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Understanding molecular mechanisms leading to reactivation derepression of gamma-globin gene

Understanding the molecular mechanisms leading to reactivation or derepression of γ-globin gene by Jim Vadolas, Cell and Gene Therapy Group, Murdoch Childrens Research Institute, Royal Children’s Hospital

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Understanding molecular mechanisms leading to reactivation derepression of gamma-globin gene

  1. 1. Understanding the molecular U d di h l lmechanisms leading to reactivation or derepression of γ-globin gene Jim Vadolas Cell and Gene Therapy Group Murdoch Childrens Research Institute Royal Children’s Hospital
  2. 2. Human -globin locus Chromosome 11p15.5 5’ HS - 3’HS1 5’ HS 5 4 3 2 1 111 Olfactory receptor (OR) genes (OR) genes LCR  G A   5’ HS‐111 3’ Heterochromatin 3’HS‐1 LCRMultiple interactions  between regulatory regions are required  to stabalise an  i d b li  active chromatin hub Adapted from Patrinos et al., Genes & Development 2004 G A
  3. 3. Genetic basis for normal variation in HbF levels Recent insight into hemoglobin switching has come from the results of three genome-wide association studies. •XmnI polymorphisms in the β-globin locus (-158 C>T) •A region between the HBS1-like gene HBS1L and the oncogene MYB •BCL11A gene (encoding the transcription factor B-cell lymphoma/leukemia 11A) •KLF1 gene (Krüppel-like factor 1)
  4. 4. Other factors involved in HbF regulation • Histone deacetylase 1 (HDAC 1) • DNA methyltransferase 1 (DNMT1) • HMG-box protein SOX6 • Friend of PRMT1 (FOP1) protein • Orphan nuclear hormone receptors TR2, TR4, and COUP-TFII • miRNAs, 15a and 16-1
  5. 5. Globin gene regulatory networksModel of BCL11A-mediated silencing of γ-globin genes. KLF1 FOP Jian Xu et al. Genes Dev. 2010;24:783-798 Xu, al. Dev. 2010;24:783- Borg J, et al. Nat Genet 2010;42(9):801-805 van Dijk TB, et al. Mol Cell Biol 2010;30:260-272
  6. 6. Common histone modifications within the human β-globin locus In primary adult human erythroid progenitorsH3K4me3 and H3K27me3 are interpreted as activating and silencing marks, Xu J et al. Genes Dev. 2010;24:783-798
  7. 7. Development of in vitro model systemsto study reactivation or d d i i derepression i of γ-globin gene γ globin
  8. 8. Modified human -globin locuspEBAC148β LCR LTR S S OR51M1 OR51B6 OR51B5 OR51B2 OR51B4 5’HS‐111  G  A     A S LTR S S G γ‐dsRED ‐EGFP OR51B6 OR51B5 OR51B2pEBACGγdsRED‐βEGFP OR51B4    B S LTR S S A ‐EGFP OR51B6 OR51B5 OR51B2pEBACAγdsRED‐βEGFP γ‐dsRED OR51B4    C 114kb 8kb 61kb 18kb 20 40 60 80 100 120 140 160 180 (kb) 180 (kb) Chan K, et al. FASEBJ (in press)
  9. 9. Measurement of DsRed/EGFP expression following treatment f ll i t t t * * * Butyrate (μM) Hydroxyurea (μM) 5-Aza-Deoxycytidine (μM) HDAC inhibitor HDAC i hibi nitric oxide activation  i i id i i DNA methyltransferase DNA h l f (DNMT1)  inhibitorHbF-inducing drugs had minimal effect unless BCL11A was reduced
  10. 10. Development of in vivo model system to study reactivation or derepression of d i i d i f γ globin γ-globin gene
  11. 11. Summary - 1• Mechanistically, Mechanistically loss of BCL11A in primary adult erythroid cells has been shown to reconfigure the human -globin locus• Chromatin occupancy of HDAC1 and the repressive H3K27me3 mark found in the region is reduced• The altered chromatin conformation of the human -globin cluster upon knockdown of BCL11A may become amenable to p y γ-globin inducers.• While BCL11A is critical for γ globin gene silencing we cannot γ-globin silencing, exclude the possible contribution of other regulatory factors or other multiprotein complexes which may act independently or in p p y p y combination with BCL11A (such as DRED or Polycomb-group proteins)
  12. 12. Summary - 2• The evaluation of in vitro and in vivo model systems which recapitulate human globin represents a promising approach to perform genetic and functional genomic studies to identify and evaluate key factors associated with γ-globin gene suppression. pp• Further understanding of the molecular mechanism regulating HbF genes holds promise for the development of targeted therapeutic approaches for HbF induction in the β- hemoglobinopathies. g p
  13. 13. AcknowledgmentsCell and Gene Therapy Group (MCRI)Cell and Gene Therapy Group (MCRI) Kasey Chan, Hady Wardan , Sara Howden , Hsiao Voon, Lucille  Voullaire  and Faten Zaibak Voullaire and Faten ZaibakThalassaemia Research Center (Mahidol University, Bangkok)Thalassaemia Research Center (Mahidol University, Bangkok) Dr Saovaros Svasti, Dr Prof. Suthat Fucharoen and Prof. Pranee Winichagoon Prof. Pranee WinichagoonHarvard Medical School (USA)Harvard Medical School (USA) Dr Jian Xu and Prof Stuart Orkin This work was funded by grants from NH&MRC, Cooley’s Anemia Foundation,  Radiomarathon Australia, The Greek Conference and Thalassaemia Australia.