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

Amigo, J.D., Yu, M., Troadec, M.B., Gwynn, B., Cooney, J.D., Lambert, A.J., Chi, N.C., Weiss, M.J., Peters, L.L., Kaplan, J., Cantor, A.B., and Paw, B.H.
Molecular and cellular biology   31(7): 1344-56 (Journal)
Registered Authors
Amigo, Julio, Paw, Barry
blood, erythropoiesis, GATA-factors, heart, slc25 solute carriers
MeSH Terms
  • Animals
  • Base Pairing/genetics
  • Base Sequence
  • Binding Sites
  • 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
21248200 Full text @ Mol. Cell. Biol.
Mitoferrin1 (Mfrn1, Slc25a37) and Mitoferrin2 (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.
Genes / Markers
Mutation and Transgenics
Human Disease / Model Data
Sequence Targeting Reagents
Engineered Foreign Genes
Errata and Notes