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FRIMEDBIO-Fri prosj.st. med.,helse,biol

Biological significance of the histone variant H3.3

Awarded: NOK 2.2 mill.

Project Number:

213795

Application Type:

Project Period:

2012 - 2015

Our DNA is packaged by special proteins called histones. Some of these proteins play an important role in protecting "open" DNA regions that would be subject to genetic mutations causing disease. Reparation of these open or damaged regions involves a specific histone called H3.3. We have in this project elucidated the mechanisms controlling the incorporation of histone H3.3 into chromosomes in human and mouse cells. We have shown that a pool of newly synthesized H3.3 is driven by a factor we have identified (called DAXX), to specific structures in the cell nucleus that are called PML bodies. There, H3.3 is coupled to cargo proteins which then bring H3.3 to specific sites in our genome: PML bodies therefore act as sorting centers for H3.3. We have also shown that damages in chromosome structure, caused by removing an onco-protein (DEK), triggers a fast deposition of H3.3 which helps repairing chromosomes. This process involves two major cargo protein complexes which we have identified here. Microscopic examination shows how dynamic these processes are. In mouse cells, using high-throughput genomic and computing techniques, we have mapped where the PML protein (which makes up PML bodies) and H3.3 are localized in the entire genome, and the protein composition of chromosomes in these regions. We have then genetically removed the PML protein (which controls H3.3 loading on DNA as mentioned above), and show that this affect not only where H3.3 is loaded, but also the global organization of chromosomes. Our results show that PML also plays an important role in maintenance of chromosome architecture by protecting specific regions in our DNA. We have also identified molecular mechanisms behind the role of PML in controlling the loading of H3.3 on DNA, and what this means for the maintenance of cellular identity. This project has resulted in several high-quality publications and a large number of data presentations in many countries.

In the eukaryotic cell nucleus, the genome is organized into chromatin domains with different physical, biochemical and functional properties influencing gene expression and developmental decisions. These domains can be distinguished by their enrichment i n combinations of post-translationally modified histones and in variants of canonical histones. While the distribution and significance of epigenetic histone modifications have been extensively examined since the discovery of the histone code 10 years ago , little is known on the significance of histone variants. Nonetheless, a recent burst of publications reflects strong interest in elucidating the role of these variants. Among these, the H3 variant H3.3 incorporates into primarily (but not solely) transc riptionally active chromatin in a replication-independent manner. The mode of insertion of H3.3 into chromatin, its positioning in the genome and its requirement for vertebrate development remain elusive. We have recently reported the epigenetic environme nt of H3.3 in human promoters and have gathered preliminary data on its deposition pathway. Here, we combine expertise in mesenchymal stem cell biology, imaging, epigenetics and cell differentiation together with state-of-the-art high-throughput sequencin g and mass spectrometry technologies, to address the biological meaning of insertion of H3.3 in the genome in a cell differentiation context. Our goal is to characterize in detail the molecular mechanism of incorporation of H3.3 into chromatin and get a h int at its function in cellular differentiation. OBJECTIVE 1 aims to provide full genome coverage of H3.3 enrichment sites, in human primary progenitor cells and differentiated cells. OBJECTIVE 2 aims to elucidate the mechanistic details of H3.3 incorpora tion into chromatin, by characterizing H3.3 'transit sites' which we have recently evidenced. Work will be done by a PhD student with technical support.

Funding scheme:

FRIMEDBIO-Fri prosj.st. med.,helse,biol

Funding Sources