RED CELLS , IRON , AND ERYTHROPOIESIS Inhibition of G 9 a methyltransferase stimulates fetal hemoglobin production by facilitating LCR / g-globin looping

In humans, the b-globin cluster contains fetal gand g-globin and adult dandb-globin genes. Around the timeof birth, fetal hemoglobin (HbF) is almost completely replaced by adult hemoglobin (HbA) containing 2 b-globin chains. Based upon this developmental transition in hemoglobin production, mutations in the b-globin gene locus can cause a variety of hemoglobinopathies including sickle cell disease and b-thalassemia. One longstanding goal for developing treatments for these b-hemoglobinopathies is the reactivation and increased expression of HbF in adult erythroid cells. Therefore, considerable research effort has been focused upon understanding the mechanisms that underlie g-globin gene repression during the developmental switch between HbF and HbA that could suggest new therapeutic approaches for these diseases. Expression of b-globin genes is regulated by physical interactions betweengenepromoters and the locus control region (LCR)enhancer. Experiments using RNA interference have shown that this interaction is facilitated by the LDB1/LMO2/GATA-1/TAL1 erythroid-specific protein complex (LDB1 complex). The LDB1 complex occupies the LCR and the b-globin gene promoter and provides chromatin loop formation between them through interaction between LDB1 homodimerization domains. Mouseb-globingenes are also regulatedby theG9a/EHMT2H3K9 histone methyltransferase. G9a contains a SET domain responsible for histone H3K9 monoand di-methylation associated with repression of gene expression. Interestingly, recent observations support the view that G9a can play a role in activation of gene expression independently from its repressive methyltransferase activity. In human cells, G9a functions as a stable heteromeric complex with a related protein, GLP (EHMT1). UNC0638 specifically inhibits methyltransferase activity of G9a and GLP, causing a strong decrease in bulk H3K9me2 and reactivation of G9a-silenced genes in mouse embryonic stem cells. UNC0638 treatment of CD34 hematopoietic progenitor cells delayed adoption of differentiated phenotypes, suggesting an important role for G9a in lineage specification. Moreover, brief treatment of these cells with UNC0638 activated fetal g-globin genes in parallel with repression of adult dand b-globin genes, reversing the normal sequence of events that occurs late in erythroid differentiation. The mechanistic role of G9a in epigenetic regulation of the b-globin locus remains unclear. Here, we investigated the role of G9a in silencing fetal g-globin genes and activation of adult dand b-globin genes during ex vivo differentiation of CD34 adult hematopoietic progenitor cells.We found thatUNC0638 treatment acts primarily upon erythroblasts as they acquire a glycophorin A positive (GPA) phenotype in response to erythropoietin, and we show that G9a is directly involved in epigenetic repression of the human g-globin genes.

[1]  Patrick J. Paddison,et al.  G9a/GLP-dependent H3K9me2 patterning alters chromatin structure at CpG islands in hematopoietic progenitors , 2014, Epigenetics & Chromatin.

[2]  Philip D. Gregory,et al.  Reactivation of Developmentally Silenced Globin Genes by Forced Chromatin Looping , 2014, Cell.

[3]  J. D. Engel,et al.  Fetal Globin Gene Repressors as Drug Targets for Molecular Therapies To Treat the β-Globinopathies , 2014, Molecular and Cellular Biology.

[4]  A. Dean,et al.  The hematopoietic regulator TAL1 is required for chromatin looping between the β-globin LCR and human γ-globin genes to activate transcription , 2014, Nucleic acids research.

[5]  R. Margueron,et al.  The histone H3 lysine 9 methyltransferases G9a and GLP regulate polycomb repressive complex 2-mediated gene silencing. , 2014, Molecular cell.

[6]  Tiziana Bonaldi,et al.  Polycomb-dependent H3K27me1 and H3K27me2 regulate active transcription and enhancer fidelity. , 2014, Molecular cell.

[7]  Paola Sebastiani,et al.  Fetal hemoglobin in sickle cell anemia: a glass half full? , 2013, Blood.

[8]  Xi-Ping Huang,et al.  Discovery of an in vivo chemical probe of the lysine methyltransferases G9a and GLP. , 2013, Journal of medicinal chemistry.

[9]  Y. T. Lee,et al.  LIN28B-mediated expression of fetal hemoglobin and production of fetal-like erythrocytes from adult human erythroblasts ex vivo. , 2013, Blood.

[10]  Hiroshi Kimura,et al.  Histone modifications for human epigenome analysis , 2013, Journal of Human Genetics.

[11]  A. Dean,et al.  Role of Ldb1 in the transition from chromatin looping to transcription activation , 2013, Epigenetics & Chromatin.

[12]  Osamu Tanabe,et al.  Lysine-specific demethylase 1 is a therapeutic target for fetal hemoglobin induction , 2013, Nature Medicine.

[13]  R. Taneja,et al.  G9a, a multipotent regulator of gene expression , 2013, Epigenetics.

[14]  Weiqun Peng,et al.  Histone H3K9 methyltransferase G9a represses PPARγ expression and adipogenesis , 2012, The EMBO journal.

[15]  Patrick J. Paddison,et al.  G9a/GLP-dependent histone H3K9me2 patterning during human hematopoietic stem cell lineage commitment. , 2012, Genes & development.

[16]  F. Dilworth,et al.  Maintenance of gene silencing by the coordinate action of the H3K9 methyltransferase G9a/KMT1C and the H3K4 demethylase Jarid1a/KDM5A , 2012, Proceedings of the National Academy of Sciences.

[17]  P. Gregory,et al.  Controlling Long-Range Genomic Interactions at a Native Locus by Targeted Tethering of a Looping Factor , 2012, Cell.

[18]  Ryan K. Dale,et al.  Distinct Ldb1/NLI complexes orchestrate γ-globin repression and reactivation through ETO2 in human adult erythroid cells. , 2011, Blood.

[19]  Peter A. DiMaggio,et al.  A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. , 2011, Nature chemical biology.

[20]  Y. Shinkai,et al.  H3K9 methyltransferase G9a and the related molecule GLP. , 2011, Genes & development.

[21]  Xiaojun Ding,et al.  Histone methyltransferase G9a contributes to H3K27 methylation in vivo , 2011, Cell Research.

[22]  S. Orkin,et al.  Update on fetal hemoglobin gene regulation in hemoglobinopathies , 2011, Current opinion in pediatrics.

[23]  M. Groudine,et al.  Multiple functions of Ldb1 required for beta-globin activation during erythroid differentiation. , 2010, Blood.

[24]  Hee June Choi,et al.  Negative regulation of hypoxic responses via induced Reptin methylation. , 2010, Molecular cell.

[25]  C. Palii,et al.  Dual role for the methyltransferase G9a in the maintenance of β-globin gene transcription in adult erythroid cells , 2009, Proceedings of the National Academy of Sciences.

[26]  S. Goh,et al.  Cytokine-mediated increases in fetal hemoglobin are associated with globin gene histone modification and transcription factor reprogramming. , 2009, Blood.

[27]  Y. T. Lee,et al.  Identification of TWSG1 as a second novel erythroid regulator of hepcidin expression in murine and human cells. , 2009, Blood.

[28]  S. Fiering,et al.  Complex developmental patterns of histone modifications associated with the human beta-globin switch in primary cells. , 2009, Experimental hematology.

[29]  H. Kimura,et al.  G9a/GLP complexes independently mediate H3K9 and DNA methylation to silence transcription , 2008, The EMBO journal.

[30]  Xiaodong Cheng,et al.  Protein lysine methyltransferase G9a acts on non-histone targets. , 2008, Nature chemical biology.

[31]  E. Avvedimento,et al.  DNA Oxidation as Triggered by H3K9me2 Demethylation Drives Estrogen-Induced Gene Expression , 2008, Science.

[32]  Chunhui Hou,et al.  A positive role for NLI/Ldb1 in long-range beta-globin locus control region function. , 2007, Molecular cell.

[33]  I. Wood,et al.  Chromatin crosstalk in development and disease: lessons from REST , 2007, Nature Reviews Genetics.

[34]  S. Lau,et al.  5-Aza-2′-deoxycytidine-mediated reductions in G9A histone methyltransferase and histone H3 K9 di-methylation levels are linked to tumor suppressor gene reactivation , 2007, Oncogene.

[35]  M. Cam,et al.  A newly discovered human alpha-globin gene. , 2005, Blood.

[36]  Tsutomu Ohta,et al.  Histone methyltransferases G9a and GLP form heteromeric complexes and are both crucial for methylation of euchromatin at H3-K9. , 2005, Genes & development.

[37]  M. Groudine,et al.  Proximity among distant regulatory elements at the beta-globin locus requires GATA-1 and FOG-1. , 2005, Molecular cell.

[38]  Beatrix Ueberheide,et al.  Histone methyltransferases direct different degrees of methylation to define distinct chromatin domains. , 2003, Molecular cell.

[39]  R. Hoffman,et al.  Effects of 5-aza-2'-deoxycytidine on fetal hemoglobin levels, red cell adhesion, and hematopoietic differentiation in patients with sickle cell disease. , 2003, Blood.

[40]  Erik Splinter,et al.  Looping and interaction between hypersensitive sites in the active beta-globin locus. , 2002, Molecular cell.

[41]  H. Kato,et al.  G9a histone methyltransferase plays a dominant role in euchromatic histone H3 lysine 9 methylation and is essential for early embryogenesis. , 2002, Genes & development.

[42]  S. Orkin,et al.  The switch from fetal to adult hemoglobin. , 2013, Cold Spring Harbor perspectives in medicine.

[43]  D. Lavelle,et al.  Effect of 5-aza-2'-deoxycytidine (Dacogen) on covalent histone modifications of chromatin associated with the epsilon-, gamma-, and beta-globin promoters in Papio anubis. , 2006, Experimental hematology.

[44]  F. Grosveld,et al.  The beta-globin nuclear compartment in development and erythroid differentiation. , 2003, Nature genetics.

[45]  5—Azacytidine selectively increases globin synthesis in a patient with thalassemia , 1983 .