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

1. Understanding the molecular
U d di h l l
mechanisms 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. 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
LCR
Multiple 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. 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. 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. Globin gene regulatory networks
Model 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

7. Common histone modifications within the
human β-globin locus
In primary adult human erythroid progenitors
H3K4me3 and H3K27me3 are interpreted as 'activating' and 'silencing' marks,
Xu J et al. Genes Dev. 2010;24:783-798

8. Development of in vitro model systems
to study reactivation or d
d i i derepression
i
of γ-globin gene
γ globin

9. Modified human -globin locus
pEBAC148β 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
OR51B2
pEBACGγdsRED‐βEGFP
OR51B4
  
B
S
LTR
S S A ‐EGFP
OR51B6
OR51B5
OR51B2
pEBACAγdsRED‐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)

12. 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)
inhibitor
HbF-inducing drugs had minimal effect unless BCL11A was reduced

13. Development of in vivo model system to
study reactivation or derepression of
d i i d i f
γ globin
γ-globin gene

14. 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)

15. 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

16. Acknowledgments
Cell 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 Zaibak
Thalassaemia Research Center (Mahidol University, Bangkok)
Thalassaemia Research Center (Mahidol University, Bangkok)
Dr Saovaros Svasti, Dr Prof. Suthat Fucharoen and
Prof. Pranee Winichagoon
Prof. Pranee Winichagoon
Harvard 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.