Autism-Associated Chromatin Remodeler CHD8 Governs the Survival and Differentiation of Hematopoietic Stem/Progenitor Cells

Hematopoiesis is a strictly regulated process which depends on regulated proliferation, differentiation, and survival of hematopoietic stem/progenitor cells (HSPCs). Multiple signaling pathways, transcription factors and epigenetic machineries are involved in this important process. Increasing evidence from recent studies indicates that ATP-dependent chromatin-remodeling genes are involved in the control of crucial gene-expression programs in stem/progenitor cell regulation. Among them, the CHD8 gene encodes a member of chromodomain helicase DNA-binding (CHD) family of SNF2H-like ATP-dependent chromatin remodeler, mutations of which define a subtype of autism spectrum disorders. Whether CHD8 plays a role in hematopoietic cells remains unknown.

In this study, to define the role of CHD8 in hematopoiesis and HSPC regulation, we carried out conditional deletion of CHD8 in the hematopoietic lineages using an interferon inducible Mx1-Cre; Chd8^F/Fmouse model. As early as one week after polyI:C injection, the CHD8 knockout mice experienced drastic pancytopenia and bone marrow failure with a reduction of bone marrow (BM) cellularity to ~1/3 of that of WT controls. In the HSPC compartment, loss of CHD8 resulted in a depletion of Lin^-c-Kit⁺ (LK) and Lin^-Sca1⁺c-Kit⁺ (LSK) progenitors and a severe reduction of the GMP, MEP, MPP and CD150⁺CD48^-LSK HSC populations. Accompanying the loss of HSPCs were dramatically increased apoptosis rate and cell cycle arrest in Chd8^-/- cells. An examination of HSPC function by transplantation found that the BM cells from Chd8^-/- mice could not engraft in the recipient mice that died ~20 days post-transplantation. The chimerism of Chd8^-/- BM cells also reduced drastically in a competitive BM transplantation assay when induction of Chd8 deletion occurred after Mx1-Cre; Chd8^F/Fcells were pre-engrafted at 1:1 ratio with WT BM cells. These results indicate that CHD8 is essential for hematopoiesis and HSPC survival and proliferation.

To elucidate the underlying mechanism of CHD8 function in HSPCs, we performed complementary biochemical, genomic and genetic analyses of the WT (Chd8^F/F) and Chd8^-/-HSPC cells. Firstly, we found blood progenitor cells dominantly express the short form of CHD8 containing residues 1 to 751 which retains the N-terminal P53 binding domain, rather than the long form of the CHD8 protein. Secondly, we revealed by RNA-seq and subsequent RT-PCR analyses that the P53 signaling pathway signatures including P53, P21, Bax and Noxa are aberrantly activated in Chd8^-/-HSPCs. At the protein level, P53 and P21 are significantly elevated in Chd8^-/- LK cells. Thirdly, ATAC-seq analysis of the LSK cells revealed increased global chromatin accessibilities after CHD8 deletion, including the promoter regions of P21 and Noxa genes. By a "Cut & Run" assay using H3K4me3 antibody in Chd8^F/Fand Chd8^-/- LSK cells we have also seen significantly enhanced epigenetic H3K4me3 mark on the promoter region of P21 and Noxa. An anti-CHD8 "Cut & Run" further detected direct binding sites of CHD8 on P53 and P21, but not Noxa or Bax gene. Fourthly, we observed a direct binding interaction by co-immunoprecipitation between P53 and CHD8 proteins. Finally, genetic deletion of one allele of P53 gene in Chd8^-/- HSPCs (P53^+/-;Chd8^-/-) was able to completely suppress the Bax/Noxa expression and apoptosis phenotype and rescue most of the HSC and early progenitor defects but not the later stage of differentiation defects and BM cellularity loss. Interestingly, deletion of both alleles of P53 gene (P53^-/-;Chd8^-/-) was able to further remove the differentiation block between LSK and LK cells observed in the P53^+/-;Chd8^-/-background which is associated with a P21 mediated G0 cell cycle phase arrest. These combined results suggest a mechanism of action that CHD8 works through a direct complex with P53 to regulate P21 gene expression which mediates cell cycle control of HSPC differentiation and an indirect CHD8 mediated P53 expression and subsequent P53 regulated Noxa/Bax expression which regulates HSPC survival.

Thus, we identify the autism-associated CHD8 as an essential chromatin suppressor in regulating hematopoiesis through restricting the expression of P53 and chromatin accessibility of P53 downstream targets, Bax/Noxa and P21, to enable selective control of HSPC survival and differentiation, respectively.