Dissecting a Multi-Step Mechanism of Beta-Globin Transcriptional Activation.

Defining factors and signals that establish and maintain the native nucleoprotein structure of endogenous chromatin domains represents a powerful approach for elucidating transcriptional mechanisms. In adult erythroid cells, the locus control region (LCR) and the adult beta-globin genes of the murine beta-globin locus are highly enriched in acetylated histones H3 and H4 (acH3, acH4) and H3 methylated at lysine 4 (H3-meK4). By contrast, the embryonic beta-globin genes reside in a broad region of reduced acetylation. Histone H3 methylated at lysine 79 (H3-meK79) is highly enriched at the adult beta-globin genes, but not at the LCR. To elucidate the molecular steps in beta-globin transcriptional activation, genetic complementation experiments were conducted in GATA-1-null G1E cells containing an estrogen receptor hormone binding domain-GATA-1 fusion protein (ER-GATA-1). Kinetic analysis of ER-GATA-1 occupancy of chromatin and establishment of the histone modification pattern by chromatin immunoprecipitation (ChIP) revealed that GATA-1 occupies multiple regions within the LCR prior to the beta-major promoter. Chromatin accessibility at the promoter was low until ER-GATA-1 assembled into regulatory complexes at the LCR. Subsequently, ER-GATA-1 accessed the beta-major promoter, induced acH3, RNA polymerase II (Pol II) recruitment, and elevated H3-meK79. Acquisition of transcriptional competence appears to require establishment of H3-meK4, which is GATA-1-independent. Blocking transcriptional elongation did not erase H3-meK79, indicating that maintenance of H3-meK79 does not require ongoing elongation. Analysis of N-terminal GATA-1 deletion mutants that retain Friend of GATA-1 (FOG-1) binding and DNA binding activities revealed that FOG-1 binding and DNA binding activities are insufficient for Pol II recruitment and chromatin modification at the promoter. These results support a model in which ER-GATA-1 binding to the LCR increases acH3 at the promoter as an early event in transcriptional activation, which is tightly coupled to ER-GATA-1 access to the promoter, increased promoter accessibility, and Pol II recruitment. Increased promoter accessibility, which likely permits ER-GATA-1 access to the promoter, precedes maximal induction of H3-meK79, a late event in activation. Given the dynamic regulation of H3-meK79 by GATA-1 and NF-E2 and the modulation of H3-meK79 levels during erythropoiesis, we propose that H3-meK79 is a crucial signal that controls the rate of beta-globin transcription. Studies are underway to test this hypothesis and to dissect mechanisms underlying the requirement of N-terminal sequences of GATA-1 for Pol II recruitment and chromatin modification. Furthermore, having identified individual steps in transcriptional activation of the endogenous beta-globin genes, we are testing whether inducers of human fetal hemoglobin affect these specific steps. GATA-1 has been reported by the Crispino group to be expressed as an N-terminally truncated species in megakaryoblastic leukemia. Defining how the N-terminus functions should therefore lead to a molecular understanding of this disorder. As the N-termini of GATA factors differ considerably, one might expect these divergent sequences to establish GATA factor-specific functions, and this prediction is being tested via detailed analysis of the activities of chimeric GATA factors.