Identification of distal cis-regulatory elements at mouse mitoferrin loci using zebrafish transgenesis.

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Base-Pairing, Base-Sequence, Binding-Sites, Chromatin-Immunoprecipitation, Enhancer-Elements-Genetic, Erythropoiesis, GATA1-Transcription-Factor, Gene-Expression-Regulation, Gene-Transfer-Techniques, Genes-Reporter, Genetic-Loci, Genome, Green-Fluorescent-Proteins, Heart, Membrane-Transport-Proteins, Mice, Molecular-Sequence-Data, Promoter-Regions-Genetic, Protein-Binding, Transcription-Factors, Transcription-Genetic, Zebrafish, Zebrafish-Proteins

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Mol Cell Biol 2011 Apr; 31(7):1344-56.


Mitoferrin 1 (Mfrn1; Slc25a37) and mitoferrin 2 (Mfrn2; Slc25a28) function as essential mitochondrial iron importers for heme and Fe/S cluster biogenesis. A genetic deficiency of Mfrn1 results in a profound hypochromic anemia in vertebrate species. To map the cis-regulatory modules (CRMs) that control expression of the Mfrn genes, we utilized genome-wide chromatin immunoprecipitation (ChIP) datasets for the major erythroid transcription factor GATA-1. We identified the CRMs that faithfully drive the expression of Mfrn1 during blood and heart development and Mfrn2 ubiquitously. Through in vivo analyses of the Mfrn-CRMs in zebrafish and mouse, we demonstrate their functional and evolutionary conservation. Using knockdowns with morpholinos and cell sorting analysis in transgenic zebrafish embryos, we show that GATA-1 directly regulates the expression of Mfrn1. Mutagenesis of individual GATA-1 binding cis elements (GBE) demonstrated that at least two of the three GBE within this CRM are functionally required for GATA-mediated transcription of Mfrn1. Furthermore, ChIP assays demonstrate switching from GATA-2 to GATA-1 at these elements during erythroid maturation. Our results provide new insights into the genetic regulation of mitochondrial function and iron homeostasis and, more generally, illustrate the utility of genome-wide ChIP analysis combined with zebrafish transgenesis for identifying long-range transcriptional enhancers that regulate tissue development.