Role of H1 and his partners in the structure and epigenetic properties of chromatin – ChromComp

Our genetic heritage goes far beyond the sequence of our genome. Our development, phenotypical characteristics and cellular functions depend critically on the way the genetic information encoded by DNA is packaged and organized into chromatin within the nucleus. The fundamental unit of this organization, the nucleosome core particle (NCP) is now well-characterized at the structural and functional level. The atomic structure of this complex formed by 147 bp of DNA wrapped around an octameric protein structure composed of 2 copies of each of the 4 core histones H2A, H2B, H3 and H4 has been determined. The function and the mode of action of the fifth histone, the linker histone H1, is far less well understood and the primary objective of this proposal is to provide new biochemical and structural information to shed new light on its fundamental biological action.
The linker histone has a crucial function in the higher order organization and compaction of nucleosomal arrays and is therefore involved in several biological processes such as transcription, DNA replication and repair. It interacts with the NCP but the exact nature of this interaction is still under debate and how this binding leads to chromatin compaction and to the formation of the 30 nm chromatin fiber remains elusive. The three-dimensional (3-D) organization of the H1-containing nucleosome and the 30 nm chromatin fiber are two major structural problems in the chromatin field that stay unanswered for more than 35 years and our proposal will address this question using new tools and imaging technologies.
The regulatory role of H1 in compacting chromatin is determined by its specific deposition to dedicated chromatin domains and by the action of protein complexes that further stabilize the compact chromatin. However the mode of deposition of H1 as well as in vivo interaction partners of H1 are currently poorly documented. Our projects will characterize the H1 interactome by using tagged cell lines to identify the H1-specific chaperones and its chromatin interaction partners.
A paramount example of a specialized chromatin is the centromere, localized at the site of the primary chromosome constriction and required for kinetochore formation. Correct centromere formation and specification is essential for cell survival and aberrations in this process cause chromosomal instability, aneuploidy and cancer. CENP-A, which replaces the conventional histone H3 in centromeric nucleosomes, constitutes the epigenetic marker of the centromere. It is currently unknown whether CENP-A nucleosomes are able to bind histone H1. The crystal structure of the CENP-A nucleosome indicates that the ends of the DNA are highly flexible and that the a-N helix of CENP-A, important for DNA end orientation, is shorter than in H3. This flexibility is likely to prevent histone H1 binding and in this case the CENP-A chromatin would exhibit a functionally important open structure. We will analyze the histone H1 binding properties of the CENP-A nucleosome and disclose the specific features that affect H1-binding.
Methylation of DNA is another major epigenetic mark required for cell differentiation and development. Binding of H1 to nucleosomes containing methylated DNA is poorly described. The human MeCP2 was the first identified methyl-DNA binding protein and its mutations cause most cases of Rett syndrome, a X-linked neurodevelopmental disease. hMeCp2 binds to nucleosomes close to the linker DNA entry-exit site where it is likely to interfere with Histone H1 interaction. Despite its central role in chromatin organization, structural insights on the interaction of MeCP2 with its natural template, the nucleosome, are missing. Our project aims at deciphering the interaction of MeCP2 with nucleosomes formed on methylated DNA and to study at the structural level the possible interference with H1-binding.