Understanding the role of histone H3.3 and its chaperone ATRX in the maintenance of chromatin integrity
thesisposted on 01.03.2017, 05:47 by Chang, Tsz Man
Histones and their associated post-translational modifications (PTM) play an essential role in regulating chromatin dynamics. In addition to the canonical histone family, histone variants have also emerged as key players in regulating chromatin structure and dynamics and governing cellular integrity. For example, recent studies have identified frequent mutations of not only histone variant H3.3 at specific key residues, but also its chaperones Alpha-Thalassemia Mental Retardation X-linked (ATRX) and DAXX (Death-associated protein 6) in human cancers. In particular, mutations of the ATRX genes is found to be closely associated (>90% mutation rate) in human cancers that maintain the telomere length by a recombination based mechanism, known as the Alternative Lengthening of Telomeres (the ALT pathway). The function of ATRX is required for mediating the deposition of H3.3 at heterochromatin including the telomeres and pericentric DNA repeats. Although it has been well documented that ATRX is important for the maintenance of a heterochromatic state at these repeats such as the telomeres, much remains unknown of the pathway and nuclear platform that promote ATRX binding and H3.3 deposition at telomeres. Furthermore, it is also unclear whether ATRX inactivation can further affect H3.3 deposition and its PTM profile at other genomic regions or in the global genome. In this study, I investigated the assembly pathway essential for binding of ATRX and deposition of H3.3 at telomeres in pluripotent mouse embryonic stem cells (mESC). I showed a novel role of Promyelocytic nuclear bodies (PML-NB) in regulating the assembly of H3.3 by ATRX at the telomeres. In mouse ES cells, the association of telomeres with these nuclear bodies is important for the propagation of a unique telomeric chromatin state which is important for maintaining the mESC pluripotent state. More importantly, my study shows that these nuclear bodies are functionally distinct to the ALT-associated PML nuclear bodies found in human ALT cancers. Considering the high inactivation frequency of ATRX reported in these telomerase deficient cancers, this project also examined the effect of ATRX loss of function on the distribution and PTM of H3.3 through the investigation of the profile of Serine 31 phosphorylation on H3.3 (H3.3S31ph). H3.3S31ph is normally a histone mark associated with heterochromatic repeats including the telomeres and pericentric DNA repeats in mitotic cells. However, in ALT cancer cells, H3.3S31 phosphorylation is found to be extremely high across the entire chromosome arms. This aberrant localisation of H3.3S31ph correlates with the loss of ATRX expression and is driven by the high level of endogenous CHK1 activity in these cancer cells. Together, this study has examined the role of ATRX and H3.3 in the regulation of chromatin integrity in two different cellular settings: the normal and disease cell state. Findings from this study have provided novel insights into the mechanism underlying H3.3 deposition at telomeres in normal cells, and the impact of ATRX inactivation on H3.3 PTM profile, and its contribution to disease development in cancer cells.