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dc.contributor.advisorJordan Vallès, Albert-
dc.contributor.authorIzquierdo Bouldstridge, Andrea-
dc.contributor.otherUniversitat de Barcelona. Departament de Genètica, Microbiologia i Estadística-
dc.description.abstract[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.eng
dc.description.abstract[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
dc.format.extent342 p.-
dc.publisherUniversitat de Barcelona-
dc.rights(c) Izquierdo, 2018-
dc.subject.classificationExpressió gènica-
dc.subject.otherGene expression-
dc.subject.otherHuman beings-
dc.titleAnalysis of the genomic distribution of linker histone H1 variants in human = Anàlisi de la distribució genòmica de les variants d'histona H1 en humans-
Appears in Collections:Tesis Doctorals - Departament - Genètica, Microbiologia i Estadística

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