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. 2004 Jan 13;101(2):476-81.
doi: 10.1073/pnas.0306315101. Epub 2003 Dec 26.

Context-dependent regulation of GATA-1 by friend of GATA-1

Danielle L Letting  1 , Ying-Yu Chen, Carrie Rakowski, Sarah Reedy, Gerd A Blobel
Affiliations

Context-dependent regulation of GATA-1 by friend of GATA-1

Danielle L Letting et al. Proc Natl Acad Sci U S A. .

"VSports" Abstract

The transcription factor GATA-1 and its cofactor, friend of GATA-1 (FOG-1), are essential for normal erythroid development. FOG-1 physically interacts with GATA-1 to augment or inhibit its activity. The mechanisms by which FOG-1 regulates GATA-1 function are unknown. By using an assay that is based on the phenotypic rescue of a GATA-1-null erythroid cell line, we found that a conditional form of GATA-1 (GATA-1-ER) strongly induced histone acetylation at the beta-major globin promoter in vivo, consistent with previous results. In contrast, GATA-1 bearing a point mutation that impairs FOG-1 binding [GATA-1(V205M)-ER] failed to induce high levels of histone acetylation at this site. However, at DNase I-hypersensitive site (HS)3 of the beta-globin locus control region, GATA-1-induced histone acetylation was FOG-1-independent. Because the V205M mutation does not disrupt GATA-1 binding to DNA templates in vitro, we were surprised to find that in vivo GATA-1(V205M)-ER fails to bind the beta-globin promoter. However, at HS3, DNA binding by GATA-1 was FOG-1-independent, thus correlating histone acetylation with GATA-1 occupancy. Examination of additional GATA-1-dependent regulatory elements showed that the interaction with FOG-1 is required for GATA-1 occupancy at select sites, such as HS2, but is dispensable at others, including the FOG-1-independent GATA-1 target gene EKLF. Remarkably, at the GATA-2 gene, which is repressed by GATA-1, interaction with FOG-1 was dispensable for GATA-1 occupancy and was required for transcriptional inhibition and histone deacetylation VSports手机版. These results indicate that FOG-1 employs distinct mechanisms when cooperating with GATA-1 during transcriptional activation and repression. .

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Fig. 1.
Fig. 1.
FOG-1 is required for GATA-1-induced histone acetylation at the β-major promoter but not at HS3. ChIP assays were performed with cells expressing GATA-1-ER or GATA-1(V205M)-ER before (–E2) and after (+E2) treatment with estradiol for 21 h. (A) Chromatin was immunoprecipitated with the anti-diacetyl H3 antibody (acH3) and analyzed with PCR primers spanning the β-major globin gene promoter. Results are plotted as percent of input chromatin. Controls include no chromatin (nc), no antibody (na), and nonimmune IgG (ctr). Results are averages of six independent experiments. (B) Experiments are as in A, but PCR primers are specific for HS3. Results are averages of four independent experiments. In all figures, error bars indicate SD.
Fig. 2.
Fig. 2.
FOG-1 is required for GATA-1 occupancy at the β-major promoter but not HS3. (A) ChIP assay as in Fig. 1 with anti-GATA-1 (G1) and anti-ER (ER) antibodies. Results are the averages of five independent experiments. (B) ChIP analysis as in A but with primers specific for HS3.
Fig. 3.
Fig. 3.
Kinetics of GATA-1 occupancy at the β-globin locus. ChIP experiments were performed at indicated time points after the addition of estradiol. Occupancy of GATA-1-ER (A) and GATA-1(V205M)-ER (B)atthe β-major promoter and at HS3 (C and D) is shown. The results of each time point are the averages of at least three independent experiments.
Fig. 4.
Fig. 4.
FOG-1 is required for GATA-1 occupancy at select erythroid regulatory regions. For ChIP assay, anti-GATA-1 (G1) and anti-ER antibodies (ER) were used. Controls are as in Fig. 1. Cells were treated with estradiol (E2) for 21 h. Primer pairs were directed against HS4 (A), HS2 (B), and the upstream enhancer of the EKLF gene (C). Results are the averages of at least four independent experiments.
Fig. 5.
Fig. 5.
Increase in GATA-1 occupancy at select sites after estradiol treatment. Results from Figs. 2 and 4 are replotted to indicate fold increase in GATA-1 occupancy after estradiol treatment. Results are shown for HS3 (A), HS4 (B), the EKLF promoter (C), the β-major promoter (D), and HS2 (E). αG1, anti-GATA-1; αER, anti-ER.
Fig. 6.
Fig. 6.
FOG-1 can function as a corepressor for GATA-1. (A) GATA-1-ER and GATA-1(V205M)-ER occupancy at the GATA-2 gene. ChIP experiments were performed as described for Fig. 2. Results are the average of four independent experiments. (B) ChIP analysis of histone acetylation levels at the GATA-2 enhancer by using the acH3 and acH4 antibodies as in Fig. 1. Results are the averages of five acH3 and two acH4 independent experiments.
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