Identification of Distal cis-Regulatory Elements at Mouse Mitoferrin Loci Using Zebrafish Transgenesis

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.

ACKNOWLEDGMENTS

We thank Christian Lawrence, Jason Best, Adam Cardullo, and Paul McBride for help with zebrafish animal husbandry; Jessica Wagner for help with confocal microscopy; Kathleen Soltis for screening cloche and Tg(Mfrn:GFP) zebrafish; Shuo Lin for the Tg(GATA-1:GFP) transgenic line; David Traver for the Tg(GATA-1:DsRed) transgenic line; and Susan Tamowski for the pronuclear injections. We thank Iman Schultz, Heidi Anderson, Adrianne Kolpak, and John Kanki for reviewing the manuscript. Confocal fluorescence microscopy was performed at the Harvard Digestive Disease Center Imaging Facility (Children's Hospital Boston).

This work was supported by grants from the American Heart Association (J.D.C.) and the March of Dimes Foundation (B.H.P.) and by National Institutes of Health grants K08 HL074891 (N.C.C.), U01 HL066611 (L.L.P.), R01 DK052380 (J.K.), R01 DK070838 (B.H.P.), P01 HL032262 (A.B.C. and B.H.P.), and P30 DK072437 (J.K. and B.H.P.).

We declare no financial conflict of interest.