|Institution:||University of Oslo|
|Full text PDF:||https://www.duo.uio.no/handle/10852/11475
The genome is organized into chromatin domains which can be distinguished by enrichment in post-translational modifications of histones and histone variants. Since the histone code has been discovered, little is known about the biological significance and more of chromatin deposition of histone variants. The histone H3 variant H3.3 marks active chromatin and in contrast to canonical histones, it is incorporated in a replication-independent manner. Deposition of H3.3 into chromatin is mediated independently by the DAXX/ATRX and HIRA/ASF1A chaperone complexes. In this study, we provide evidence, by immunofluorescence and fluorescence recovery after photobleaching (FRAP), for the deposition of epitope-tagged H3.3 into chromatin through a recruitment to PML bodies. H3.3 co-localizes with DAXX, ATRX, HIRA and ASF1A chaperones at PML bodies. FRAP analysis shows that DAXX is necessary for H3.3 recruitment to PML bodies and is also required for enrichment of ATRX in these structures. However, HIRA and ASF1A accumulate at PML bodies independently of DAXX. ASF1A facilitates H3.3 targeting to PML bodies, but is not essential in this process. H4 overexpression also facilitates H3.3 recruitment to PML bodies. Finally, enhanced recruitment to PML bodies of an H3.3(H113A) mutant which in silico prevents (H3.3-H4) tetramerization supports a model where H3 and H4 are preferentially recruited to PML as an (H3.3-H4) dimer rather than as a tetramer. Our data support a working model of H3.3 deposition mechanism where (H3.3-H4) dimers are recruited to PML bodies by DAXX, prior to incorporation into chromatin.