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.

[1]  T. Townes,et al.  KLF1 regulates BCL11A expression and γ- to β-globin gene switching , 2010, Nature Genetics.

[2]  A. Bank,et al.  Multiple hematopoietic defects and delayed globin switching in Ikaros null mice , 2002, Proceedings of the National Academy of Sciences of the United States of America.

[3]  H. Cao.  Pharmacological Induction of Fetal Hemoglobin Synthesis using Histone Deacetylase Inhibitors , 2004, Hematology.

[4]  J. D. Engel,et al.  Embryonic and fetal β‐globin gene repression by the orphan nuclear receptors, TR2 and TR4 , 2007, The EMBO journal.

[5]  Antoine H. F. M. Peters,et al.  LSD1 demethylates repressive histone marks to promote androgen-receptor-dependent transcription , 2005, Nature.

[6]  Min Gyu Lee,et al.  Functional Interplay between Histone Demethylase and Deacetylase Enzymes , 2006, Molecular and Cellular Biology.

[7]  E. Seto,et al.  The orphan nuclear receptor TR2 interacts directly with both class I and class II histone deacetylases. , 2001, Molecular endocrinology.

[8]  Stuart H. Orkin,et al.  A protein interaction network for pluripotency of embryonic stem cells , 2006, Nature.

[9]  Li-Na Wei,et al.  Cloning and Characterization of Mouse RIP140, a Corepressor for Nuclear Orphan Receptor TR2 , 1998, Molecular and Cellular Biology.

[10]  S. Baylin,et al.  DNMT1 binds HDAC2 and a new co-repressor, DMAP1, to form a complex at replication foci , 2000, Nature Genetics.

[11]  Ping-Chih Ho,et al.  HDAC3 as a Molecular Chaperone for Shuttling Phosphorylated TR2 to PML: A Novel Deacetylase Activity-Independent Function of HDAC3 , 2009, PloS one.

[12]  E. Lander,et al.  MicroRNA-15a and -16-1 act via MYB to elevate fetal hemoglobin expression in human trisomy 13 , 2011, Proceedings of the National Academy of Sciences.

[13]  G. Stamatoyannopoulos,et al.  Histone acetylation at the human β-globin locus changes with developmental age , 2004 .

[14]  Hendrik G. Stunnenberg,et al.  MBD2/NuRD and MBD3/NuRD, Two Distinct Complexes with Different Biochemical and Functional Properties , 2006, Molecular and Cellular Biology.

[15]  Prim B. Singh,et al.  KAP-1 Corepressor Protein Interacts and Colocalizes with Heterochromatic and Euchromatic HP1 Proteins: a Potential Role for Krüppel-Associated Box–Zinc Finger Proteins in Heterochromatin-Mediated Gene Silencing , 1999, Molecular and Cellular Biology.

[16]  C. Gooding,et al.  LCR/MEL: a versatile system for high-level expression of heterologous proteins in erythroid cells. , 1992, Nucleic acids research.

[17]  R. Losson,et al.  Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family , 1999, The EMBO journal.

[18]  Laurent Kiger,et al.  Ex vivo generation of fully mature human red blood cells from hematopoietic stem cells , 2005, Nature Biotechnology.

[19]  S. Schreiber,et al.  Nuclear Receptor Repression Mediated by a Complex Containing SMRT, mSin3A, and Histone Deacetylase , 1997, Cell.

[20]  Henriette O'Geen,et al.  ZNF274 Recruits the Histone Methyltransferase SETDB1 to the 3′ Ends of ZNF Genes , 2010, PloS one.

[21]  A. Shevchenko,et al.  Mass spectrometric sequencing of proteins silver-stained polyacrylamide gels. , 1996, Analytical chemistry.

[22]  S. Berger,et al.  In vivo dual cross-linking for identification of indirect DNA-associated proteins by chromatin immunoprecipitation. , 2006, BioTechniques.

[23]  P. Kingsley,et al.  "Maturational" globin switching in primary primitive erythroid cells. , 2005, Blood.

[24]  C. Glass,et al.  Sensors and signals: a coactivator/corepressor/epigenetic code for integrating signal-dependent programs of transcriptional response. , 2006, Genes & development.

[25]  E. Seto,et al.  The Rpd3/Hda1 family of lysine deacetylases: from bacteria and yeast to mice and men , 2008, Nature Reviews Molecular Cell Biology.

[26]  S. Kato,et al.  p54nrb acts as a transcriptional coactivator for activation function 1 of the human androgen receptor. , 2003, Biochemical and biophysical research communications.

[27]  En Li,et al.  Suv 39 h-Mediated Histone H 3 Lysine 9 Methylation Directs DNA Methylation to Major Satellite Repeats at Pericentric Heterochromatin , 2003 .

[28]  C. Glass,et al.  A complex containing N-CoR, mSln3 and histone deacetylase mediates transcriptional repression , 1997, nature.

[29]  S. Pradhan,et al.  Epigenetic mechanisms in mammals , 2008, Cellular and Molecular Life Sciences.

[30]  Arthur W. Nienhuis,et al.  Hemoglobin switching , 1978, Cell.

[31]  S. Kato,et al.  Transrepressive Function of TLX Requires the Histone Demethylase LSD1 , 2008, Molecular and Cellular Biology.

[32]  S. Orkin,et al.  Transcriptional silencing of {gamma}-globin by BCL11A involves long-range interactions and cooperation with SOX6. , 2010, Genes & development.

[33]  Dario Neri,et al.  In vivo protein biotinylation for identification of organ-specific antigens accessible from the vasculature , 2005, Nature Methods.

[34]  Luyang Sun,et al.  LSD1 Is a Subunit of the NuRD Complex and Targets the Metastasis Programs in Breast Cancer , 2009, Cell.

[35]  R. Neades,et al.  Silencing of Aγ-Globin Gene Expression during Adult Definitive Erythropoiesis Mediated by GATA-1-FOG-1-Mi2 Complex Binding at the −566 GATA Site , 2008, Molecular and Cellular Biology.

[36]  Li-Na Wei,et al.  A Novel Nuclear Receptor Heterodimerization Pathway Mediated by Orphan Receptors TR2 and TR4* , 1998, The Journal of Biological Chemistry.

[37]  G. Blobel,et al.  FOG‐1 recruits the NuRD repressor complex to mediate transcriptional repression by GATA‐1 , 2005, The EMBO journal.

[38]  M. Sutton,et al.  Role of epigenetic modifications in normal globin gene regulation and butyrate-mediated induction of fetal hemoglobin. , 2007, Blood.

[39]  F. Grosveld,et al.  Fetal globin expression is regulated by Friend of Prmt1. , 2010, Blood.

[40]  E. Bresnick,et al.  Developmentally dynamic histone acetylation pattern of a tissue-specific chromatin domain. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[41]  Philip R. Gafken,et al.  A Chicken Ovalbumin Upstream Promoter Transcription Factor I (COUP-TFI) Complex Represses Expression of the Gene Encoding Tumor Necrosis Factor α-induced Protein 8 (TNFAIP8)*♦ , 2009, Journal of Biological Chemistry.

[42]  Chawnshang Chang,et al.  Recent advances in the TR2 and TR4 orphan receptors of the nuclear receptor superfamily , 2002, The Journal of Steroid Biochemistry and Molecular Biology.

[43]  F. Jeanmougin,et al.  A possible involvement of TIF1 alpha and TIF1 beta in the epigenetic control of transcription by nuclear receptors. , 1996, The EMBO journal.

[44]  Drew T. Erickson,et al.  Sox6 Directly Silences Epsilon Globin Expression in Definitive Erythropoiesis , 2005, PLoS genetics.

[45]  Chawnshang Chang,et al.  Disruption of TR4 Orphan Nuclear Receptor Reduces the Expression of Liver Apolipoprotein E/C-I/C-II Gene Cluster* , 2003, Journal of Biological Chemistry.

[46]  Sandip K. Mishra,et al.  Transcriptional repression of oestrogen receptor by metastasis-associated protein 1 corepressor , 2000, Nature Cell Biology.

[47]  James Douglas Engel,et al.  The TR2 and TR4 orphan nuclear receptors repress Gata1 transcription. , 2007, Genes & development.

[48]  W. Herr,et al.  The VP16 accessory protein HCF is a family of polypeptides processed from a large precursor protein , 1993, Cell.

[49]  Patrick Rodriguez,et al.  Isolation of transcription factor complexes by in vivo biotinylation tagging and direct binding to streptavidin beads. , 2006, Methods in molecular biology.

[50]  J. D. Engel,et al.  An embryonic/fetal β‐type globin gene repressor contains a nuclear receptor TR2/TR4 heterodimer , 2002, The EMBO journal.

[51]  H. Samuels,et al.  PSF Is a Novel Corepressor That Mediates Its Effect through Sin3A and the DNA Binding Domain of Nuclear Hormone Receptors , 2001, Molecular and Cellular Biology.

[52]  Paul A. Khavari,et al.  BRG1 contains a conserved domain of the SWI2/SNF2 family necessary for normal mitotic growth and transcription , 1993, Nature.

[53]  D. Lavelle,et al.  siDNMT1 increases γ-globin expression in chemical inducer of dimerization (CID)-dependent mouse βYAC bone marrow cells and in baboon erythroid progenitor cell cultures. , 2011, Experimental hematology.

[54]  Peter L. Jones,et al.  DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters , 2000, Nature Genetics.

[55]  Chawnshang Chang,et al.  Loss of TR4 Orphan Nuclear Receptor Reduces Phosphoenolpyruvate Carboxykinase–Mediated Gluconeogenesis , 2007, Diabetes.

[56]  G. Patrinos,et al.  A comparative study of Greek nondeletional hereditary persistence of fetal hemoglobin and β-thalassemia compound heterozygotes , 2002, Journal of Molecular Medicine.

[57]  K. Chin,et al.  SCF induces gamma-globin gene expression by regulating downstream transcription factor COUP-TFII. , 2009, Blood.

[58]  F. Lan,et al.  Regulation of LSD1 histone demethylase activity by its associated factors. , 2005, Molecular cell.

[59]  D. Na,et al.  Molecular Cloning and Characterization of CAPER, a Novel Coactivator of Activating Protein-1 and Estrogen Receptors* , 2002, The Journal of Biological Chemistry.

[60]  Min Gyu Lee,et al.  An essential role for CoREST in nucleosomal histone 3 lysine 4 demethylation , 2005, Nature.

[61]  Yang Shi,et al.  Histone Demethylation Mediated by the Nuclear Amine Oxidase Homolog LSD1 , 2004, Cell.

[62]  S. Fiering,et al.  Developmental- and differentiation-specific patterns of human γ- and β-globin promoter DNA methylation , 2007 .

[63]  J. Davie,et al.  Epigenetic control , 2009, Journal of cellular physiology.

[64]  M. Groudine,et al.  A Complex Chromatin Landscape Revealed by Patterns of Nuclease Sensitivity and Histone Modification within the Mouse β-Globin Locus , 2003, Molecular and Cellular Biology.

[65]  R. Morimoto.  Dynamic Remodeling of Transcription Complexes by Molecular Chaperones , 2002, Cell.

[66]  Y. Saunthararajah,et al.  DNA hypo‐methylating agents and sickle cell disease , 2004, British journal of haematology.

[67]  F. Grosveld,et al.  Haploinsufficiency for the erythroid transcription factor KLF1 causes Hereditary Persistence of Fetal Hemoglobin , 2010, Nature Genetics.

[68]  J. Drouin,et al.  TIF1β/KAP-1 Is a Coactivator of the Orphan Nuclear Receptor NGFI-B/Nur77* , 2009, Journal of Biological Chemistry.

[69]  David I. K. Martin,et al.  CpG Hypomethylation in a Large Domain Encompassing the Embryonic β-Like Globin Genes in Primitive Erythrocytes , 2007, Molecular and Cellular Biology.

[70]  C. Glass,et al.  The coregulator exchange in transcriptional functions of nuclear receptors. , 2000, Genes & development.

[71]  J. D. Engel,et al.  The Orphan Nuclear Receptor TR4 Is a Vitamin A-activated Nuclear Receptor*♦ , 2010, The Journal of Biological Chemistry.

[72]  Gratien G. Prefontaine,et al.  Histone Methylation-Dependent Mechanisms Impose Ligand Dependency for Gene Activation by Nuclear Receptors , 2007, Cell.

[73]  Yi Zhang,et al.  Dynamic Regulation of Histone H3 Methylated at Lysine 79 within a Tissue-specific Chromatin Domain* , 2003, The Journal of Biological Chemistry.

[74]  J. Hirschhorn,et al.  Supporting Online Material Materials and Methods Figs. S1 to S10 Tables S1 to S7 References Human Fetal Hemoglobin Expression Is Regulated by the Developmental Stage-specific Repressor Bcl11a , 2022 .

[75]  J. D. Engel,et al.  Fine Tuning of Globin Gene Expression by DNA Methylation , 2006, PloS one.

[76]  J. Strouboulis,et al.  Methylated DNA and MeCP2 recruit histone deacetylase to repress transcription , 1998, Nature Genetics.

[77]  G. Stamatoyannopoulos,et al.  Regulation of embryonic/fetal globin genes by nuclear hormone receptors: a novel perspective on hemoglobin switching , 1999, The EMBO journal.

[78]  M. Wasik,et al.  STAT3- and DNA methyltransferase 1-mediated epigenetic silencing of SHP-1 tyrosine phosphatase tumor suppressor gene in malignant T lymphocytes. , 2005, Proceedings of the National Academy of Sciences of the United States of America.

[79]  Henriette O'Geen,et al.  Genome-wide binding of the orphan nuclear receptor TR4 suggests its general role in fundamental biological processes , 2010, BMC Genomics.

[80]  J. D. Engel,et al.  Context-dependent EKLF responsiveness defines the developmental specificity of the human epsilon-globin gene in erythroid cells of YAC transgenic mice. , 2000, Genes & development.

[81]  Ann Dean,et al.  Epigenetics of beta-globin gene regulation. , 2008, Mutation research.

[82]  Cameron S. Osborne,et al.  Intergenic Transcription, Cell-Cycle and the Developmentally Regulated Epigenetic Profile of the Human Beta-Globin Locus , 2007, PloS one.

[83]  J. Ni,et al.  TR4 nuclear receptor functions as a fatty acid sensor to modulate CD36 expression and foam cell formation , 2009, Proceedings of the National Academy of Sciences.

[84]  J. D. Engel,et al.  Adult Stage γ-Globin Silencing Is Mediated by a Promoter Direct Repeat Element , 2005, Molecular and Cellular Biology.

[85]  E. Li,et al.  The lysine demethylase LSD1 (KDM1) is required for maintenance of global DNA methylation , 2009, Nature Genetics.

[86]  Pawan Gupta,et al.  Activation of testicular orphan receptor 4 by fatty acids. , 2009, Biochimica et biophysica acta.

[87]  R. Ware,et al.  Quantitative analysis of erythrocytes containing fetal hemoglobin (F cells) in children with sickle cell disease , 1997, American journal of hematology.

[88]  Jonghwan Kim,et al.  Epigenetic regulation of hematopoietic differentiation by Gfi-1 and Gfi-1b is mediated by the cofactors CoREST and LSD1. , 2007, Molecular cell.

[89]  Jeroen Krijgsveld,et al.  GATA‐1 forms distinct activating and repressive complexes in erythroid cells , 2005, The EMBO journal.

[90]  Patrick Rodriguez,et al.  Efficient biotinylation and single-step purification of tagged transcription factors in mammalian cells and transgenic mice , 2003, Proceedings of the National Academy of Sciences of the United States of America.

[91]  Luke Hughes-Davies,et al.  DNA methyltransferase Dnmt1 associates with histone deacetylase activity , 2000, Nature Genetics.

[92]  Y. Hung,et al.  Subfertility with defective folliculogenesis in female mice lacking testicular orphan nuclear receptor 4. , 2008, Molecular endocrinology.

[93]  Colin A. Johnson,et al.  Transcriptional repression by the methyl-CpG-binding protein MeCP2 involves a histone deacetylase complex , 1998, Nature.