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. 2011 May 5;117(18):4769-72.
doi: 10.1182/blood-2010-10-313031. Epub 2011 Mar 11.

Context-dependent function of "GATA switch" sites in vivo

Jonathan W Snow  1 , Jennifer J Trowbridge, Kirby D Johnson, Tohru Fujiwara, Nikla E Emambokus, Jeffrey A Grass, Stuart H Orkin, Emery H Bresnick
Affiliations

Context-dependent function of "GATA switch" sites in vivo

Jonathan W Snow et al. Blood. .

Abstract

Master transcriptional regulators of development often function through dispersed cis elements at endogenous target genes. While cis-elements are routinely studied in transfection and transgenic reporter assays, it is challenging to ascertain how they function in vivo. To address this problem in the context of the locus encoding the critical hematopoietic transcription factor Gata2, we engineered mice lacking a cluster of GATA motifs 2.8 kb upstream of the Gata2 transcriptional start site. We demonstrate that the -2.8 kb site confers maximal Gata2 expression in hematopoietic stem cells and specific hematopoietic progenitors. By contrast to our previous demonstration that a palindromic GATA motif at the neighboring -1.8 kb site maintains Gata2 repression in terminally differentiating erythroid cells, the -2.8 kb site was not required to initiate or maintain repression. These analyses reveal qualitatively distinct functions of 2 GATA motif-containing regions in vivo.

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Figures

Figure 1
Figure 1
−2.8 kb GATA switch site regulates Gata2 expression in hematopoietic stem and progenitor cells, but is dispensible in erythroid cells. Gata2 expression within LT-HSC and MPP populations (LT-HSC defined as Lindimckit+Sca1+CD150+ and MPP defined as Lindimckit+Sca1+CD34+ or Lindimckit+Sca1+CD150−) from E12.5 fetal livers normalized to β-actin (A). Frequencies of LT-HSCs and MPPs in wild-type and Δ-2.8 fetal livers (B). Gata2 expression within CMP and MEP populations (with CMP defined as LindimSca1−,c-kit+CD34+, FcγR− and MEP defined as Lindim Sca1−c-kit+CD34−, FcγR−) from E12.5 fetal livers normalized to β-actin (C). Frequencies of CMPs and MEPs from wild-type and Δ-2.8 fetal livers (D). Gata2 expression was assessed by qPCR in Stage I through Stage IV sorted erythroid cells, corresponding to CD71loTer119− (committed erythroid progenitors, Stage I), CD71hiTer119− (proerythroblasts, Stage II), CD71hiTer119+ (basophilic erythroblasts, Stage III), and CD71loTer119+ (late erythroblasts, Stage IV) from wild-type and Δ-2.8 embryos. (E). Relative number of cells in Stage I-IV was determined in wild-type and Δ-2.8 E13.5 fetal liver cells, based on CD71 and Ter119 expression (F). Mean ± SEM. Statistical significance was assessed by 2-sided Student t test. *P ≤ .05 and **P ≤ .01.
Figure 2
Figure 2
The −2.8 kb GATA switch site establishes trimeH3K27 marks at the Gata2 locus. Quantitative ChIP analysis of the Gata2 locus in E14.5 fetal liver cells using antibodies to GATA-1 (A), dimeH3K4 (B), trimeH3K27 (C), and Total H3 (D). Updated model of the molecular regulation of Gata2 repression (E). Mean ± SEM. Statistical significance was assessed by 2-sided Student t test and *P ≤ .05 and **P ≤ .01.
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