., and Grant BFU2008-00460 to P.S.), and by the Regional Government of Catalonia (Generalitat de Catalunya; Grant 2009-SGR-1222 to A.J.). J.-M.T. received a JAE-Doc contract from the Spanish National Research Council (CSIC)-MICINN; R.M. a TA contract from CSIC-MICINN; and L.M.-A. an FPU predoctoral fellowship from MICINNPeer 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

Dynamics and dispensability of variant-specific histone H1 Lys-26/Ser-27 and Thr-165 post-translational modifications

Jean-Michel Terme et al.In mammals, the linker histone H1, involved in DNA packaging into chromatin, is represented by a family of variants. H1 tails undergo post-translational modifications (PTMs) that can be detected by mass spectrometry. We developed antibodies to analyze several of these as yet unexplored PTMs including the combination of H1.4 K26 acetylation or trimethylation and S27 phosphorylation. H1.2-T165 phosphorylation was detected at S and G2/M phases of the cell cycle and was dispensable for chromatin binding and cell proliferation; while the H1.4-K26 residue was essential for proper cell cycle progression. We conclude that histone H1 PTMs are dynamic over the cell cycle and that the recognition of modified lysines may be affected by phosphorylation of adjacent residues. © 2014 Federation of European Biochemical Societies. Published by Elsevier B.V. All rights reserved.This work was supported by the Spanish Ministry of Science and Innovation (MICINN) and European Regional Development Fund (Grant BFU2011-23057 to A.J., and Grant BFU2008-00460 to P.S.), and by the Regional Government of Catalonia (Generalitat de Catalunya; Grant 2009-SGR-1222 to A.J.). J.-M.T. received a JAE-Doc contract from the Spanish National Research Council (CSIC)-MICINN; R.M. a TA contract from CSIC-MICINN; and L.M.-A. an FPU predoctoral fellowship from MICINNPeer Reviewe

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

Analysis of the genomic distribution of linker histone H1 variants in human = Anàlisi de la distribució genòmica de les variants d'histona H1 en humans

[eng] Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Using variant-specific antibodies to H1 and hemagglutinin-tagged recombinant H1 variants expressed in breast cancer cells, their genomic distribution was assessed. Specifically, ChIP-Seq data was obtained for two replication-dependent (H1.2 and H1.4) and replication-independent H1 variants (H1.0 and H1X) together with core histone H3. Briefly, we have previously reported that H1.2 is the H1 variant that better correlates with gene repression. It was found enriched at GC-poor, gene-poor and intergenic chromosomal domains in addition to lamin-associated domains (LADs). We further explored linker histone H1 variant distribution and strikingly, we found that distribution of replication-independent H1 variants (H1.0 and H1X) is distinct. H1.0 was found enriched at nucleolar features such as nucleolus-associated domains (NADs), nucleolus organizer regions (NORs) encoding for the 45S rDNA, specifically at non-transcribed spacers and also in 5S rDNA. Specific repetitive sequences such as SINE-VNTR-Alu (SVA) retrotransposons and telomeric and ACRO1 satellites showed also a specific enrichment of H1.0. On the other hand, H1X has been associated to actively transcribed chromatin indicated by a colocalization with RNAPII-enriched regions and an enrichment towards the 3’ end of active genes. In addition, constitutive exons, included alternatively spliced exons and retained introns are enriched in H1X. Further, specific non-coding RNA (miRNA and snoRNA), mainly found at introns showed a H1X enrichment. Our results point to a potential role of H1X in elongation, splicing or non-coding RNA regulation, which might be prompting gene transcription without changes in core histone post-translational modifications. Furthermore, depletion of multiple H1 variants (H1.2 and H1.4) triggers an interferon response due to an aberrant transcription of repetitive elements in breast cancer cells. Transcription of repetitive elements was observed by an increase in their RNA levels (RT-qPCR), increase in cytoplasmic dsRNA (immunofluorescence) and transcription of intergenic regions (RNA-Seq). Variants H1.2 and H1.4 seem to be critical in the observed phenotype but rescue experiments showed redundant functions for H1 variants. The molecular mechanism that leads to transcription of repetitive elements upon multiH1 KD, as happens for DE genes upon single or multiple H1 variants KD, is still unsolved. We were able to show an increase in nucleosome accessibility genome-wide (ATAC-Seq) that did not fully correlate with the observed transcriptional changes in multiple H1 depleted cells. Surprisingly, post-translational modifications of core histone remained unchanged as happens for single H1X depletion. Specific molecular mechanisms, involved in transcriptional modulation, that might be regulated by a particular H1 variant (or H1 variant combinations) are appealing possibilities. Among them, establishment, maintenance or organization of nuclear domains (lamin-, nucleolus- or topologically associated domains), chromosome structures (centromeres) or localised heterochromatin regions (transposons). Beyond promoters where histone H1 content clearly correlate with repression, other transcription-related processes might be regulated by specific H1 variants. Processes influenced by RNAPII (elongation or splicing) and other regulatory elements (non-coding RNAs or enhancers) need to be certainly explored in a histone H1 variant(s) depletion context. Upon single and multiple H1 variants depletion, H1.0 is induced in a regulated manner that may depend in histone acetylation, assessed by ChIP-qPCR at promoter regions and by treatments with histone deacetylase inhibitor (TSA). Further experiments are needed to elucidate relocation of histone replication-independent H1 variants, mainly H1.0 upon changing H1 stoichiometry and during differentiation, reprogramming and cancer.[spa] Existen siete variantes de histona H1 presentes en células somáticas humanas con una prevalencia diferente según el tipo celular. Usando anticuerpos específicos contra las variantes de H1 y variantes de H1 recombinantes etiquetadas con hemaglutinina, evaluamos su distribución genómica en células de cáncer de mama. Concretamente, obtuvimos datos de ChIP-Seq de dos variantes de H1 dependientes de replicación (H1.2 y H1.4) y las dos variantes independientes de replicación (H1.0 y H1X). Anteriormente, observamos que H1.2 es la variante que mejor correlaciona con la represión génica y se encuentra enriquecida en dominios cromosómicos pobre en GC, pobres en genes e intergénicos, además de en los dominios asociados a lamin. Después exploramos con más profundidad la distribución de las variantes de H1 independientes de replicación. H1.0 se encontraba enriquecida en regiones asociadas al nucléolo como los dominios asociados al nucléolo, las regiones organizadoras del nucléolo que codifican para el ARN ribosomal 45S, específicamente en las regiones espaciadoras no transcritas y, también, en el 5S DNA ribosomal. Elementos repetitivos como los retrotransposones SINE-SVA-Alu y los satélites teloméricos y ACRO1 también mostraron un enriquecimiento específico de H1.0. Por otro lado, encontramos H1X asociada a cromatina activa transcripcionalmente, demostrado por una colocalización con regiones asociadas a RNAPII y un enriquecimiento hacia el extremo 3’ de genes activos. Además, todas las regiones codificantes que se incluyen en el transcrito final (exones constitutivos, exones incluidos alternativamente e intrones retenidos) mostraron un enriquecimiento en H1X. Algunas especies de ARN no codificante (miRNA y snoRNA), que se encuentran principalmente en intrones, estaban enriquecidas en H1X. Nuestros resultados apuntan a que H1X puede tener un papel en la regulación de la elongación, splicing o el ARN no codificante, que podría estar induciendo la transcripción de genes sin cambios en las modificaciones post-traduccionales de histonas. La depleción de varias variantes de H1 (H1.2 y H1.4) desencadena una respuesta de interferón debido a una transcripción aberrante de elementos repetitivos en cáncer de mama. La transcripción de elementos repetitivos se observó por un aumento de sus niveles de ARN, un aumento de los ARN de doble cadena en el citoplasma y por la transcripción de regiones intergénicas. El mecanismo molecular que conduce a su transcripción, tal como sucede con los genes desregulados en células deplecionadas de una sola variante, aún no está resuelto. Mostramos un aumento global en la accesibilidad a la cromatina que no correlaciona completamente con los cambios transcripcionales observados al deplecionar múltiples variantes de H1. Sorprendentemente, las modificaciones post-traduccionales de las histonas se mantienen intactas

Anàlisi de la distribució genòmica de les variants d'histona H1 en humans

Tesis llevada a cabo para conseguir el grado de Doctor por la Universidad de Barcelona.--2018-03-13.--Excelente[ES] Existen siete variantes de histona H1 presentes en células somáticas humanas con una prevalencia diferente según el tipo celular. Usando anticuerpos específicos contra las variantes de H1 y variantes de H1 recombinantes etiquetadas con hemaglutinina, evaluamos su distribución genómica en células de cáncer de mama. Concretamente, obtuvimos datos de ChIP-Seq de dos variantes de H1 dependientes de replicación (H1.2 y H1.4) y las dos variantes independientes de replicación (H1.0 y H1X). Anteriormente, observamos que H1.2 es la variante que mejor correlaciona con la represión génica y se encuentra enriquecida en dominios cromosómicos pobre en GC, pobres en genes e intergénicos, además de en los dominios asociados a lamin. Después exploramos con más profundidad la distribución de las variantes de H1 independientes de replicación. H1.0 se encontraba enriquecida en regiones asociadas al nucléolo como los dominios asociados al nucléolo, las regiones organizadoras del nucléolo que codifican para el ARN ribosomal 45S, específicamente en las regiones espaciadoras no transcritas y, también, en el 5S DNA ribosomal. Elementos repetitivos como los retrotransposones SINE-SVA-Alu y los satélites teloméricos y ACRO1 también mostraron un enriquecimiento específico de H1.0. Por otro lado, encontramos H1X asociada a cromatina activa transcripcionalmente, demostrado por una colocalización con regiones asociadas a RNAPII y un enriquecimiento hacia el extremo 3’ de genes activos. Además, todas las regiones codificantes que se incluyen en el transcrito final (exones constitutivos, exones incluidos alternativamente e intrones retenidos) mostraron un enriquecimiento en H1X. Algunas especies de ARN no codificante (miRNA y snoRNA), que se encuentran principalmente en intrones, estaban enriquecidas en H1X. Nuestros resultados apuntan a que H1X puede tener un papel en la regulación de la elongación, splicing o el ARN no codificante, que podría estar induciendo la transcripción de genes sin cambios en las modificaciones post-traduccionales de histonas. La depleción de varias variantes de H1 (H1.2 y H1.4) desencadena una respuesta de interferón debido a una transcripción aberrante de elementos repetitivos en cáncer de mama. La transcripción de elementos repetitivos se observó por un aumento de sus niveles de ARN, un aumento de los ARN de doble cadena en el citoplasma y por la transcripción de regiones intergénicas. El mecanismo molecular que conduce a su transcripción, tal como sucede con los genes desregulados en células deplecionadas de una sola variante, aún no está resuelto. Mostramos un aumento global en la accesibilidad a la cromatina que no correlaciona completamente con los cambios transcripcionales observados al deplecionar múltiples variantes de H1. Sorprendentemente, las modificaciones post-traduccionales de las histonas se mantienen intactas.[EN] [eng] Seven linker histone H1 variants are present in human somatic cells with distinct prevalence across cell types. Using variant-specific antibodies to H1 and hemagglutinin-tagged recombinant H1 variants expressed in breast cancer cells, their genomic distribution was assessed. Specifically, ChIP-Seq data was obtained for two replication-dependent (H1.2 and H1.4) and replication-independent H1 variants (H1.0 and H1X) together with core histone H3. Briefly, we have previously reported that H1.2 is the H1 variant that better correlates with gene repression. It was found enriched at GC-poor, gene-poor and intergenic chromosomal domains in addition to lamin-associated domains (LADs). We further explored linker histone H1 variant distribution and strikingly, we found that distribution of replication-independent H1 variants (H1.0 and H1X) is distinct. H1.0 was found enriched at nucleolar features such as nucleolus-associated domains (NADs), nucleolus organizer regions (NORs) encoding for the 45S rDNA, specifically at non-transcribed spacers and also in 5S rDNA. Specific repetitive sequences such as SINE-VNTR-Alu (SVA) retrotransposons and telomeric and ACRO1 satellites showed also a specific enrichment of H1.0. On the other hand, H1X has been associated to actively transcribed chromatin indicated by a colocalization with RNAPII-enriched regions and an enrichment towards the 3’ end of active genes. In addition, constitutive exons, included alternatively spliced exons and retained introns are enriched in H1X. Further, specific non-coding RNA (miRNA and snoRNA), mainly found at introns showed a H1X enrichment. Our results point to a potential role of H1X in elongation, splicing or non-coding RNA regulation, which might be prompting gene transcription without changes in core histone post-translational modifications. Furthermore, depletion of multiple H1 variants (H1.2 and H1.4) triggers an interferon response due to an aberrant transcription of repetitive elements in breast cancer cells. Transcription of repetitive elements was observed by an increase in their RNA levels (RT-qPCR), increase in cytoplasmic dsRNA (immunofluorescence) and transcription of intergenic regions (RNA-Seq). Variants H1.2 and H1.4 seem to be critical in the observed phenotype but rescue experiments showed redundant functions for H1 variants. The molecular mechanism that leads to transcription of repetitive elements upon multiH1 KD, as happens for DE genes upon single or multiple H1 variants KD, is still unsolved. We were able to show an increase in nucleosome accessibility genome-wide (ATAC-Seq) that did not fully correlate with the observed transcriptional changes in multiple H1 depleted cells. Surprisingly, post-translational modifications of core histone remained unchanged as happens for single H1X depletion. Specific molecular mechanisms, involved in transcriptional modulation, that might be regulated by a particular H1 variant (or H1 variant combinations) are appealing possibilities. Among them, establishment, maintenance or organization of nuclear domains (lamin-, nucleolus- or topologically associated domains), chromosome structures (centromeres) or localised heterochromatin regions (transposons).Peer reviewe

Specificities and genomic distribution of somatic mammalian histone H1 subtypes BBA Gene Regulatory Mechanisms

Histone H1 is a structural component of chromatin that may have a role in the regulation of chromatin dynamics. Unlike core histones, the linker histoneH1 family is evolutionarily diverse andmany organisms have multiple H1 variants or subtypes, distinguishable between germ-line and somatic cells. In mammals, the H1 family includes seven somatic H1 variantswith a prevalence that varies between cell types and over the course of differentiation, H1.1 to H1.5 being expressed in a replication-dependent manner, whereas H1.0 and H1X are replicationindependent. Until recently, it has not been known whether the different variants had specific roles in the regulation of nuclear processes or were differentially distributed across the genome. To address this, an increasing effort has been made to investigate divergent features among H1 variants, regarding their structure, expression patterns, chromatin dynamics, post-translational modifications and genome-wide distribution. Although H1 subtypes seem to have redundant functions, several reports point to the idea that they are also differently involved in specific cellular processes. Initial studies investigating the genomic distribution of H1 variants have started to suggest that despite a wide overlap, different variants may be enriched or preferentially located at different chromatin types, but this may depend on the cell type, the relative abundance of the variants, the differentiation state of the cell, orwhether cells are derived from a neoplastic process. Understanding the heterogeneity of the histone H1 family is crucial to elucidate their role in chromatin organization, gene expression regulation and other cellular processes. This article is part of a Special Issue entitled: Histone H1, edited by Dr. Albert Jordan.This work was supported by the Spanish Ministry of Science and Innovation (MICINN) and the European Regional Development Fund (grant BFU2014-52237).Peer Reviewe

Epigenetic mechanisms in health and disease: BCEC 2017

The Barcelona Conference on Epigenetics and Cancer (BCEC) entitled “Epigenetic Mechanisms in Health and Disease” was held in Barcelona, October 26-26, 2017. The 2017 BCEC was the fifth and last edition of a series of annual conferences organized as a joint effort of five leading Barcelona research institutes together with B-Debate. This edition was organized by Albert Jordan from the Molecular Biology Institute of Barcelona (IBMB-CSIC) and Marcus Bushbeck from the Josep Carreras Leukaemia Research Institute (IJC). Jordi Bernués, Marian Martínez-Balbás, and Ferran Azorín were also part of the scientific committee. In 22 talks and 51 posters, researchers presented their latest results in the fields of histone variants, epigenetic regulation, and chromatin 3D organization to an audience of around 250 participants from 16 countries. This year, a broad number of talks focused on the epigenetic causes and possible related treatments of complex diseases such as cancer. Participants at the 2017 BCEC elegantly closed the series, discussing progress made in the field of epigenetics and highlighting its role in human health and diseaseRF was supported by a FPU fellowship [grant number FPU13/01384]. AI- B was supported by a grant from Spanish Ministry of Economy and Com- petitiveness (MINECO) and European Regional Development Fund [grant number BFU2014–52237-P] to Dr. Albert Jordan. CM was supported by grants from Instituto de Salud Carlos III [grant number PIE16-00011]; and Fundaci on Olga Torres to Dr. Sonia-Vanina Forcales. AC was sup- ported by grants from MINECO [grant number BFU2012-30724] and [grant number BFU2015-65082P], the Generalitat de Catalunya [grant number SGR2009-1023] and [grant number SGR2014-204], and the Euro- pean Community FEDER program to Dr. Ferran Azor ın.Peer reviewe

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