Chd7 Is a Cell Autonomous Regulator of Chromatin In Hematopoietic Stem Cells.

Abstract 1596

Hematopoietic stem cells (HSC) are specified during embryogenesis, and the induction process involves not only transcription factors but also epigenetic factors that modulate chromatin to regulate the hematopoietic transcriptional programs. Here, we performed a reverse genetic screen to identify all the chromatin factors that are required for HSC induction in zebrafish. The zebrafish homologs of 350 human chromatin factors were identified by reciprocal BLAST and knocked down by injecting morpholinos designed against each homolog into the single cell embryo. Morphants were then analyzed for changes in blood formation by in situ hybridization for β-globin e3 expression in primitive erythrocytes at 16 somite stage and for c-myb and runx1 in definitive stem cells at 36 hours post fertilization. From the screen, we have identified known regulators of hematopoiesis such as bmi1, in which knock down results in loss of stem cell formation. We have also identified one novel HSC regulator chd7. Chd7 is a member of the chromodomain helicase DNA-binding domain family that functions at gene enhancer elements and in ribosomal RNA synthesis. Zebrafish embryos injected with chd7 morpholino had higher levels of β-globin e3 and c-myb/runx1 expression. Additional markers such as scl, gata1, fli1, and lmo2 were also upregulated, although vascular markers flk1 and ephrinB2 were downregulated. Early mesodermal markers eve1 and ntl expression appeared normal, suggesting that the effects of chd7 knock down occurs when the mesodermal precursor cell population becomes an HSC. Transplants of chd7 deficient Tg(c-myb:GFP) blastomeres into Tg(lmo2:DsRed) blastulas resulted in more chimeric embryos compared to controls, demonstrating that the phenotype is cell autonomous. In humans, haploinsufficiency for CHD7 is the main cause of CHARGE syndrome, and it has been recognized more recently that these patients are immunodeficient, though the etiology remains unknown. Our studies indicate a new role for chd7 in hematopoiesis in which it functions to repress HSC formation during embryogenesis.