An enhancer with amalgamated E-box and GATA motifs (+9.5) controls expression of the regulator of hematopoiesis GATA-2. While similar GATA-2-occupied elements are common in the genome, occupancy does not predict function, and GATA-2-dependent genetic networks are incompletely defined. A “+9.5-like” element resides in an intron of Samd14 (Samd14-Enh) encoding a sterile alpha motif (SAM) domain protein. Deletion of Samd14-Enh in mice strongly decreased Samd14 expression in bone marrow and spleen. Although steady-state hematopoiesis was normal, Samd14-Enh−/− mice died in response to severe anemia. Samd14-Enh stimulated stem cell factor/c-Kit signaling, which promotes erythrocyte regeneration. Anemia activated Samd14-Enh by inducing enhancer components and enhancer chromatin accessibility. Thus, a GATA-2/anemia-regulated enhancer controls expression of an SAM domain protein that confers survival in anemia. We propose that Samd14-Enh and an ensemble of anemia-responsive enhancers are essential for erythrocyte regeneration in stress erythropoiesis, a vital process in pathologies, including β-thalassemia, myelodysplastic syndrome, and viral infection.

Establishment of the hematopoietic system requires the transcription factor GATA-2, and human GATA-2 mutations cause immunodeficiency, myelodysplastic syndrome, acute myeloid leukemia and vascular/lymphatic dysfunction. GATA-2-regulated enhancers differentially control Gata2 expression in hematopoietic stem/progenitor cells to ensure normal hematopoiesis. The enhancer +9.5 kb downstream of the transcription start site activates Gata2 transcription in endothelium and hematopoietic stem cells (HSCs), and its deletion in mice abrogates HSC generation. The -77 kb enhancer activates transcription in myeloid progenitors, and its deletion impairs progenitor differentiation. Since +9.5-/- embryos are HSC-deficient, it was unclear whether the +9.5 enhancer functions in HSC-derived progenitors or if GATA-2 expression in progenitors solely requires the -77. We dissected relationships between the enhancers using -77;+9.5 compound heterozygous (CH) mice. The CH mutation was embryonic lethal and quantitatively depleted MEPs, differing from -77+/- and +9.5+/- mutants. While the +9.5 suffices to trigger HSC generation, both the -77 and +9.5 enhancers must reside on one Gata2 allele to induce MEPs. The -77enhancer generated BFU-E through the induction of GATA-1 and other GATA-2 target genes. The enhancer circuits established developmental signaling that orchestrates a blood development program. The +9.5, but not the -77, consists of an E-box-8 bp spacer-AGATAA composite element, and human germ-line mutations in and near this sequence are pathogenic. While hundreds of GATA-2-occupied composite elements reside in the genome, GATA-2 occupancy does not predict function. One of the “+9.5-like” elements resides in an intron of Samd14 (Samd14-Enh) that encodes a sterile alpha motif (SAM) domain protein implicated in Stem Cell Factor (SCF)/c-Kit signaling. Targeted deletion of Samd14-Enh in mice strongly decreased Samd14 expression in bone marrow and spleen, without affecting expression in brain. Although we hypothesized that Samd14-Enh controls c-Kit signaling as a key step in developmental hematopoiesis, Samd14-Enh-/- mice had normal steady-state hematopoiesis. However, acute anemia was lethal in Samd14-Enh-/- mice. Anemia activated Samd14-Enh through a multi-component signaling and transcriptional mechanism. Thus, by controlling Samd14 expression, a GATA-2/anemia-regulated enhancer confers survival in severe anemia. Samd14-Enh is the founding member of an ensemble of anemia-sensing enhancers essential for red cell regeneration in stress erythropoiesis, a vital process in diverse pathologies. The strategies deployed to identify developmental and regenerative enhancers are being used to elucidate GATA-2 function in physiological and disease contexts.

While thousands of cis-regulatory elements are predicted to control critical cellular processes, identifying elements with essential, non-redundant functions in vivo remains challenging. Using sequence and molecular attributes shared with the Gata2 +9.5 cis-element site, which activates Gata2 expression during embryogenesis, is required for hematopoiesis, and is mutated in a primary immunodeficiency syndrome, we defined a cohort of similar sites ("+9.5-like"). GATA-2 and Scl/TAL1 occupy a related cis-element (Samd14-Enh) within intron 1 of the poorly-studied Samd14 gene, which consists of a canonical E-box-spacer-GATA motif. We demonstrated the Samd14-Enh regulates Samd14 expression, and Samd14 promotes Stem Cell Factor (SCF)/c-Kit signaling in fetal liver erythroid precursor cells. Since nothing is known about Samd14 function in vivo, we generated a Samd14 enhancer knockout mouse. Samd14-Enh-/- mice had ∼30-fold lower Samd14 expression in bone marrow, but not in brain. Whereas the Samd14- Enh-/- mice exhibited normal steady-state hematopoiesis, the mutant mice were hypersensitive to stress, which yielded lethality ∼4 days after two administrations of 60 mg/kg phenylhydrazine (PHZ). Following a milder PHZ dose, Samd14-Enh-/- mice had smaller spleens, reduced hematocrit, and fewer circulating red blood cells. PHZ induced Samd14 transcription 33-fold and 3.2-fold in splenic CD71+Ter119- and CD71+Ter119+ cells, respectively. Stress-induced Samd14 and Kit upregulation was abrogated in Samd14-Enh-/- mice. Samd14-Enh-/- erythroid precursors in the PHZ-stressed spleen exhibited attenuated SCF-mediated AKT and ERK phosphorylation. These results demonstrate that Samd14-Enh has an essential function to control stress erythropoiesis in vivo and promote stress-dependent SCF/c-Kit signaling. Based on this mechanism, one might predict that Samd14-regulated c-Kit signaling would impact more broadly on the hematopoietic system. Unraveling the respective mechanisms, which is in progress, may reveal new therapeutic approaches for hematologic diseases including anemias.

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 (CD71low, Ter119-) and R2 (CD71high, 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.

Thousands of cis-elements in genomes are predicted to have vital functions. Although conservation, activity in surrogate assays, polymorphisms, and disease mutations provide functional clues, deletion from endogenous loci constitutes the gold-standard test. A GATA-2-binding, Gata2 intronic cis-element (+9.5) required for hematopoietic stem cell genesis in mice is mutated in a human immunodeficiency syndrome. Because +9.5 is the only cis-element known to mediate stem cell genesis, we devised a strategy to identify functionally comparable enhancers ("+9.5-like") genome-wide. Gene editing revealed +9.5-like activity to mediate GATA-2 occupancy, chromatin opening, and transcriptional activation. A +9.5-like element resided in Samd14, which encodes a protein of unknown function. Samd14 increased hematopoietic progenitor levels/activity and promoted signaling by a pathway vital for hematopoietic stem/progenitor cell regulation (stem cell factor/c-Kit), and c-Kit rescued Samd14 loss-of-function phenotypes. Thus, the hematopoietic stem/progenitor cell cistrome revealed a mediator of a signaling pathway that has broad importance for stem/progenitor cell biology.

Cis-regulatory mechanisms exert precise spatiotemporal control of genes during development and homeostasis. Elucidating cis-element function in specific cellular contexts has led to the discovery of genomic loci important in human disease. Mutations in a composite E-box-GATA element of GATA2 (+9.5 site) can cause the human immunodeficiency disorder MonoMAC syndrome that is associated with myelodysplastic syndrome and acute myeloid leukemia. The +9.5 site is critical for generating hematopoietic stem cells (HSCs) in the aorta gonad mesonephros region of the mouse embryo. By utilizing the chromatin regulators LDB1 and BRG1, the +9.5 site positively regulates Gata2 transcriptional activity. We implemented a multifactorial strategy to identify cis-elements with structural/functional similarity to the +9.5 site. As the "+9.5-like" sites share diverse attributes with the +9.5 site, and genes harboring these elements are GATA-2-regulated, we propose that certain +9.5-like sites have important functions to generate and/or regulate HSCs. We used TALEN-mediated genetic editing to delete two prioritized elements and analyzed the functional consequences in heterozygous cells using an allele-specific transcription assay. The deletions abrogated primary transcript expression from the mutant allele of the +9.5-like element-associated genes. Disrupting the E-box of the +9.5-like elements reduced TAL1 and GATA-2 occupancy, as well as chromatin accessibility. Loss-of-function analysis of a gene encoding a previously unstudied Sterile Alpha Motif (SAM)-domain-containing protein, which contains an intronic +9.5-like element, revealed a role of the protein to confer hematopoietic progenitor levels in fetal liver. Functional analyses with the SAM domain protein are ongoing in primary hematopoietic cells and mutant mice, and additional prioritized +9.5-like elements are being analyzed. Thus, the multifactorial prioritization strategy revealed important cis-elements operational at endogenous loci and a novel determinant of fetal liver hematopoiesis. Cis-regulatory mechanisms exert precise spatiotemporal control of genes during development and homeostasis. Elucidating cis-element function in specific cellular contexts has led to the discovery of genomic loci important in human disease. Mutations in a composite E-box-GATA element of GATA2 (+9.5 site) can cause the human immunodeficiency disorder MonoMAC syndrome that is associated with myelodysplastic syndrome and acute myeloid leukemia. The +9.5 site is critical for generating hematopoietic stem cells (HSCs) in the aorta gonad mesonephros region of the mouse embryo. By utilizing the chromatin regulators LDB1 and BRG1, the +9.5 site positively regulates Gata2 transcriptional activity. We implemented a multifactorial strategy to identify cis-elements with structural/functional similarity to the +9.5 site. As the "+9.5-like" sites share diverse attributes with the +9.5 site, and genes harboring these elements are GATA-2-regulated, we propose that certain +9.5-like sites have important functions to generate and/or regulate HSCs. We used TALEN-mediated genetic editing to delete two prioritized elements and analyzed the functional consequences in heterozygous cells using an allele-specific transcription assay. The deletions abrogated primary transcript expression from the mutant allele of the +9.5-like element-associated genes. Disrupting the E-box of the +9.5-like elements reduced TAL1 and GATA-2 occupancy, as well as chromatin accessibility. Loss-of-function analysis of a gene encoding a previously unstudied Sterile Alpha Motif (SAM)-domain-containing protein, which contains an intronic +9.5-like element, revealed a role of the protein to confer hematopoietic progenitor levels in fetal liver. Functional analyses with the SAM domain protein are ongoing in primary hematopoietic cells and mutant mice, and additional prioritized +9.5-like elements are being analyzed. Thus, the multifactorial prioritization strategy revealed important cis-elements operational at endogenous loci and a novel determinant of fetal liver hematopoiesis.