Mechanism Governing Heme Synthesis Reveals a GATA Factor-Heme Circuit That Controls Differentiation

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 reduced GATA-1-induced Alas2 transcription and heme biosynthesis.  Surprisingly, the cis-element ablation impaired GATA-1 regulation of other vital constituents of the erythroid cell transcriptome, including components mediating autophagy and nutrient transport. Bypassing ALAS-2 function in Alas2 cis-element-mutant cells by providing its catalytic product 5-aminolevulinic acid rescued heme biosynthesis and a sector of the GATA-1-dependent genetic network. A component of the gene cohort deregulated in heme deficiency could not be reactivated by rescuing heme biosynthesis, and we are testing whether irreversible changes in their chromatin states upon heme deficiency confer stable repression. Heme amplified GATA-1 function by downregulating the heme-sensing transcriptional repressor Bach1 and via a Bach1-insensitive mechanism. Based on this dual mechanism, we developed a paradigm in which a fundamentally critical process in hematology, heme biosynthesis, is intricately linked to the actions of a quintessential regulator of a cell type-specific transcriptional program that controls cellular differentiation. As known mechanistic insights into GATA factor mechanisms do not predict whether a GATA-1 target gene will be heme- or Bach1-sensitive, these studies revealed a new mode of GATA factor function that controls erythroid cell development and function. We will discuss how this mechanism is being dissected in biologically important murine and human systems.