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

Mol Cell Biol. 2011 Apr;31(7):1344-56. doi: 10.1128/MCB.01010-10. Epub 2011 Jan 19.

Abstract

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.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

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

Substances

  • Cation Transport Proteins
  • GATA1 Transcription Factor
  • Membrane Transport Proteins
  • Slc25a28 protein, mouse
  • Transcription Factors
  • Zebrafish Proteins
  • mitoferrin protein, mouse
  • zfpm1 protein, zebrafish
  • Green Fluorescent Proteins