Hematopoietic Signaling Mechanism Revealed from a Stem/Progenitor Cell Cistrome

Abstract

SUMMARY Thousands of cis-elements in genomes are predicted to have vital functions. While 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. As +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, 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.

GATA1 Activity Governed by Configurations of cis-Acting Elements

“…One plausible explanation for the difference in SCL occupancy is that differences in undesignated sequences in the composite E-box–GATA motif may influence complex formation, depending on whether GATA1 or GATA2 is present. Binding analyses utilizing naked DNA have indicated that SCL preferentially binds to CAGGTG (33) or CAGATG (42) motifs in essential hematopoiesis genes, whereas CATCTG sequences are enriched in GATA2-occupied loci in lineage-negative hematopoietic progenitors (43). Furthermore, the spacer lengths and sequences between E-box and GATA-binding motif vary at individual loci.…”

GATA Factor-Regulated Samd14 Enhancer Confers Red Blood Cell Regeneration and Survival in Severe Anemia

“…Insufficient GATA-2 levels/activity resulting from GATA2 coding or +9.5 enhancer mutations underlie hematologic diseases including primary immunodeficiencies that frequently progress to myelodysplastic syndrome (MDS), acute myeloid leukemia (AML) (Dickinson et al, 2011; Hahn et al, 2011; Hsu et al, 2011; Ostergaard et al, 2011) and pediatric MDS/AML independent of immunodeficiency (Wlodarski et al, 2016). Since GATA factor occupancy of a GATA motif in chromatin does not predict GATA factor-dependent regulation of the linked gene (DeVilbiss et al, 2014; Hewitt et al, 2015; Sanalkumar et al, 2014), many questions remain unanswered regarding mechanisms conferring GATA-2 activity.…”

“…Prioritization of GATA-2-occupied E-box-GATA elements with attributes resembling the +9.5 enhancer, and gene editing in G1E mouse erythroid precursor cells revealed sites that regulate their endogenous loci and seemingly similar sites that do not (Hewitt et al, 2015; Tanimura et al, 2015). The functional sites included a conserved intronic enhancer (Samd14-Enh) at the largely unstudied, GATA-2-occupied Samd14 gene (Hewitt et al, 2015).…”

“…Prioritization of GATA-2-occupied E-box-GATA elements with attributes resembling the +9.5 enhancer, and gene editing in G1E mouse erythroid precursor cells revealed sites that regulate their endogenous loci and seemingly similar sites that do not (Hewitt et al, 2015; Tanimura et al, 2015). The functional sites included a conserved intronic enhancer (Samd14-Enh) at the largely unstudied, GATA-2-occupied Samd14 gene (Hewitt et al, 2015). SAMD14 is expressed in HSC, megakaryocyte erythrocyte progenitor (MEP) and committed erythroid progenitors (Chen and Lodish, 2014; Hewitt et al, 2015), downregulated in colorectal cancer and adenocarcinoma (Shen et al, 2012a; Sun et al, 2008), and polymorphisms associated with blood platelet volume are linked to altered SAMD14 expression (Fehrmann et al, 2011).…”

“…The functional sites included a conserved intronic enhancer (Samd14-Enh) at the largely unstudied, GATA-2-occupied Samd14 gene (Hewitt et al, 2015). SAMD14 is expressed in HSC, megakaryocyte erythrocyte progenitor (MEP) and committed erythroid progenitors (Chen and Lodish, 2014; Hewitt et al, 2015), downregulated in colorectal cancer and adenocarcinoma (Shen et al, 2012a; Sun et al, 2008), and polymorphisms associated with blood platelet volume are linked to altered SAMD14 expression (Fehrmann et al, 2011). A genome-wide screen for human genetic variants linked to hematologic phenotypes correlated a polymorphism within the SAMD14 5’ UTR with platelet distribution width (Astle et al, 2016).…”