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 ␤-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 ␤-type globin promoters but not to the adult ␤-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 TIF1␤, 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 ␤-type globin promoters but, upon terminal differentiation, they specifically dissociate only from the adult ␤-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 ␤-type globin genes through coordinated epigenetic chromatin modifications.Regulatory pathways that control development through temporally specified gene activation and repression mechanisms have been recognized as "epigenetic" (i.e., heritable changes not involving alterations in the primary DNA code) for decades, although the molecules that elicit those developmental programs through epigenetic means have only been elucidated during the past several years. It is currently widely accepted that metazoan transcription factors (both activators and repressors) elicit their specific transcriptional responses through an enormous variety of cofactor molecules whose major purpose is to modulate chromatin structure (8, 31). Many such cofactors have been shown to chemically modify histones, transcription factors, and cofactors, as well as DNA, in order to elicit the required transcriptional responses.The ␤-globin locus has been extensively studied as a paradigm for epigenetic regulation of lineage-specific and developmentally specific gene expression (29), as well as for its clinical relevance to ␤-globin disorders such as sickle cell disease and ␤-thalassemia. The human ␤-globin locus is composed of ε-(embryonic), G␥-and A␥-(fetal), and ␦-and ␤-globin (adult) genes, which are spatially arranged from 5Ј to 3Ј and developmentally expressed in the same order (72). The elucidation of the molecular basis for ␥-globin silencing in the adult stage in particular has been the focus of intense investigation, since it has been observed that coinheritance of genetic conditions that confer elevated ␥-globin synthesis can significantly alleviate the symptoms of ␤-globin disorders (44, 56). Previously, several adul...

Pomalidomide reverses γ-globin silencing through the transcriptional reprogramming of adult hematopoietic progenitors

“…Among these, BCL11A is recognized as a master regulator of g-globin repression, 3 but other studies have also implicated SOX6, IKZF1 (Ikaros), KLF1, LSD1, RCOR-1 (CoREST), MI2B, and GATA-1 in the regulation of the fetalto-adult globin switch. [26][27][28][29][30] qRT-PCR analyses revealed that BCL11A, SOX6, GATA1, KLF1, and LSD1 were downregulated by pomalidomide, whereas levels of IKZF1, MI2B, GATA2, HDAC1, and CoREST transcripts were unaffected at day 4 of differentiation ( Figure 3A). By examining the protein levels at day 4 and day 11 of culture, we found that these proteins could be categorized into 3 different groups: those for which pomalidomide decreased their expression at first (day 4) and maintained decreased expression during differentiation (IKZF1, BCL11A, and SOX6; Figure 3B); those that were decreased at day 4 but normalized at day 11 (KLF1, GATA1, and LSD1; Figure 3C); and those that were not affected by pomalidomide (GATA2, CoREST, HDAC1; Figure 3D).…”

The LSD1 inhibitor RN-1 recapitulates the fetal pattern of hemoglobin synthesis in baboons (P. anubis)

“…46 Our results show that the LSD1 inhibitor RN-1 induces high levels of HbF in baboons and are consistent with previous studies showing that LSD1 plays a critical role in γ-globin silencing as a component of the DRED complex. 25,31 However, the possibility of a role for perturbation of erythroid differentiation in the mechanism of HbF reactivation cannot be excluded, and the in vivo mechanism of action will be investigated in future studies. The level of the HbF response varies between different baboon species, 47 and, therefore, the HbF response in man and baboons may differ somewhat due to differences between species and/or physiological differences between the experimental animal model and patients with hemoglobinopathies.…”

“…22 Recognition of the role of Direct Repeat (DR) elements in the e-and γ-globin gene promoters in repression of these respective genes led to the identification of the TR2 and TR4 non-steroidal nuclear receptor proteins that specifically bind these elements and subsequently recruit a multi-protein co-repressor complex that includes DNA methyltransferase 1 (DNMT1), lysine specific demethylase 1 (LSD1), and histone deacetylases (HDACs) to repress gene expression. [23][24][25] Both DNMT1 and LSD1 have also been identified as components of co-repressor complexes also recruited by Bcl11a. 26,27 LSD1, the first histone demethylase identified, demethylates lysine 4 (H3K4) and lysine 9 (H3K9) residues of histone H3 and represses gene expression during hematopoietic differentiation.…”

The Switch from Fetal to Adult Hemoglobin

“…By examining proteins that bind the direct repeat elements found within the promoter of the g-globin genes, the orphan nuclear hormone receptors TR2 and TR4 have been suggested to play a role as repressors of the expression of the g-globin genes (Tanabe et al 2002(Tanabe et al , 2007Cui et al 2011). Paradoxically, overexpression of TR2 and TR4 in transgenic mouse models results in elevated expression of g-globin and when overexpressed in sickle cell disease mouse models can result in partial amelioration of hematologic and pathologic symptoms of these mice (Campbell et al 2011).…”