CHD7 Negatively Regulates Hematopoietic Stem Cell Potency

Abstract 850

The molecular mechanisms governing HSC homeostasis and potency are incompletely understood. Runx1-CBFb is required for HSC formation in the embryo and for maintaining normal HSC and progenitor cell frequencies in the adult. To identify chromatin regulatory proteins that function with Runx1-CBFb during hematopoiesis, we performed co-immunoprecipitation and mass spectrometry using a FLAG-tagged CBFb protein, and as a control a FLAG-CBFb (G61A/N104A) mutant that cannot bind Runx1. Multiple unique peptides of the ATP-dependent chromatin remodeling enzyme Chromodomain Helicase DNA binding protein 7 (CHD7) were immunoprecipitated specifically with FLAG-CBFb. Autosomal dominant CHD7 mutations are associated with CHARGE syndrome (Coloboma of the eye, Heart defects, Atresia of the choanae, Retardation of growth and/or development, Genital and/or urinary abnormalities, and Ear abnormalities and deafness). Several subunits of the nucleosome remodeling and histone deacetylase (NuRD) complex were also identified, including p66a, p66b, MTA2, and histone binding protein RBBP4. Hence Runx1-CBFb may associate with distinct CHD7 and NuRD containing complexes to regulate HSC function.

The CHD7 interaction with Runx1 requires the Runx1 transactivation domain, which is critical for all stages of hematopoiesis. The interaction of CHD7 with Runx1 does not require heterodimer formation with CBFb. Runx1^+/−; Chd7^+/− double heterozygous mice are born in a sub-Mendelian ratio, indicating that Runx1 and Chd7 interact genetically. Knockdown of chd7 in zebrafish embryos increased the number of runx1 expressing cells specified in the AGM, and this effect is cell autonomous as determined by blastula transplantation. Downstream lineages such as myeloid and erythroid cells were also expanded. Consistent with these results, conditional pan-hematopoietic deletion of Chd7 in mice with Vav1-Cre did not cause mature lineage differentiation defects. Methylcellulose colony forming assays of CHD7 deficient cells resulted in increased granulocyte/monocyte progenitors. Although CHD7 deficient bone marrow had a normal frequency of CD48⁻ CD150⁺ Lin⁻Sca-1⁺c-kit⁺(LSK) phenotypic long term repopulating HSCs (LT-HSCs), it has a three fold higher frequency of functional LT-HSCs as determined by whole bone marrow limiting dilution transplants. By serial transplantation, a higher percentage of tertiary recipient mice were engrafted with CHD7 deficient bone marrow cells compared to wild type. In summary, our study identifies CHD7 as a novel epigenetic regulator of HSC and progenitor function that functions with Runx1 and members of the NuRD complex to negatively regulate HSC numbers and potency.