1. Role of LDB1 in the transition from chromatin looping
to transcription activation
Ivan Krivega
Laboratory of Cellular and Developmental Biology, NIDDK, NIH

2. Models of long-range interaction between enhancer and promoter
RNApolII
as a linker
TF
Transcription factors
as a linker
Krivega and Dean, Curr Opin Genet Dev, 2012
PolII
PolII
PolII PolII

3. HS5
HS4
HS3
HS2
HS1
3’HS1
βmaj βminβh1εy
LCR
DD DD
LDB1LDB1
β-globin gene
promoter
HS2
β-globin gene
activation
10 kb
?
Long-range interaction in the mouse β-globin gene cluster
Song et al., MolCell 2007

4. αLDB1
αHA
αTub
LDB1 FL
LDB1 FL rescued β-globin expression in LDB1 KD MEL cells
LDB1 FL
DD NLS LIDHA
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Relativeexpression
β-globin
Endogenous
Ldb1
LDB1 FL
LDB1

5. DD stabilizes LDB1
LDB1 FL
DD NLS LIDHA
LDB1ΔDD
αHA
αTub
αHA
αTub
αHA
αTub
LDB1ΔDD
0
0.2
0.4
0.6
0.8
1
1.2
Relativeexpression
Transgenic mRNA
level

6. The DD is required for β-globin gene activation
LDB1 FL
DD NLS LIDHA
LDB1ΔDD K365R
LDB1 FL K365R
0
0.2
0.4
0.6
0.8
1
1.2
1.4
β-globin expressionRelativeexpression

7. Relativeexpression
0
0.2
0.4
0.6
0.8
1
1.2
The LMO-DD fusion is sufficient for β-globin gene activation
DD-LMO
LMO-DD
LMO2
DD
DDHA LMO2
DD
LDB1
DD
LMO/DD
β-globin expression

8. 0
1
2
3
4
5
6
103659323 103669323 103679323 103689323 103699323 103709323 103719323 103729323
Empty
LDB1 FL
LMO-DD
DD
Empty
uninduced
εyβH1βmajβmin
HS2
Relativecrosslinkingfrequency
chr 7
DD of LDB1 is sufficient for chromatin looping

9. 0
0.2
0.4
0.6
0.8
1
1.2
1.4
LDB1Δ1
LDB1Δ2
LDB1Δ3
LDB1Δ4/5
1 3 4 5
1 2 4 5
1 2 3
4 52 3
4 52 31 LDB1 FL αHA
αTub
LDB1Δ1 LDB1Δ2
LDB1Δ3 LDB1Δ4/5 FL
FL
αHA
αTub
The LDB1 DD can be functionally sub-divided
β-globin expression
Relativeexpression

10. Mx1Cre
Cre
Ldb1fl
Ldb1Δ
Ldb1
IFN-β
-
- + +
primers
Cre- -+ + +
-
Virus
infection
Time (h) 0 24 72
Cre
induction
The DD is required for β-globin gene activation in vivo
E14.5 fetal liver cells
with floxed Ldb1
• with Mx1Cre
• without Mx1Cre
0
0.2
0.4
0.6
0.8
1
1.2
Relativeexpression
Ldb1
Mx1CRE Ldb1
fl/fl
fl/fl
0
0.2
0.4
0.6
0.8
1
1.2
β-globin expression
Relativeexpression
Ldb1 β-globin

11. DD
DD Δ14 5
1 2 3 4 5
2 3
DD Δ4/51 2 3
0
0.2
0.4
0.6
0.8
1
1.2
αLMO2
αETO2
αLDB1
αTAL1
αHA
WT MEL cells
Input
DD
Input
DDΔ1
Input
DDΔ4/5
αHA
αLDB1
αHA
αLDB1
The complete DD is not required for LDB1 homodimerization.
β-globin transcripts
WT MEL cells

12. Dimerization of LDB1 is required for chromatin looping
0
1
2
3
4
5
6
7
103659323 103669323 103679323 103689323 103699323 103709323 103719323 103729323
Empty
LDB1 FL
LDB1 Δ1
LDB1 Δ2
LDB1 Δ3
LDB1 Δ4/5
Empty
uninduced
HS2
εyβH1βmajβmin
chr 7
Relativecrosslinkingfrequency

13. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
HS2 β necdin
Empty
Ldb1 FL
Ldb1 Δ4/5
LMO-DD
*
*
Enrichment
LDB1Δ4/5
0%
10%
20%
30%
40%
50%
60%
70%
80%
90%
100%
Interior
Lamin B1LAMB1
0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
LDB1 FL Uninduced
LDB1 FL Induced
Empty Induced
LDB1 Δ4/5
LMO-DD Induced
LAD HS2 βTrem151a
*
Enrichment
*
LDB1Δ4/5 Induced
LDB1 FL
Chromatin looping is not sufficient for β-globin gene activation and β-
globin locus intra-nuclear migration.
Pol II occupancy
LAMB1 occupancy
N=110
* - p<0.05

14. 0
0.05
0.1
0.15
0.2
0.25
0.3
0.35
0.4
0.45
Empty
Ldb1 FL
Ldb1 Δ4/5
LMO-DD
*
*
Enrichment
HS2 β necdin
Enrichment
0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
HS2 β necdin
Empty
FL Ldb1
Ldb1 Δ4/5
LMO-DD*
*
*
The LDB1 4/5 region is required for SWI/SNF complex occupancy at the
β-globin promoter.
BRG1 occupancy acH3 occupancy
* - p<0.05
Enrichment
0
0.5
1
1.5
2
2.5
3
HS2 β necdin
Empty
Ldb1 FL
Ldb1 Δ4/5
LMO-DD*
*
LDB1Δ4/5
LDB1 FL
GATA1 occupancy
LDB1 FL LDB1 FL
LDB1Δ4/5 LDB1Δ4/5
0
0.5
1
1.5
2
2.5
3
3.5
4
HS2 β necdin
Empty
Ldb1 FL
Ldb1 Δ4/5
LMO-DD
*
*
Enrichment
CBP occupancy
LDB1Δ4/5
LDB1 FL
0
0.1
0.2
0.3
0.4
0.5
0.6
HS2 β necdin
Empty
Ldb1 FL
Ldb1 Δ4/5
LMO-DD
* *
Enrichment
HDAC1 occupancy
LDB1Δ4/5
LDB1 FL

15. The DD 4/5 region interacts with FOG1
0
0.1
0.2
0.3
0.4
0.5
0.6
HS2 β necdin
Enrichment
Empty
Ldb1 FL
Ldb1 Δ4/5
*
*
*
*
0
0.05
0.1
0.15
0.2
0.25
HS2 β necdin
Enrichment
Empty
Ldb1 FL
Ldb1 Δ4/5
*
*
*
*
LDB1 FL
LDB1Δ4/5
LDB1 FL
LDB1Δ4/5
MTA2 occupancy FOG1 occupancy
αFOG1
αΤub
αFOG1
αLDB1
αGATA1
αLMO2
WT MEL cells
αFOG1
αGATA1
αLMO2
αHA
LDB1 KD
HA-LDB1 FL
LDB1 KD
HA-LDB1Δ4/5
αFOG1
αLMO2
αHA
E47
LID
LIM1 LIM2
* - p<0.05

16. 4/5 region of LDB1 is required for regulation of FOG1-dependent genes.
4/5-dependent
genes
4/5-independent
genes

17. 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
Empty
Ldb1 FL
Ldb1 Δ4/5
0
0.2
0.4
0.6
0.8
1
1.2
1.4
1.6
1.8
GATA1
Empty
Ldb1 FL
Ldb1 Δ4/5
LDB1 FL
LDB1 Δ4/5
496 LDB1-activated genes rescued by
LDB1 FL expression in LDB1 KD cells
147 genes
4/5-dependent
349 genes
4/5-independent
FOG1
4/5-dependent 4/5-independent
*
* *
* *
* *
Relativeexpression
EnrichmentEnrichment
0
0.2
0.4
0.6
0.8
1
1.2
1.4
Empty
LDB1 FL
LDB1Δ4/5
4/5 region of LDB1 is required for regulation of FOG1-dependent genes.
*
LDB1 FL
LDB1Δ4/5
* - p<0.05

18. 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

19. • Enhancer-promoter looping can be
uncoupled from transcriptional activation.
• Chromatin looping is not sufficient for
β-globin locus intra-nuclear migration.
• 4/5 region of DD domain of LDB1 is
required for the recruitment of the co-
regulators FOG1 and NuRD complex,
promoter remodeling and activation of
transcription.
Conclusions

20. Gene Regulation and Development Section
Laboratory of Cellular and Developmental Biology, NIDDK
Francine Katz
Undergraduate
Student
Ryan Dale
Bioinformatician
LiQi Li,
Paul Love
(NICHD, NIH)
Karen Meaburn,
Tom Misteli
(NCI, NIH)