Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1.

Recent evidence suggests that long-range enhancers and gene promoters are in close proximity, which might reflect the formation of chromatin loops. Here, we examined the mechanism for DNA looping at the beta-globin locus. By using chromosome conformation capture (3C), we show that the hematopoietic transcription factor GATA-1 and its cofactor FOG-1 are required for the physical interaction between the beta-globin locus control region (LCR) and the beta-major globin promoter. Kinetic studies reveal that GATA-1-induced loop formation correlates with the onset of beta-globin transcription and occurs independently of new protein synthesis. GATA-1 occupies the beta-major globin promoter normally in fetal liver erythroblasts from mice lacking the LCR, suggesting that GATA-1 binding to the promoter and LCR are independent events that occur prior to loop formation. Together, these data demonstrate that GATA-1 and FOG-1 are essential anchors for a tissue-specific chromatin loop, providing general insights into long-range enhancer function.

Identification of cyclin D3 as a direct target of E2A using DamID.

The transcription factor E2A can promote precursor B cell expansion, promote G(1) cell cycle progression, and induce the expressions of multiple G(1)-phase cyclins. To better understand the mechanism by which E2A induces these cyclins, we characterized the relationship between E2A and the cyclin D3 gene promoter. E2A transactivated the 1-kb promoter of cyclin D3, which contains two E boxes. However, deletion of the E boxes did not disrupt the transactivation by E2A, raising the possibility of indirect activation via another transcription factor or binding of E2A to non-E-box DNA elements. To distinguish between these two possibilities, promoter occupancy was examined using the DamID approach. A fusion construct composed of E2A and the Escherichia coli DNA adenosine methyltransferase (E47Dam) was subcloned in lentivirus vectors and used to transduce precursor B-cell and myeloid progenitor cell lines. In both cell types, specific adenosine methylation was identified at the cyclin D3 promoter. Chromatin immunoprecipitation analysis confirmed the DamID findings and localized the binding to within 1 kb of the two E boxes. The methylation by E47Dam was not disrupted by mutations in the E2A portion that block DNA binding. We conclude that E2A can be recruited to the cyclin D3 promoter independently of E boxes or E2A DNA binding activity.

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

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. These results indicate that FOG-1 employs distinct mechanisms when cooperating with GATA-1 during transcriptional activation and repression.

GATA-1-mediated proliferation arrest during erythroid maturation.

Transcription factor GATA-1 is essential for erythroid and megakaryocytic maturation. GATA-1 mutations are associated with hematopoietic precursor proliferation and leukemogenesis, suggesting a role in cell cycle control. While numerous GATA-1 target genes specifying mature hematopoietic phenotypes have been identified, how GATA-1 regulates proliferation remains unknown. We used a complementation assay based on synchronous inducible rescue of GATA-1(-) erythroblasts to show that GATA-1 promotes both erythroid maturation and G(1) cell cycle arrest. Molecular studies combined with microarray transcriptome analysis revealed an extensive GATA-1-regulated program of cell cycle control in which numerous growth inhibitors were upregulated and mitogenic genes were repressed. GATA-1 inhibited expression of cyclin-dependent kinase (Cdk) 6 and cyclin D2 and induced the Cdk inhibitors p18(INK4C) and p27(Kip1) with associated inactivation of all G(1) Cdks. These effects were dependent on GATA-1-mediated repression of the c-myc (Myc) proto-oncogene. GATA-1 inhibited Myc expression within 3 h, and chromatin immunoprecipitation studies indicated that GATA-1 occupies the Myc promoter in vivo, suggesting a direct mechanism for gene repression. Surprisingly, enforced expression of Myc prevented GATA-1-induced cell cycle arrest but had minimal effects on erythroid maturation. Our results illustrate how GATA-1, a lineage-determining transcription factor, coordinates proliferation arrest with cellular maturation through distinct, interrelated genetic programs.

Formation of a tissue-specific histone acetylation pattern by the hematopoietic transcription factor GATA-1.

One function of lineage-restricted transcription factors may be to control the formation of tissue-specific chromatin domains. In erythroid cells, the beta-globin gene cluster undergoes developmentally regulated hyperacetylation of histones at the active globin genes and the locus control region (LCR). However, it is unknown which transcription factor(s) governs the establishment of this erythroid-specific chromatin domain. We measured histone acetylation at the beta-globin locus in the erythroid cell line G1E, which is deficient for the essential hematopoietic transcription factor GATA-1. Restoration of GATA-1 activity in G1E cells led to a substantial increase in acetylation of histones H3 and H4 at the beta-globin promoter and the LCR. Time course experiments showed that histone acetylation occurred rapidly after GATA-1 activation and coincided with globin gene expression, indicating that the effects of GATA-1 are direct. Moreover, increases in histone acetylation correlated with occupancy of GATA-1 and the acetyltransferase CBP at the locus in vivo. Together, these results suggest that GATA-1 and its cofactor CBP are essential for the formation of an erythroid-specific acetylation pattern that is permissive for high levels of gene expression.

Control of megakaryocyte-specific gene expression by GATA-1 and FOG-1: role of Ets transcription factors.

The transcription factor GATA-1 and its cofactor FOG-1 are essential for the normal development of erythroid cells and megakaryocytes. FOG-1 can stimulate or inhibit GATA-1 activity depending on cell and promoter context. How the GATA-1-FOG-1 complex controls the expression of distinct sets of gene in megakaryocytes and erythroid cells is not understood. Here, we examine the molecular basis for the megakaryocyte-restricted activation of the alphaIIb gene. FOG-1 stimulates GATA-1-dependent alphaIIb gene expression in a manner that requires their direct physical interaction. Transcriptional output by the GATA-1-FOG-1 complex is determined by the hematopoietic Ets protein Fli-1 that binds to an adjacent Ets element. Chromatin immunoprecipitation experiments show that GATA-1, FOG-1 and Fli-1 co-occupy the alphaIIb promoter in vivo. Expression of several additional megakaryocyte-specific genes that bear tandem GATA and Ets elements in their promoters also depends on the physical interaction between GATA-1 and FOG-1. Our studies define a molecular context for transcriptional activation by GATA-1 and FOG-1, and may explain the occurrence of tandem GATA and Ets elements in the promoters of numerous megakaryocyte-expressed genes.