The Role of Histone Variant H3.3 Regulation By Hira in Hematopoiesis

A large proportion of adult hematopoietic stem cells (HSCs) remain quiescent throughout life, rarely undergoing cell division. These quiescent HSCs must undergo global changes in gene expression patterns in order to leave their dormant state and differentiate. Chromatin modifiers that act independent of cell division may therefore be of particular importance in regulating the behavior of quiescent HSCs through alterations in the chromatin landscape.

One such chromatin modifier is the histone variant H3.3 chaperone, Hira, which is responsible for incorporating H3.3 independent of DNA replication at poised and transcriptionally active promoters. Despite the fact that this histone variant only differs from the canonical H3 histones by four or five amino acids, its gene-specific association is required for the proper establishment of gene expression and lineage specification in murine embryonic stem cells (mESCs), post-mitotic neurons, and oocytes. Since Hira deposits H3.3 outside of S phase, we hypothesize that Hira plays a critical role in the maintenance of quiescent HSCs through the regulation of H3.3 independent of cell division.

HSCs divide rapidly in the fetal liver but become quiescent in adulthood, providing an interesting opportunity to study HSCs that are vastly different in terms of cell cycle properties. In order to study the role of Hira in HSCs, we used a conditional knockout (cKO) mouse model to delete Hira in hematopoietic cells in early development (Vav-iCre; Hira^fl/fl). Importantly, we observed severe depletion of adult, but not fetal, HSCs upon loss of Hira. This suggests that Hira is important for the maintenance of quiescent adult HSCs, but not rapidly dividing fetal HSCs, and supports our model of Hira as a regulator of HSC quiescence through the DNA replication-independent incorporation of H3.3. We additionally observed altered differentiation potential, reduced reconstitution capacity, and increased cycling in adult HSCs upon Hira cKO. These results demonstrate that Hira is an essential regulatory factor for the control of adult HSC function.

To obtain adult Hira cKO HSCs for further analysis, we also used an inducible model of Hira deletion in the hematopoietic system upon polyinosinic-polycytidylic (pIpC) treatment (Mx1-Cre; Hira^fl/fl). We performed RNA-seq analysis on adult Hira cKO HSCs, which showed widespread alterations in gene expression patterns and upregulation of PRC2 target genes. Since Hira is known to recruit polycomb repressive complex 2 (PRC2) to bivalent promoters in mESCs in an H3.3-dependent manner (Banaszynski et. al. Cell 2013), chromatin-immunoprecipitation and sequencing (ChIP-seq) for H3K27me3 will be used to show how loss of Hira affects PRC2 activity in HSCs. We have additionally performed assay for transposase-accessible chromatin and sequencing (ATAC-seq) in order to determine how patterns of DNA accessibility in HSCs are affected by Hira cKO. Altered nucleosome positioning and PRC2 activity will be discussed as potential mechanisms for the role of Hira in regulating the behavior of quiescent HSCs.

Our findings reveal a novel paradigm for epigenetic state regulation in adult HSCs. Knowledge gained from this study may inform future research for clinical applications, such as for improved methods of in vitro HSC expansion. Characterizing the importance of H3.3 regulation by Hira in normal HSC activity will therefore provide invaluable information regarding the epigenetic mechanisms underlying hematopoietic homeostasis.