Structural and functional analysis of the histone variant H2AZ deposition/exchange mechanisms. – VARIZOME

To study the machineries responsible for the deposition of the histone variant H2AZ, we have used biochemical approaches based upon the purification of protein complexes associated to this histone variant followed by mass spectrometry characterization. This was allowed by the expression in cells of tagged histone variant that carried a specific motif allowing its immunopurification. We have also used this tagged variant to identify the genomic regions where it is enriched. This was achieved through immunopurification of the chromatin and high-throughput sequencing of the corresponding DNA. We have also studied the consequence of the inactivation of the gene coding for one of the deposition factors to identify genomic regions where this factor was playing a key role in the deposition of this variant.

We have purified protein complexes associated to both standard histone H2A, as well as its variant H2AZ that is specifically associated with gene promoters. We have identified proteins that are specifically associated to each of these histones, in particular AN32 for H2AZ and NAP-1 for H2A and have shown that they were playing a chaperon function to favor either eviction or deposition. We have mapped sites of H2AZ enrichment within the genome using high-throughput sequencing and have observed enrichment in silenced or poised promoter. Interestingly H2AZ is released from these promoters during transcription activation a prerequisite for the recruitment of PolII. We have also identified large genomic domains where H2AZ enrichment depends on AN32. We have also characterized the structure of the H2AZ/H2B dimer in association with AN32 and dissected their mode of interaction. We have also analyzed the structural and functional consequences of AN32 binding to H2AZ nucleosomes that results in the eviction of H2AZ from the nucleosome. Altogether, these characterizations allow a better understanding of how the structural alterations of the nucleosome have an impact on genome function.

Our observations that different sets of genomic regions are affected by mutation of these different factors improve our understanding of the differences between the various tumoral phenotypes. Our structural and functional characterization of certain nucleosomal states offers a mechanistic perspective to better understand the consequences of modifications of the epigenetic memory in both normal and pathologic situations. Understanding the mechanisms involved improves our ability to devise targeted therapeutic approaches.

Most of our results have resulted in a scientific publication that is in revision for publication. The additional obtained results are being prepared for publication.

An32, a histone chaperone specialized in the removal of H2A.Z from chromatin. Arnaud Obri1*, Khalid Ouararhni1*, Marie-Laure Diebold2, Kiran Padmanabhan3, Christophe Papin1, Martin Marek2, Isabelle Stoll1, Stefan Dimitrov3,§, Christophe Romier2, § and Ali Hamiche1,§.

Histone variants are non-allelic isoforms of conventional histones, which are deposited to chromatin by highly specialized deposition complexes containing histone chaperones and chromatin remodeling factors. This project aims at a global understanding of the role of the histone variant H2AZ and its deposition/exchange machineries in the epigenetic control of mammalian genome activity. From a functional perspective, H2AZ is one of the most studied histone variants in recent years. The functional information gathered to date, points to an important physiological role of H2AZ in the establishment and maintenance of chromatin states. H2AZ is a highly conserved protein, which has been shown to be necessary for survival in a broad range of organisms. However, the function of this histone variant has been, and still remains, extremely controversial. H2AZ plays an important role in chromatin remodeling during transcription and DNA repair. However, its role is not clear and much remains to be done to understand its function in these processes. In this project we propose to study the biological function of H2AZ using the state of the art biochemical approaches combined with structural and functional studies. We are aiming at understanding the role played by H2AZ in gene regulation, genome integrity and epigenetics phenomena. We are particularly interested to clarify the mechanism of deposition/exchange of H2AZ at specific loci and to study how H2AZ chaperones are fulfilling this function in vivo. The novelty of the project lies in the combination of the various expertises that should allow addressing long-standing questions that have received up-to-now only partial fragmentary answers. It is believed that such a comprehensive view of histone H2AZ deposition/exchange mechanisms is necessary to gain a relevant understanding of its function, in particular in the epigenetic control of gene expression and genome integrity. This project should allow key advances in a fundamental knowledge of importance for human health. We also expect fundamental insights into epigenetic phenomena that are involved in genetic diseases and other human diseases. This knowledge can on the long-term supply new possibilities for therapeutic developments.