The role of histone H1 and CENP-C in the structure and epigenetic properties of chromatin – Chrom3D

The methods and technologies used include: molecular biology, protein expression and purification, nucleosome reconstitution, reconstitution of nucleosomal arrays, cryo-electron microscopy, X-ray crystallography, analytical ultracentrifugation, small-angle scattering (SAXS), cross-linking, hydroxylradical footprinting, electrophoretic mobility shift assays, fluorescence microscopy, proteomics and co-immunoprecipitation experiments.

We determined the 3D structure of an H1-bound six-nucleosome array at 9.5 Å resolution by X-ray crystallography and validated the structure by cryo-EM and biochemical analysis. The structure reveals a surprisingly flat conformation whose nucleosome density is only half that of the 30-nm chromatin fiber. Using cryo-EM and biophysical analysis we showed that a minor change in ionic conditions induces the array to adopt a more compact, twisted conformation that corresponds to that of the 30-nm fiber, shedding important light on the structural plasticity of chromatin. This work was recently published (Garcia-Saez et al., Mol Cell 2018, PMID 30392928). In addition we reconstituted a CENP-A nucleosome core particle (NCP) using 601 Widom DNA sequence and determined its structure by phase-plate cryo-EM at 4 Å resolution. Surprisingly, the structure revealed that the right DNA arm is considerably more disordered (more flexible) than the left arm. A manuscript describing this result is currently in preparation. We investigated the two-residue insertion within the L1 loop that differentiates CENP-A from histone H3. We have shown that in vivo replacement of CENP-A by L1 mutants compromises the association of multiple kinetochore components with CENP-A nucleosomes and induces severe mitotic and cytokinetic defects, highlighting the L1 insert as a critical epitope for kinetochore recruitment. Moreover, siRNA-mediated depletion of CENP-A in naïve HeLa cells led to a massive delocalization of CENP-C from centromeres. CENP-C localization could be rescued by WT CENP-A but not by CENP-A L1 mutants, demonstrating that the L1 loop is critical for proper CENP-C localization and function at the centromeres.

The finding that a minor change in ionic conditions induces the 6-nucleosome array to switch from a flat, extended conformation to a more compact, twisted conformation that corresponds to that of the 30-nm fiber is an exciting discovery. These results provide insights into a possible assembly pathway for the 30-nm fiber. Moreover, they reveal how a minor change in local environment (which could for example bey generated by the post-translational modification of histones) can induce a radical change in chromatin conformation, providing insights into the structural plasticity of chromatin that is central to the regulation of gene expression.

1. Garcia-Saez I, Menoni H, Boopathi R, Shukla MS, Soueidan L, Noirclerc-Savoye M, Le Roy A, Skoufias DA, Bednar J, Hamiche A, Angelov D, Petosa C, Dimitrov S. (2018) Structure of an H1-Bound 6-Nucleosome Array Reveals an Untwisted Two-Start Chromatin Fiber Conformation. Molecular Cell 72:902-915
doi.org/10.1016/j.molcel.2018.09.027

2. Sharma AB, Dimitrov S, Hamiche A, Van Dyck E. (2018) Centromeric and ectopic assembly of CENP-A chromatin in health and cancer: old marks and new tracks. Nucleic Acids Res 47:1051-1069.
doi.org/10.1093/nar/gky1298

The structure of a complete nucleosome (including linker DNA and linker histone H1), the three-dimensional organization of the 30-nm chromatin fiber, and the structure of centromeric chromatin represent key problems in the chromatin and epigenetics field. Moreover, how centromeric chromatin, which is characterized by nucleosomes bearing the H3 histone variant CENP-A, recruits the centromere protein CENP-C to mediate assembly of an active kinetochore is poorly understood. Research into these questions will advance our understanding of fundamental chromatin biology and shed light on the molecular mechanisms underlying specific genetic and epigenetic disorders.

Objectives: The aims of this project are to obtain three-dimensional structural data for the H1-containing nucleosome, the 30-nm chromatin fiber and CENP-A chromatin, and to decipher how CENP-C specifically recognizes CENP-A chromatin.

Methodology: We will reconstitute mono-nucleosomes using recombinant core histones, different isoforms of the H1 linker histone and DNA bearing a strong nucleosome positioning sequence. We will determine structures by X-ray crystallography and electron cryo-microscopy (cryo-EM) exploiting the latest technological developments. We will validate these structures using DNA-footprinting and a novel cross-linking/qPCR technology developed by the consortium called Identification of Closest Neighbouring Nucleosomes (ICNN).

Consortium: The project is an interdisciplinary collaboration involving five research teams located in Grenoble, Lyon and Strasbourg. The work envisaged is a comprehensive effort that combines expertise in the structural biology, biochemistry, biophysics and cell biology of chromatin.

Expected results and impact: This project is expected to generate new knowledge regarding the structure of chromatin, the roles of linker histone H1 and of CENP-A in determining chromatin structure, and the interactions of CENP-A chromatin with CENP-C. This foundational knowledge is indispensable for understanding the organization of the genome as well as the genetic and epigenetic mechanisms that underlie major nuclear processes such as gene expression, DNA replication, DNA repair and mitosis.

Biomedical relevance: Understanding the higher-order structure of chromatin and the regulatory role played by linker histones and CENP-A is critical for understanding how several severe diseases arise at the molecular level. Alterations in CENP-A chromatin are associated with severe chromosome and segregation defects as well as perturbed cytokinesis. These lead to meiotic aneuploidy, a major cause of spontaneous abortions, infertility and birth defects, including Down, Edwards and Patau syndromes that affect a significant proportion of newborns every year. This project is expected to advance our understanding of the detailed molecular mechanisms that give rise to the above disorders.