Investigation into the roles of HP1γ, H3.3 and HIRA in gene regulation in vivo
Creators: name: Liang, Bing
Publisher: name: Imperial College London
Year: 2019

description: This thesis addresses two distinct aspects of chromatin regulation in vivo in mice; the chromatin remodeler HIRA and the heterochromatin binding protein HP1γ. To date, H3.3 has been largely studied in vitro and reported to be enriched at promoters, bodies of active genes and some regulatory elements throughout the cell cycle by its chaperone HIRA and has been described as an ‘active mark’ for transcription. Moreover, H3.3 is also targeted to telomeric and pericentric heterochromatin regions by the DAXX/ATRX complex. However, the in vivo function of H3.3 in mammalian systems is still largely unknown. In this thesis, we showed that in the HIRA KO compared to WT, there was an apparent abnormal presence of a memory T cell population in the mouse thymus indicated by CD44 expression on CD4 and CD8 single positive T cells, and an increased population of CD44 expressing cells in the lymph node and spleen. Thus, either CD44 gene expression is directly affected by HIRA KO or the T cell differentiation program is altered or both. Moreover, TCRα V(D)J recombination seems to be impaired by HIRA KO. This might imply that HIRA chromatin remodeling or the presence of H3.3 might be important for TCR recombination. Recently the importance of Heterochromatin Protein 1 γ (HP1γ) in regulating sex differences was demonstrated in our lab. In this thesis, sex dimorphism in proliferation was investigated and we showed that male embryonic fibroblasts (MEFs) proliferate faster than female MEFs, which is consistent with previous observations that the embryonic growth rate is higher in male compared with female mammals. HP1γ KO completely abolished the sex difference in proliferation. To determine whether this sex-regulatory effect of HP1γ was a more general phenomenon, recently, RNA-seq analysis was performed in the lab on both male and female MEFs, with or without HP1γ. Strikingly, males showed a higher dependency on HP1γ in maintaining their normal gene expression profile compared to females. RNA splicing analysis of the RNA-seq data suggested that the sex difference in gene expression was not a result of difference in splicing and vice versa. Similarly, HP1γ KO exerted a bigger impact in males in terms of alternative splicing where there are many more genes found to be alternatively spliced upon HP1γ KO in males than females. This was consistent with the results of Mass Spectrometry following pulldown of MEF-derived proteins with HP1γ antibody which revealed splicing factors in the male but not the female samples. Consistent for a role for HP1γ in regulating sex dimorphism in alternative splicing, HP1γ KO ameliorated this dimorphism. The emerging evidence of an activating role of HP1γ in euchromatin brings the possibility that HP1γ and HIRA/H3.3 may interact to regulate gene expression in euchromatin. Especially with the recent observation in our lab that shows co-localization between HP1γ and H3.3 in MEFs by co-immunoprecipitation. Therefore, it is important to further investigate the interaction of HP1γ with H3.3 and the role of HP1γ as a key epigenetic modifier in defining sexually dimorphic gene expression in males and females and its role in cellular proliferation in order to shed light on the largely unexplained sex bias previously reported in normal physiology and diseases.