Cistrome Control of Hematopoieitic Stem/Progenitor Cell Function

Cis-regulatory mechanisms control chromatin structure and cellular identity. At the GATA2 locus, two cis-elements are linked to human pathologies, including a primary immunodeficiency (MonoMAC syndrome) associated with multiple complex phenotypes, myelodysplastic syndrome, and acute myeloid leukemia (AML). Mutations that disrupt the function of an intronic GATA2 +9.5 element cause MonoMAC syndrome, while an inversion that relocates the distal GATA2 -77 element to the EVI1 locus induces AML. The +9.5 and -77 cis-elements are GATA-2-occupied and confer context-dependent enhancer activities in select hematopoietic cell types in vivo. In knockout mouse models, the Gata2 +9.5 cis-element is required for hematopoietic stem cell (HSC) genesis, whereas the Gata2 -77 cis-element governs a unique sector of the myeloid progenitor cell transcriptome without impacting HSC genesis. Three other GATA-2-occupied cis-elements (-1.8, -2.8 and -3.9) were not individually required for hematopoietic development, and had relatively mild effects on Gata2 expression; the -1.8 site was required to maintain Gata2 repression in late-stage erythroblasts, the -2.8 conferred maximal Gata2 expression, and the -3.9  had no effect on Gata2 expression. We predict that additional cis-elements exist in the genome with functions resembling the +9.5 and -77, and their analysis will provide important mechanistic and biological insights. We utilized the known properties of Gata2 cis-elements as learning tools to identify prospective constituents of a hematopoietic stem/progenitor cell (HSPC) regulatory cistrome genome-wide. Using sequence attributes shared with the critically-important +9.5 element, namely a CATCTG-8bp spacer-AGATAA, we generated a list of 797 candidate cis-elements (“+9.5-like” elements). This list was prioritized using chromatin occupancy by GATA-2 and Scl/TAL-1, among others, chromatin accessibility, evolutionary conservation, and histone modifications in a multitude of biologically-relevant cell types. Gene editing was used to delete three high-ranked elements (Samd14 +2.5, Bcl2l1 +12.2, and Dapp1 +23.5), revealing their importance for transcriptional activation, GATA-2 occupancy and chromatin accessibility, while deletion of two low-ranked elements (Mrps9 +17.6 and Mgmt +182) had no effect on gene transcription. One such cis-element (Samd14 +2.5) resided in Samd14, a gene with undescribed biological function. Samd14 has a conserved sterile α-motif and coiled-coil domain, and is highly expressed in hematopoietic progenitors and differentiated progeny. Mouse knockout of the Samd14 +2.5 element dramatically lowered expression of Samd14 in hematopoietic progenitors. We conducted loss-of-function analysis to elucidate Samd14 function in lineage-depleted (Lin^-) E14.5 fetal liver cells infected with control or Samd14 shRNA-expressing retrovirus. In a CFU assay, Samd14 knockdown reduced BFU-E and CFU-GM colonies 3.4-fold. Early erythroid precursor R1 (CD71^low, Ter119^-) and R2 (CD71^high, Ter119^-) cell populations decreased ~2-fold, concomitant with increases in more mature R3 and R4/5 populations (Ter119⁺). In R1/R2 cells, Samd14 knockdown reduced surface c-Kit expression by 1.6-fold and prevented Stem Cell Factor/c-Kit activation of AKT. Cellular deficits resulting from Samd14 knockdown could be rescued by c-Kit. In -77^-/- common myeloid progenitors, Samd14 was ~20-fold downregulated. Thus, the importance of Samd14 and the Samd14 +2.5 element on progenitor function and SCF/c-Kit signaling validates our strategy for identifying cis-elements relevant for hematopoiesis. Our findings demonstrate that +9.5-like elements control cell signaling (Samd14 +2.5) and apoptosis (Bcl2l1 +12.2), and we predict that additional cistrome constituents will control these and other important HSPC processes. I will discuss the mechanistic and biological properties of additional cis-elements analyzed from a cohort of 68 GATA-2-occupied elements and general principles arising from the HSPC cistrome analysis, which provide unique insights into the control of hematopoiesis and GATA-2-linked pathologies.