The β-hemoglobinopathies are the most common monogenic disorders in humans, with symptoms arising after birth when the fetal γ-globin genes are silenced and the adult β-globin gene is activated. There is a growing appreciation that genome organization and the folding of chromosomes are key determinants of gene transcription. Underlying this function is the activity of transcriptional enhancers that increase the transcription of target genes over long linear distances. To accomplish this, enhancers engage in close physical contact with target promoters through chromosome folding or looping that is orchestrated by protein complexes that bind to both sites and stabilize their interaction. We find that enhancer activity can be redirected with concomitant changes in gene transcription. Both targeting the β-globin locus control region (LCR) to the γ-globin gene in adult erythroid cells by tethering and epigenetic unmasking of a silenced γ-globin gene lead to increased frequency of LCR/γ-globin contacts and reduced LCR/β-globin contacts. The outcome of these manipulations is robust, pancellular γ-globin transcription activation with a concomitant reduction in β-globin transcription. These examples show that chromosome looping may be considered a therapeutic target for gene activation in β-thalassemia and sickle cell disease.
Module for Grade 9 for Asynchronous/Distance learning
Chromatin looping as a target for altering gene expression
1. Chromatin looping as a target for altering erythroid
gene expression
Ivan Krivega, PhD
Laboratory of Cellular and Developmental Biology, NIDDK, NIH
2. How do different cell and tissue types arise?
Changes in gene expression drive developmental progression
The activity of enhancers underlies cell-specific transcription patterns
Enhancers contact genes over long distances
protein
complex
gene
enhancer
Krivega, Dean. Curr Opin Genet Dev. 2012.
3. Questions:
1. How do enhancers loop to target genes?
2. Can loops be manipulated to change disease-associated
gene expression?
4. Questions:
1. How do enhancers loop to target genes?
• role of looping factor LDB1
2. Can loops be manipulated to change disease-associated
gene expression?
5. The mammalian β-globin loci
Locus control region (LCR) enhancer loops to genes
determining which one is active as development proceeds
Tolhuis et al, Mol. Cell, 2002
Palstra et al, Nat. Genet., 2003
adult embryo
mouse
bmajbmin bh1 ey 10 kb
LCR
eGgAgdb
fetusadult OR genesembryo
human
LCR
Drissen et al, Genes Dev., 2004
Vakoc et al, Cell, 2005
Song et al. Mol. Cell, 2007
Yun et al, NAR, 2014
LDB1 complex
6. LDB1
NLSdimerization domain (DD) LIM interaction domain
(LID)
N C
Orthologue of D.melanogaster Chip, cloned in a screen for enhancer facilitators
Highly conserved, no DNA-binding or enzymatic activity, widely expressed
Morcillo et al, Genes Dev., 1997
Muhopadhyay et al, Development, 2003
Wadman et al, EMBO J, 1997
Required for erythropoiesis
WT Ldb1 null
E8.5 embryo
no blood
LID
LIM1 LIM2LMO2
LDB1
23. LDB1 DD 4/5 region is required for proper activation of
blood disease-associated genes
V205M mutation of GATA-1 abolishes
its interaction with FOG1 and causes
X-linked dyserythropoietic anemia
(Nichols et al., Nature Genetics, 2000)
Human disease-associated
homologs from OMIM
4/5-dependent
genes
4/5-independent
genes
Mouse gene
Human homolog
Disease association
(OMIM base)
0
5
10
15
20
25
30
35
4/5-dependent
4/5-independent
all
p=.001
p=.93
%ofdiseaseassociatedgenes
blood related diseases others
Krivega, Dale, Dean. Genes Dev. 2014
24. Questions:
1. How do enhancers loop to target genes?
• role of looping factor LDB1
2. Can loops be manipulated to change disease-
associated gene expression?
• forced looping
25. 0
0.01
0.02
0.03
0.04
0.05
0.06
0.07
0.08
HS3 HS2 HS1 εy βh1 βmaj βmin mCD4
IgG
HA
enrichment
0
0.5
1
1.5
2
2.5
3
103658500 103678500 103698500 103718500
BglII
Control
βZF-DD
Induced Control
βmin βmaj βh1 εy
HS2
interactionfrequency
Forced LCR looping using ZF-DD
G1E+GATA1
0
1
2
3
4
5
6
7
relativeexpression
βmaj
G1E
Deng et al, Cell, 2012
DD
βZF-DD
βZF HA
G1E cells (GATA1 null)
no βmaj expression
26. Forced LCR looping using dCas9-DDΔ4/5
0
0.02
0.04
0.06
0.08
0.1
0.12
0.14
0.16
HS5 HS4 HS3 HS2 HS1 ey βh1 β-major β-minor 3'HS1 necdin
HA Control
HA βmaj-DDΔ4/5
enrichment
0
2
4
6
8
10
12
14
16
0 10000 20000 30000 40000 50000 60000 70000
BglII
Control
dCas9-DΔ4/5
Induced Control
0
2
4
6
8
10
12
14
16
relativeexpression
βmaj
HS2
βmin βmaj βh1 εy
interactionfrequency
βmaj-DDΔ4/5
UMEL IMEL
Krivega and Dean, Submitted
4/5DD
dCas9-DDΔ4/5
dCas9 HA Uninduced MEL
no βmaj expression
27. Globin gene expression during development
HbF HbA
There are 2 ‘switches’ in globin gene expression
After the γ to b switch, β-thalassemia and sickle cell disease become manifest
Elevated fetal hemoglobin in adults moderates severity of the b-hemoglobinopathies
28. Forced chromatin looping to activated γ-globin genes
expression
e d bS
fetus adultOR genes embryo
human LCR
OR genes
Gg Ag
γZF
LDB1
DD
adult stage
29. Reversal of chromatin looping by γZF-LDB1-DD in
primary human adult erythroid cells
adult
CD34(+)
progenitor cells expansion
5-6 days
differentiation
10-12 days
lentiviral infection GFP sort
γZF DD
Deng*, Rupon*, Krivega et al, Cell, 2014
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
HS2 ε pro γ pro δ pro β pro HS40 α pro CD4
enrichment
IgG
HA
HA
30. interactionfrequency
Reversal of chromatin looping by γZF-LDB1-DD in
primary human adult erythroid cells
εy
0
0.2
0.4
0.6
0.8
1
1.2
5220000 5230000 5240000 5250000 5260000 5270000 5280000 5290000 5300000 5310000 5320000
EcoRI
Control
γZF-DD
Ag Ggδβ
LCR
31. Reactivation of the γ-globin gene in primary human
erythroid cells by γZF-DD
* - p<0.05 by Student’s t-test
0
10
20
30
40
50
60
70
80
90
γZF-DD γZF Control
γ-globin/(γ-globin+β-globin)
% γ-globin of total
* *
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
γZF-DD γZF Control
γ-globin/α-globin
*
*
0
0.5
1
1.5
2
2.5
γZF-DD γZF Control
β-globin/α-globin
γ-globin β-globin
*
*
32. γZF-DD Activates HbF Production
Control
Control
Control Control
Control Control
γZF-DD γZF-DD γZF-DD
γZF-DD γZF-DD γZF-DD
* - p<0.05 by Student’s t-test
34. Model: Manipulation of chromatin loops can overcome
developmental silencing to activate gene expression
γZF DD
Adult erythroid cells
+++
AdultFetal
LCR
LDB1
Adult
+
LCR
Deng*, Rupon*, Krivega et al, Cell, 2014
35. Questions:
1. How do enhancers loop to target genes?
• role of looping factor LDB1
2. Can loops be manipulated to change disease-associated
gene expression?
• forced looping
• pharmacological inhibition of epigenetic factors activity
36. Epigenetic reversal of chromatin looping
g-globin repression
H3K9me2 G9a
MTX
UNC0638
eGgAgdb
LCR
H3K9me2
Phase I-III
UNC0638
UNC0638
UNC0638
0 21
Control
Phase I
7 14
Phase II
Phase III
UNC0638
Days
UNC0638 UNC0638
CD34(+) cells from three
healthy donors were
differentiated ex vivo in 3 phase
serum free culture system
Krivega*, Byrnes* et al, Blood. 2015
expansion differentiation
37. Inhibition of G9a methyltransferase activity in adult human
erythrocytes stimulates fetal hemoglobin production
* - p<0.05 by Student’s t-test
0
5
10
15
20
25
30
35
40
Control Phase I Phase II Phase III Phase I-III
HbF(%)
*
*
*
0
5
10
15
20
25
30
35
0 0.031 0.062 0.12 0.25 0.5 1
HbF(%)
UNC0638 (μM)
*
*
*
*
*
*
0
10
20
30
40
50
60
70
80
90
100
0 0.031 0.062 0.12 0.25 0.5 1
HbF(%)
UNC0638 (μM)
*
43. Model: Epigenetic changes can relieve silencing by allowing
chromatin loops to reactivate gene expression
Adult erythroid cells
G9a
MT
+++
AdultFetal
H3K9me2
LCR
LDB1
Adult
++
LCR
X
Krivega*, Byrnes* et al, Blood. 2015
44. Summary
Enhancer mechanisms: Homodimerization of LDB1 protein through DD domain is
required for looping and transcription activation of b-globin genes.
Expression of blood disease-associated genes depends on interaction between LDB1 and
FOG1 proteins.
Enhancer looping manipulation: In adult erythroid cells, the LCR can be targeted to the
fetal g-globin genes by an ZF-DD based peptide resulting in their re-activation.
Inhibiting G9a methyltransferase activity relieves y-globin silencing in adult erythroid cells
resulting in redistribution of LDB1, LCR looping and g-globin re-activation.
These experiments suggest that chromosome looping can be considered a
therapeutic target for gene activation in b-hemoglobinopathies.
g-globin expression is pan-cellular and is increased to levels
potentially therapeutic in b-thalassemia and sickle cell disease
with balanced decrease in b-globin expression
45. Gene Regulation and Development Section
Laboratory of Cellular and Developmental Biology, NIDDK
Ann Dean
Xiang Guo
Luis Diaz
Ben Leadem
Maria Soledad Ivaldi
Guo-you Liu
Jun Zhang
LCDB Bioinformatics
Ryan Dale
NIDDK Genomics Core
Harold Smith
Collaborators
Jeffery Miller, Colleen Byrnes, Jaira F. de Vasconcellos
NIDDK, NIH
Gerd Blobel, Wulan Deng, Jeremy Rupon
CHOP, Philadelphia, PA
Stefano Rivella, Laura Breda
CHOP, Philadelphia, PA
Acknowledgements