Adult Hematopoietic Stem and Progenitor Cell Maintenance Requires Histone Variant H3.3 Regulation By Hira

The adult hematopoietic system is sustained by a balance of self-renewal and differentiation in a small pool of stem and progenitor cells. This balance must be maintained to ensure a continuous supply of blood cells throughout life and prevent malignancy from arising. There are many facets of epigenetic regulation that are well known to be key components of healthy and diseased hematopoiesis, such as DNA methylation and histone post-translational modifications. However, the role of histone variant incorporation in hematopoiesis remains relatively unexplored. In this study, we explore the role of histone variant H3.3 regulation in the hematopoietic system by assessing the function of the histone H3.3 chaperone, Hira.

Toward this goal, we use inducible and early developmental conditional knockout (cKO) mouse models to assess the role of Hira within the hematopoietic system. Following Hira cKO early in hematopoietic development (Vav-iCre; Hira^fl/fl), we find that HSPCs are unaffected in the fetal liver but deplete quickly after homing to the bone marrow. Using polyinosinic-polycytidylic (pIpC) inducible Hira cKO mice (Mx1-Cre; Hira^fl/fl), we find a similarly severe depletion of HSPCs in adult mice within 1 month after Hira loss. In contrast, differentiated cells remain largely unaffected following Hira cKO, demonstrating that Hira is especially important in the hematopoietic stem and progenitor compartment. Since Hira is known to incorporate H3.3 throughout the cell cycle and not just during S-phase like H3.1/2, we hypothesized that adult HSPCs are more dependent upon Hira to regulate histone H3 dynamics since they are slowly dividing. The loss of Hira-mediated H3.3 deposition would also be particularly detrimental to the function of these cells given its association with actively transcribed and bivalent genes.

To test the role of Hira in maintaining gene expression patterns, we performed bulk RNA-seq on adult HSPCs and found that hematopoietic differentiation genes are dysregulated after Hira cKO with increased erythroid lineage and decreased lymphoid lineage gene expression. We then assessed gene expression changes in Hira cKO HSPCs in a doxycycline-inducible H2B-GFP background (Mx1-Cre; Hira^fl/fl; R26-M2rtTa; TetOP-H2B-GFP) to distinguish between the gene expression changes caused by Hira loss before and after cell division. In the absence of Hira-mediated H3.3 incorporation, we expect some highly expressed genes in slowly dividing adult HSPCs to be affected by Hira loss prior to cell division due to nucleosome turnover in the wake of RNA Polymerase II. At other loci, like bivalent promoters, H3.3 would be diluted after cell division by H3.1/2 during S-phase in Hira cKO HSPCs. In support of this hypothesis, we found that increased expression of the erythroid differentiation gene Klf1 in Hira cKO MPPs after cell division (H2B-GFP^Low) relative to Hira cKO MPPs before division (H2B-GFP^High) and WT MPPs that have divided (H2B-GFP^Low). The findings from both of these transcriptome analyses point toward a role of Hira in regulating HSPC differentiation genes and are supported by our in vitro and in vivo data showing increased differentiation of Hira cKO HSPCs and decreased self-renewal.

In order to more fully understand the H3.3-dependent gene expression changes after Hira cKO in HSPCs, we correlated H3.3 enrichment patterns from chromatin-immunoprecipitation and sequencing (ChIP-seq) with our data from assay for transposase-accessible chromatin and sequencing (ATAC-seq). Our results demonstrate that Hira cKO HSPCs have more open chromatin and fewer H3.3 peaks, suggesting that loss of Hira-mediated H3.3 deposition increases DNA accessibility.

This study identifies a novel epigenetic mechanism required for adult HSPC maintenance and elucidates a previously unappreciated regulator of normal hematopoietic homeostasis. Further understanding how Hira-mediated H3.3 regulation maintains adult HSPCs will provide greater depth to our current understanding of the epigenetic regulators essential for hematopoiesis.