Identification of distal cis-regulatory elements at mouse mitoferrin loci using zebrafish transgenesis.
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.
Department of Medicine, Division of Hematology, Brigham and Women’s Hospital, and Harvard Medical School, Boston, Massachusetts 02115, USA.
Julio D
JD
Amigo
Ming
M
Yu
Marie-Berengere
MB
Troadec
Babette
B
Gwynn
Jeffrey D
JD
Cooney
Amy J
AJ
Lambert
Neil C
NC
Chi
Mitchell J
MJ
Weiss
Luanne L
LL
Peters
Jerry
J
Kaplan
Alan B
AB
Cantor
Barry H
BH
Paw
D000818:Animals; D020029:Base Pairing / Q000235:genetics; D001483:Base Sequence; D001665:Binding Sites; D027682:Cation Transport Proteins; D047369:Chromatin Immunoprecipitation; D004742:Enhancer Elements, Genetic / Q000235:genetics; D004920:Erythropoiesis / Q000235:genetics; D050982:GATA1 Transcription Factor / Q000378:metabolism; D005786:Gene Expression Regulation; D018014:Gene Transfer Techniques; D017930:Genes, Reporter; D056426:Genetic Loci / Q000235:genetics*; D016678:Genome / Q000235:genetics; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006321:Heart / Q000196:embryology; D026901:Membrane Transport Proteins / Q000235:genetics* / Q000378:metabolism; D051379:Mice; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D011485:Protein Binding; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D015027:Zebrafish / Q000235:genetics*; D029961:Zebrafish Proteins / Q000378:metabolism
D027682:Cation Transport Proteins; D050982:GATA1 Transcription Factor; D026901:Membrane Transport Proteins; C486056:Slc25a28 protein, mouse; D014157:Transcription Factors; D029961:Zebrafish Proteins; C509051:mitoferrin protein, mouse; C508768:zfpm1 protein, zebrafish; D049452:Green Fluorescent Proteins
EMBO J. 2000 Jun 1;19(11):2492-502
10835348
J Biol Chem. 1999 Apr 30;274(18):12819-26
10212268
Proc Natl Acad Sci U S A. 2008 Mar 4;105(9):3333-8
18303114
Development. 1995 Oct;121(10):3141-50
7588049
Blood. 2010 Jul 29;116(4):628-30
20427704
Proc Natl Acad Sci U S A. 2003 Jul 22;100(15):8811-6
12857954
Mol Cell Biol. 1997 Mar;17(3):1642-51
9032291
Development. 1995 Apr;121(4):957-74
7743939
Development. 2003 Dec;130(25):6187-99
14602685
Proc Natl Acad Sci U S A. 2009 Sep 22;106(38):16263-8
19805291
Genome Res. 2003 Jan;13(1):64-72
12529307
J Biol Chem. 2000 Dec 15;275(50):38949-52
11042222
EMBO J. 1991 Jul;10(7):1891-902
2050125
J Biol Chem. 1991 Dec 5;266(34):22948-53
1744088
Cell. 2005 Jan 28;120(2):169-81
15680324
Proc Natl Acad Sci U S A. 1997 Jun 10;94(12):6267-72
9177206
Exp Biol Med (Maywood). 2003 Dec;228(11):1282-90
14681544
J Biol Chem. 2010 Aug 27;285(35):26753-26759
20522548
Mol Cell. 2009 Nov 25;36(4):682-95
19941827
Genome Res. 2009 Dec;19(12):2172-84
19887574
Proc Natl Acad Sci U S A. 2009 Dec 29;106(52):22510-5
20018698
Mol Cell Biol. 2000 Jan;20(2):713-23
10611250
FEBS Lett. 2001 Apr 6;494(1-2):79-84
11297739
Oncogene. 2002 May 13;21(21):3368-76
12032775
Nature. 2006 Mar 2;440(7080):96-100
16511496
Semin Cell Dev Biol. 2005 Feb;16(1):95-106
15659344
PLoS Biol. 2008 May 13;6(5):e109
18479184
Cell Mol Life Sci. 2003 Aug;60(8):1647-58
14504655
Mol Cell Biol. 2009 Feb;29(4):1007-16
19075006
Nat Genet. 2010 Sep;42(9):806-10
20729851
Genome Res. 2006 Jan;16(1):123-31
16344561
J Biol Chem. 1997 Oct 17;272(42):26585-94
9334239
Curr Opin Genet Dev. 2002 Aug;12(4):416-22
12100886
Annu Rev Cell Dev Biol. 2002;18:747-83
12142270
Blood. 2009 Nov 19;114(21):4654-63
19729519
J Cell Physiol. 2005 Oct;205(1):1-9
15887235
Blood. 2008 Jul 15;112(2):219-30
18606887
Cell. 2005 Aug 26;122(4):505-15
16122419
Blood. 2004 Nov 15;104(10):3136-47
15297311
EMBO J. 2004 Jul 21;23(14):2841-52
15215894
Nat Biotechnol. 2004 Nov;22(11):1467-73
15529174
J Biol Chem. 1998 Jul 3;273(27):16798-809
9642238
Mol Cell Biol. 2009 Mar;29(5):1163-75
19103751
Dev Dyn. 2007 Nov;236(11):3077-87
17948311
Cell. 2004 Feb 20;116(4):499-509
14980218
Semin Cell Dev Biol. 2005 Feb;16(1):117-28
15659346
Mol Cell. 1999 Feb;3(2):219-28
10078204
Proc Natl Acad Sci U S A. 2004 Jan 13;101(2):476-81
14695898
Dev Biol. 1999 Jun 15;210(2):305-21
10357893
Nature. 2009 Feb 12;457(7231):854-8
19212405
J Biol Chem. 2010 Oct 8;285(41):31087-93
20670937
Mol Cell. 2009 Nov 25;36(4):667-81
19941826
Proc Natl Acad Sci U S A. 2004 Jan 27;101(4):980-5
14715908
J Exp Med. 2008 Mar 17;205(3):611-24
18299398