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. 2005 Jul 6;24(13):2354-66.
doi: 10.1038/sj.emboj.7600702. Epub 2005 May 26.

GATA-1 forms distinct activating and repressive complexes in erythroid cells

Patrick Rodriguez  1 , Edgar Bonte, Jeroen Krijgsveld, Katarzyna E Kolodziej, Boris Guyot, Albert J R Heck, Paresh Vyas, Ernie de Boer, Frank Grosveld, John Strouboulis
Affiliations

GATA-1 forms distinct activating and repressive complexes in erythroid cells

Patrick Rodriguez et al. EMBO J. .

Abstract

GATA-1 is essential for the generation of the erythroid, megakaryocytic, eosinophilic and mast cell lineages. It acts as an activator and repressor of different target genes, for example, in erythroid cells it represses cell proliferation and early hematopoietic genes while activating erythroid genes, yet it is not clear how both of these functions are mediated. Using a biotinylation tagging/proteomics approach in erythroid cells, we describe distinct GATA-1 interactions with the essential hematopoietic factor Gfi-1b, the repressive MeCP1 complex and the chromatin remodeling ACF/WCRF complex, in addition to the known GATA-1/FOG-1 and GATA-1/TAL-1 complexes. Importantly, we show that FOG-1 mediates GATA-1 interactions with the MeCP1 complex, thus providing an explanation for the overlapping functions of these two factors in erythropoiesis. We also show that subsets of GATA-1 gene targets are bound in vivo by distinct complexes, thus linking specific GATA-1 partners to distinct aspects of its functions. Based on these findings, we suggest a model for the different roles of GATA-1 in erythroid differentiation.

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Figures

Figure 1
Figure 1
Confirmation by streptavidin pull-downs (A–C) and immunoprecipitations (D–G) of proteins identified copurifying with GATA-1. (A) Streptavidin pull-downs of transcription factors. Biotinylated GATA-1 (top panel) is detected by streptavidin–HRP and is absent from the BirA-only transfected cells. (B) Pull-downs of the MeCP1 complex. (C) Pull-downs of ISWI-containing complexes. SB: streptavidin-bound. (D) Immunoprecipitations (IP) using antibodies against GATA-1, TAL-1, FOG-1 (lanes 2, 3 and 4, respectively) and nucleophosmin as negative control (lane 5). (E) IP of the MeCP1 complex by antibodies against GATA-1 and MTA2 (lanes 2 and 3) and nucleophosmin (lane 4). (F) GATA-1 can be specifically immunoprecipitated by an antibody against MTA2. (G) IP of the ACF/WCRF complex by GATA-1 antibodies. Nuclear extract equivalent to 5% used in each pull-down or IP was loaded as control for input material. IP: immunoprecipitating antibody. Arrows show the detecting antibodies.
Figure 2
Figure 2
Distinct GATA-1 complexes by sequential immunoprecipitations (IP). (A) Experimental procedure. (B) Efficiency of immunoprecipitating antibodies (also used to detect the immunoprecipitated protein). Sup: supernatant after IP. (C) FOG-1 immunodepletion, and FOG-1 IP (lane 2) followed by IP of supernatant with GATA-1 or control antibodies (lanes 3 and 4). (D) TAL-1 immunodepletion; same as (C), using TAL-1 antibodies in first IP. (E) MTA2 immunodepletion; same as (C) and (D), using MTA2 antibodies in first IP. The MTA2 antibody used in panels B and E is different to that used in (C) and (D) (see Supplementary data). IP: immunoprecipitating antibody. Arrows show the detecting antibodies.
Figure 3
Figure 3
(A–C) Differential interactions mediated by the GATA-1 zinc-fingers. GATA-1 zinc-finger deletions were expressed as biotin-tagged proteins in MEL cells and interactions were assessed by streptavidin pull-downs and Western blots. (A) FOG-1 and the MeCP1 complex require N-ZnF for interactions. (B) TAL-1 requires both zinc-fingers. (C) Gfi-1b and SNF2h require C-ZnF for interactions with GATA-1. The TAL-1 antibody used is different from that used in Figure 1.
Figure 4
Figure 4
(A–B) FOG-1 bridges GATA-1 and MeCP1. Nuclear extracts from HeLa cells transfected with the FOG-1 and GATA-1 combinations indicated were immunoprecipitated with FOG-1 (A) or MTA2 antibodies (B) and detected with FOG-1 and GATA-1 antibodies. ΔZn-N and ΔZn-C: GATA-1 N- and C-terminal zinc-finger deletion mutants. Arrows: crossreacting IgG. Asterisk (B): GATA-1 signal. (C) Real-time PCR transcription assays in transfected HeLa cells. GATA-1 activates transcription of pG-OVEC, whereas cotransfection of FOG-1 represses to basal levels. (D) Specific recruitment of Mi-2β by cotransfected GATA-1 and FOG-1 by ChIP assays in HeLa cells. Mi-2β recruitment to the repressed gene requires GATA-1 binding to the promoter.
Figure 5
Figure 5
Binding of GATA-1 repressive and activating complexes to target genes by ChIP assays in induced MEL cells. Binding patterns of GATA-1, FOG-1, Mbd2, TAL-1 and Gfi-1b to the −2.8 kb element of the GATA-2 locus (A), the MBP promoter in eosinophils (B) and in MEL cells (C), the myb (D) and myc (E) promoters and at the EKLF upstream enhancer (F). Relative enrichment has been normalized to input and corrected for background binding of species- and isotype-matched immunoglobulins. Antibodies: GATA-1, N6 (Santa Cruz); FOG-1 as in Tsang et al (1997); Mbd2 S923 (Ng et al, 1999); TAL-1 as in Porcher et al (1996); Gfi-1b, D19 (Santa Cruz).
Figure 6
Figure 6
Silencing of GATA-2 requires recruitment of FOG-1 and MeCP1 by GATA-1. (A) The V205M GATA-1 mutation fails to repress GATA-2. GATA-2 mRNA was measured by real-time PCR in G1E GATA-1-ER and G1E GATA-1(V205M)-ER cells before (0 h) and after 24 h of estradiol induction. Expression at 0 h was normalized against GAPDH expression and set as 1. (B) ChIP to show binding of GATA-1, Mi-2β and FOG-1 in G1E GATA-1-ER (left panels) and in G1E GATA-1(V205M)-ER (right panels) at time 0 and 24 h of estradiol induction. Relative enrichment has been normalized to input and corrected for background binding of species- and isotype-matched immunoglobulins. Data represent mean of two independent IPs and three PCRs with duplicate samples. Antibodies used were as in the legend of Figure 5, except Mi-2β antibody (Fujita et al, 2003).
Figure 7
Figure 7
Model for the distinct GATA-1 complexes and their role in erythropoiesis. The broken arrow indicates unknown function and timing. See text for explanation.
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