Dissecting Mammalian Replication-Independent Chromatin Assembly – Biochemical and Structural Studies on the H3.3-Specific Histone Chaperones Hira and Daxx

Histones are architectural proteins that wrap approximately two turns of DNA around their octameric core structure, constituting the fundamental packaging unit of eukaryotic chromatin – the nucleosome. Beyond their structural role, they regulate virtually all processes that act on or depend on DNA, such as replication, gene expression and maintenance of centromeres and telomeres. Despite the high conservation of the four core histones, H3, H4, H2A and H2B, throughout all eukaryotes, histone variants have emerged with variable degree of divergence from their canonical counterparts. These variants are thought to expand the regulatory repertoire of chromatin. The histone replacement variant H3.3 is implicated in the formation and maintenance of specialized chromatin structure in metazoan cells. H3.3-containing nucleosomes are assembled in a replication-independent manner by means of dedicated histone chaperone proteins. By purifying H3.3-containing nuclear complexes from HeLa cells, I have identified proteins that mediate this replicationindependent deposition of H3.3. I have been able to single out the death-associated protein Daxx as an bona fide histone H3.3 chaperone. Furthermore, I show that Daxx cooperates with an ATP-dependent chromatin remodeler, ATRX, in depositing histone H3.3 at telomeres in vivo (Chapter 2). I show that an evolutionary conserved histone binding domain of Daxx interacts directly with variant-specific residues of the H3.3 core. I further identify homologies with the centromere-specific histone chaperone HJURP that hint to a conserved mechanism used across replicationindependent chromatin assembly pathways (Chapter 3). I report crystallization of a complex of the Daxx histone binding domain and histones H3.3-H4 with the aim of solving the cocrystal structure to reveal how Daxx can tell the subtle differences in H3 variants apart. (Chapter 4). Through a more detailed biochemical fractionation and genetic analysis, I found that Daxx constitutes a histone deposition pathway independent of the previously described HIRA histone chaperone complex (Chapter 5), corroborating the notion that distinct replication-independent chromatin assembly pathways maintain specialized chromatin at distinct genomic locations. To facilitate the study of histone H3 variants across biological model organisms, I have developed universal, variantspecific antibodies (Chapter 6). I showcase their application in deriving a semiquantitative histone variant landscape in murine embryonic stem cells, on the basis of which the functions of H3.3 and replication-independent chromatin assembly can be studied in new detail. In the light of recent findings that place Daxx and ATRX in the roles of potent tumor suppressors for a subset of neuroendocrine cancers, my studies will help to elucidate the molecular etiology of replication-independent chromatin assembly pathways in human disease. (Simon Elsasser