Chrom3D

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.