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The fundamental building blocks of chromatin are the nucleosomes. Each such unit is composed of about 200 bp of DNA, the well-conserved core histones (H2A, H2B, H3 and H4) and a linker histone (H1). The DNA is wound around two dimers of H2A–H2B and a tetramer comprising two molecules each of H3 and H4, and there is approximately one linker histone molecule positioned on the exterior of the DNA–protein octamer complex. The nucleosome directs the various structural transitions in chromatin that are needed for proper transcriptional regulation during differentiation and development of the organism in question. The gene activity can be regulated by different histone variants, DNA–protein interactions, and protein–protein interactions, all of which are influenced by the enormous amounts of post-translational modifications that occur in the histone tails. The research underlying this thesis focused on different aspects of post-translational modifications during aging, differentiation, and progression of the cell cycle, and also on expression of linker histone variants and linker histone-chromatin interactions in a variety of cells and tissues. The present results are the first to show that H4 can be trimethylated at lysine 20 in mammalian cells. The trimethylated H4K20 was found in rat kidney and liver at levels that rose with increasing age of the nimals, and it was also detected in trace amounts in human cell lines. Furthermore, in differentiating MEL cells, trimethylated H4K20 was localized to heterochromatin, and levels of trimethylated H4K20 increased during the course of cell differentiation and were correlated with the increasing compaction of the chromatin. The chromatin of terminally differentiated chicken and frog erythrocytes is highly condensed, and the linker histone variants it contains vary between the two species. Cytofluorometric analyses revealed that the linker histones in the chicken erythrocytes exhibited higher affinity for chromatin than did those in the frog erythrocytes. Characterization of the H1° in frog erythrocytes proved it to be the H1°-2 subvariant. Other experiments demonstrated that normal human B lymphocytes expressed the linker histone variants H1.2, H1.3, H1.4, and H1.5, and that B cells from patients with B-CLL expressed the same variants although in different amounts. The most striking dissimilarity was that amounts of H1.3 in the cells were decreased or undetectable in some samples. Sequencing did not discern any defects in the H1.3 gene, and thus the absence of H1.3 is probably regulated at the post-translational level. It was also observed that the levels of linker histone phosphorylation in EBV-transformed B lymphocytes were already increased in the G1 phase of the cell cycle, which is earlier than previously thought. This increase in phosphorylation is probably responsible for the lower affinity of linker histones for chromatin in EBV-transformed cells in the G1 phase of the cell cycle.

H1 linker histone facilitates the formation of higher order chromatin structure and is essential for mammalian development. Mice have 11 H1 variants which are differentially regulated and conserved in human. Previous research indicates that H1 regulates the expression of specific genes in mouse embryonic stem cells (ESCs). However, whether individual variants have distinct functions and how H1 participates in gene regulation remain elusive. An investigation of the precise localization of individual H1 variants in vivo would facilitate the elucidation of mechanisms underlying chromatin compaction regulated gene expression, while it has been extremely difficult due to the lacking of specific antibodies toward H1 variants. In this dissertation, I have generated a knock-in system in ESCs and shown that the N-terminally tagged H1 proteins are functionally interchangeable to their endogenous counterparts in vivo. H1d and H1c are depleted from GC- and gene-rich regions and active promoters, inversely correlated with H3K4me3, but positively correlated with H3K9me3 and associated with characteristic sequence features. Surprisingly, both H1d and H1c are significantly enriched at major satellites, which display increased nucleosome spacing compared with bulk chromatin. While also depleted at active promoters and enriched at major satellites, overexpressed H10 displays differential binding patterns in specific repetitive sequences compared with H1d and H1c. Depletion of H1c, H1d ,and H1e causes pericentric chromocenter clustering and de-repression of major satellites. Collectively, these results integrate the localization of an understudied type of chromatin proteins, namely the H1 variants, into the epigenome map of mouse ESCs, and demonstrate significant changes at pericentric heterochromatin upon depletion of this epigenetic mark.

We did functional characterisation of Drosophila melanogaster linker histone, dH1. In the mutant state for this protein, we observed structural changes in polytene chromosomes chromocenter and nucleoli of mutant larvae. In addition, we performed a microarray analysis in H1 mutant background in order to determine contribution of dH1 to gene expression regulation. We determined effects of dH1 loss in different types of chromatin and we identified groups of differentially expressed (DE) genes, groups in sense of physical clusters of genes and genomic elements rather than groups of functionally related genes. We found that dH1 affects in greater extent expression of heterochromatin genes compared to its effect on euchromatin genes; that dH1 regulates transcription in a regional manner, since the genes physically nearest to the most DE genes tend to be upregulated as well; and that dH1 is negatively regulating expression of transposable elements and members of certain gene families. In addition, we found that dH1 is necessary for preserving genome stability. Among DE transposable elements we detected R1 and R2 retrotransposons, elements that are integrating specifically in rRNA locus. We showed that activation of their transcription is also upregulating expression of aberrant, transposon-inserted, rDNA units of the locus. In this regard we observed an accumulation of extra-chromosomal rDNA circles, increased γ-H2Av content, stop in cell proliferation and activation of apoptosis. Altogether, these results are revealing so far unknown role of histone H1 in preserving genome stability and its effects on cell proliferation.

No description available.

At least eleven histone H1 variants exist in mammalian somatic cells that bind to the linker DNA and stabilize the nucleosome particle contributing to higher order chromatin compaction. In addition of playing a structural role, H1 seems to be involved in the activation and repression of gene expression. It is not well known whether the different variants have specific roles or 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 by changes of expression of other variants. A different, reduced subset of genes is altered in each H1 knock-down. Interestingly, H1.2 depletion represses expression of a number of cell cycle genes. This is concomitant with a G1 arrest phenotype observed in this cell line. In addition, H1.2 depletion caused decreased global nucleosome spacing. These effects are specific of H1.2 depletion as they are not complemented by overexpression of other variants and they do not occur in knock-downs for the other variants. Moreover, H1.4 depletion caused cell death in T47D, being the first report of the essentiality of an H1 variant for survival in a human cell type. In addition to this, we have also investigated specificities of H1 subtypes location in particular promoters of interest in our laboratory, as well as specific interactions with other factors by generating HA-tagged H1 variant expressing cell lines. / Al menos once variantes de la histona H1 han sido identificadas en mamíferos, todas ellas se unen al ADN entre nucleosomas contribuyendo así, a la estabilización de la partícula nucleosómica y a la compactación de la cromatina en estructuras de alto orden. Además de jugar un papel estructural, H1 parece estar implicada en la activación y represión de la expresión génica. Se desconoce si las diferentes variantes de H1 tienen funciones específicas o regulan promotores específicos. Con el objetivo de investigar esta hipótesis se han generado líneas celulares que inhiben de forma inducible, mediante la tecnología de ARN interferente, la expresión de cada una de las variantes de forma específica. La inhibición de cada una de las variantes no es compensada por cambios en la expresión del resto de subtipos. Distintos grupos de genes resultan alterados con la depleción de cada una de las variantes de H1. La inhibición de H1.2 reprime la expresión de una serie de genes de ciclo celular, correlacionando con un fenotipo de arresto celular en fase G1 observado en esta línea. Además, la inhibición de H1.2 causa una disminución global del espaciamiento entre nucleosomas. Todos estos efectos parecen ser específicos para la falta de H1.2 ya que no son complementados por la sobreexpresión de otras variantes. Por otro lado, la inhibición de H1.4 causa muerte celular en T47D. Ésta es la primera vez que se describe que una variante de H1 es esencial para la supervivencia de una línea celular humana.En un segundo plano, se han construido líneas celulares con expresión de las variantes de H1 fusionadas al péptido HA, con el objetivo de estudiar la especificidad de su localización en promotores de interés para el grupo, así como interacciones específicas con otros factores celulares.

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Notre objectif était de mieux comprendre la régulation de la biosynthèse des parois secondaires lors de la formation du bois chez l'Eucalyptus, le feuillu le plus planté au monde et le deuxième dont le génome est séquencé. Nous avons caractérisé trois facteurs de transcription de la famille MYB-R2R3 et montré que EgMYB137 était un nouveau régulateur de la biosynthèse des parois secondaires. Nous avons aussi démontré que l'activité transcriptionnelle de EgMYB1, un répresseur de la biosynthèse des lignines, était régulée par une interaction protéine-protéine impliquant une histone linker (EgH1.3). Enfin, nous avons mis au point une méthode de transformation homologue chez l'Eucalyptus via A. rhizogenes. Les " hairy roots " transgéniques sont adaptées à la caractérisation fonctionnelle de gènes reliés à la formation du xylème. Nos résultats ont permis de découvrir de nouveaux acteurs impliqués dans la régulation des parois secondaires, mettant en lumière la complexité de ce processus mais aussi offrant de nouvelles perspectives pour l'amélioration du bois pour des applications industrielles comme la production de bioéthanol de deuxième génération. / Our objective was to better understand the regulation of the biosynthesis of the lignified secondary cell walls during wood formation in Eucalyptus, the most planted hardwood tree, and the second whose genome has been sequenced. We functionally characterized three Eucalyptus transcription factors of the R2R3-MYB family and identified EgMYB137 as a new regulator of secondary cell wall deposition. We also showed that the transcriptional activity of EgMYB1, a repressor of lignin biosynthesis was modulated by protein-protein interactions involving a linker histone (EgH1.3). Finally, we set up a homologous transformation system for Eucalyptus using Agrobacterium rhizogenes. The transgenic hairy roots are suitable for high throughput functional characterization of cell wall-related genes. Our findings not only allowed getting new insights into the complexity of the network regulating secondary cell walls but also open new avenues to improve wood quality for industrial applications such as second-generation bioethanol.