Abstract
The human embryonic, fetal and adult β-like globin genes provide a paradigm for tissue- and developmental stage-specific gene regulation. The fetal γ-globin gene is expressed in fetal erythroid cells but is repressed in adult erythroid cells. The molecular mechanism underlying this transcriptional switch during erythroid development is not completely understood. Here, we used a combination of in vitro and in vivo assays to dissect the molecular assemblies of the active and the repressed proximal γ-globin promoter complexes in K562 human erythroleukemia cell line and primary human fetal and adult erythroid cells. We found that the proximal γ-globin promoter complex is assembled by a developmentally regulated, general transcription activator NF-Y bound strongly at the tandem CCAAT motifs near the TATA box. NF-Y recruits to neighboring DNA motifs the developmentally regulated, erythroid transcription activator GATA-2 and general repressor BCL11A, which in turn recruit erythroid repressor GATA-1 and general repressor COUP-TFII to form respectively the NF-Y/GATA-2 transcription activator hub and the BCL11A/COUP-TFII/GATA-1 transcription repressor hub. Both the activator and the repressor hubs are present in both the active and the repressed γ-globin promoter complexes in fetal and adult erythroid cells. Through changes in their levels and respective interactions with the co-activators and co-repressors during erythroid development, the activator and the repressor hubs modulate erythroid- and developmental stage-specific transcription of γ-globin gene.
Highlights
The human b-like globin genes, consisting of embryonic e, fetal Gc- and Ac- and adult d- and b-globin genes, are expressed in erythroid cells and undergo an ordered developmental switching program: e-globin gene is expressed in early embryos until,7 weeks in gestation, when it is switched off and c-globin genes are switched on; at the time of birth, c-globin genes are switched off and b-globin gene is switched on
We show that the developmentally-regulated, general activator NF-Y stably bound at the tandem CCAAT motifs served as an anchor to assemble a developmentally-regulated activator hub, NF-Y/GATA-2, which recruited co-activators CBP and MLL2 to modify histones [21,22] and members of the Mediator complex [23] and basal transcription machinery to transcribe c-globin mRNA and a repressor hub, BCL11A/COUP-TFII/GATA-1, which recruited co-repressor HDAC1 to antagonize the activities of the activator hub
We showed the pivotal role of NF-Y in assembling the active and the repressed proximal c-globin promoter complexes (Fig. 7): Transcription activator NF-Y stably bound at the CCAAT motifs with affinity reported to be among the highest for DNA binding proteins [3,7,8] recruited and stabilized binding of activator GATA-2 to the neighboring GATA motif to form the activator hub, NF-Y/GATA-2; NF-Y recruited and stabilized binding to the neighboring GGCCGG motif of repressor BCL11A, which in turn interacted with COUP-TFII bound at its cognate site overlapping the distal CCAAT motif and with GATA-1 recruited by GATA-2 to form the repressor hub, BCL11A/COUP-TFII/GATA-1
Summary
The human b-like globin genes, consisting of embryonic e-, fetal Gc- and Ac- and adult d- and b-globin genes, are expressed in erythroid cells and undergo an ordered developmental switching program: e-globin gene is expressed in early embryos until ,7 weeks in gestation, when it is switched off and c-globin genes are switched on; at the time of birth, c-globin genes are switched off and b-globin gene is switched on. Base mutations in the DNA motifs in the proximal c-globin promoter cause hereditary persistence of fetal hemoglobin (HPFH) [16,17,18,19,20], indicating the functional importance of these DNA motifs and the transcription factors recruited by them in regulating c-globin gene expression. How these DNA-binding transcription factors interact to assemble the active c-globin proximal promoter complex in fetal erythroid cells and the inactive promoter complex in adult erythroid cells is largely unknown
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