Nuclear Receptors TR2 and TR4 Recruit Multiple Epigenetic Transcriptional Corepressors That Associate Specifically with the Embryonic β-Type Globin Promoters in Differentiated Adult Erythroid Cells

Abstract

Nuclear receptors TR2 and TR4 (TR2/TR4) were previously shown to bind in vitro to direct repeat elements in the mouse and human embryonic and fetal β-type globin gene promoters and to play critical roles in the silencing of these genes. By chromatin immunoprecipitation (ChIP) we show that, in adult erythroid cells, TR2/TR4 bind to the embryonic β-type globin promoters but not to the adult β-globin promoter. We purified protein complexes containing biotin-tagged TR2/TR4 from adult erythroid cells and identified DNMT1, NuRD, and LSD1/CoREST repressor complexes, as well as HDAC3 and TIF1β, all known to confer epigenetic gene silencing, as potential corepressors of TR2/TR4. Coimmunoprecipitation assays of endogenous abundance proteins indicated that TR2/TR4 complexes consist of at least four distinct molecular species. In ChIP assays we found that, in undifferentiated murine adult erythroid cells, many of these corepressors associate with both the embryonic and the adult β-type globin promoters but, upon terminal differentiation, they specifically dissociate only from the adult β-globin promoter concomitant with its activation but remain bound to the silenced embryonic globin gene promoters. These data suggest that TR2/TR4 recruit an array of transcriptional corepressors to elicit adult stage-specific silencing of the embryonic β-type globin genes through coordinated epigenetic chromatin modifications.

ACKNOWLEDGMENTS

This study was supported by NIH grants RC1 DK086956 (O.T. and J.D.E.), R01 HL24415 (J.D.E.), Cooley's Anemia Foundation Postdoctoral Fellowships (O.T. and N.O.), and an American Heart Association Postdoctoral Fellowship (L.S.).

We are grateful to our laboratory coworkers for many helpful discussions and to Gerd Blobel for initially suggesting the use of EGS in the ChIP assays. We thank Shoko Kobayashi and Carmen Yu for technical assistance.