Mechanism governing heme synthesis reveals a GATA factor/heme circuit that controls differentiation

Abstract

Metal ion-containing macromolecules have fundamental roles in essentially all biological processes throughout the evolutionary tree. For example, iron-containing heme is a cofactor in enzyme catalysis and electron transfer and an essential hemoglobin constituent. To meet the intense demand for hemoglobin assembly in red blood cells, the cell type-specific factor GATA-1 activates transcription of Alas2, encoding the rate-limiting enzyme in heme biosynthesis, 5-aminolevulinic acid synthase-2 (ALAS-2). Using genetic editing to unravel mechanisms governing heme biosynthesis, we discovered a GATA factor-and heme-dependent circuit that establishes the erythroid cell transcriptome. CRISPR/Cas9-mediated ablation of two Alas2 intronic cis elements strongly reduces GATA-1-induced Alas2 transcription, heme biosynthesis, and surprisingly, GATA-1 regulation of other vital constituents of the erythroid cell transcriptome. Bypassing ALAS-2 function in Alas2 cis element-mutant cells by providing its catalytic product 5-aminolevulinic acid rescues heme biosynthesis and the GATA-1-dependent genetic network. Heme amplifies GATA-1 function by downregulating the heme-sensing transcriptional repressor Bach1 and via a Bach1-insensitive mechanism. Through this dual mechanism, heme and a master regulator collaborate to orchestrate a cell type-specific transcriptional program that promotes cellular differentiation.

Integrating Enhancer Mechanisms to Establish a Hierarchical Blood Development Program

“…The G1E-ER-GATA-1 data was derived from RNA-seq analysis 48 h post-ER-GATA-1 activation (Tanimura et al, 2016) and microarray analysis 24 h after ER-GATA-1 activation (DeVilbiss et al, 2013; Pope and Bresnick, 2013) (Datasets 1 and 2, respectively) (Figure 6A). In R1 cells, −77 loss decreased Gata2 expression, which reduced Gata1 expression.…”

GATA1 mutations in red cell disorders

“…89,90 Further investigation showed that deletion of the int-1-GATA site disrupts the recruitment of the GATA1/TAL1/LMO2/LDB1 complex and fails to fully activate ALAS2 transcription, leading to a failure of developing mature red blood cells and embryonic lethality of mutant males at E12.5. 91,92 A fourth example can be found in the PKLR gene, which encodes pyruvate kinase, an enzyme that catalyzes the final step of glycolysis. Pyruvate kinase deficiency (PKD) is one of the most common causes of hereditary nonspherocytic hemolytic anemia.…”

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

“…In the context of GATA factors, many prospective enhancers bearing these attributes are either not GATA factor-occupied or can be deleted from a genome with little to no functional consequence (Bresnick et al, 2010; DeVilbiss et al, 2014; Fujiwara et al, 2009; Sanalkumar et al, 2014; Snow et al, 2010; Snow et al, 2011; Tanimura et al, 2015). Previously, we used a multi-parametric prioritization strategy to identify GATA-2-regulated enhancers containing E-box-GATA elements in G1E cells (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).…”