1. In collaboration with Mikkelsen Lab
Dissection of regulatory motifs in 2,000 human enhancers using a massively parallel reporter assay
Pouya Kheradpour
Kellis Lab
In collaboration with Mikkelsen Lab

2. Dissecting motifs in 2000+ enhancers
Predict activator and repressor motifs in specific cell lines
Motif instances using comparative genomics
Activator/repressor prediction using chromatin state dynamics
Motif disruption experiments in human enhancers
Selecting 5 activators and 2 repressors in two cell lines (x160)
Massively parallel reporter assay: 200,000+ expr measurements
Experimental results:
Activators: Disrupting, enhancing, and neutral motif changes
Context matters: sequence features of functional WT enhancers
Repressor disruption: aberrant enhancer activity in other cells

3. Dissecting motifs in 2000+ enhancers
Predict activator and repressor motifs in specific cell lines
Motif instances using comparative genomics
Activator/repressor prediction using chromatin state dynamics
Motif disruption experiments in human enhancers
Selecting 5 activators and 2 repressors in two cell lines (x160)
Massively parallel reporter assay: 200,000+ expr measurements
Experimental results:
Activators: Disrupting, enhancing, and neutral motif changes
Context matters: sequence features of functional WT enhancers
Repressor disruption: aberrant enhancer activity in other cells

4. Identifying conserved motif instances
movement
mutations
missing short
branches
CTCF_8mer chr
Overlaps with CTCF experimental regions
BLS = 2.23sps (78%)
Allows for:
Mutations permitted by motif degeneracy
Misalignment/movement of motifs within window (up to hundreds of nucleotides)
Missing motif in dense species tree
CTCF

5. Conserved instances more likely to be bound by corresponding factor
Conservation of motif match significantly increases enrichment in ChIP regions for factor
Enrichment in bound regions also bound in orthologous mouse region significantly higher

6. Dissecting motifs in 2000+ enhancers
Predict activator and repressor motifs in specific cell lines
Motif instances using comparative genomics
Activator/repressor prediction using chromatin state dynamics
Motif disruption experiments in human enhancers
Selecting 5 activators and 2 repressors in two cell lines (x160)
Massively parallel reporter assay: 200,000+ expr measurements
Experimental results:
Activators: Disrupting, enhancing, and neutral motif changes
Context matters: sequence features of functional WT enhancers
Repressor disruption: aberrant enhancer activity in other cells

7. Combinations of chromatin marks define Chromatin States
Ernst, Kheradpour, et al. Nature 2011

8. Coordinated activity reveals activators/repressors
Oct4 predicted activator of embryonic stem cells
Gfi1 predicted repressor K562/GM12878 cells
Ernst, Kheradpour, et al. Nature 2011

9. Coordinated activity reveals activators/repressors

10. HNF1 and HNF4 are predicted activator of HepG2 enhancers
Model: Disruption of the motif site would abolish enhancer state

11. Dissecting motifs in 2000+ enhancers
Predict activator and repressor motifs in specific cell lines
Motif instances using comparative genomics
Activator/repressor prediction using chromatin state dynamics
Motif disruption experiments in human enhancers
Selecting 5 activators and 2 repressors in two cell lines (x160)
Massively parallel reporter assay: 200,000+ expr measurements
Experimental results:
Activators: Disrupting, enhancing, and neutral motif changes
Context matters: sequence features of functional WT enhancers
Repressor disruption: aberrant enhancer activity in other cells

12. Systematically looking at HepG2 and K562: all motifs with at least 1
Systematically looking at HepG2 and K562: all motifs with at least 1.3-fold enrichment/depletion
Zfp161_3
Nrf-2_3
HNF4_6
GATA_14
Gfi1_1
HNF1_1
Foxa_2

13. Motifs predicted to be functional in HepG2 and K562 cells selected

14. Model for cell line specific motifs

15. Dissecting motifs in 2000+ enhancers
Predict activator and repressor motifs in specific cell lines
Motif instances using comparative genomics
Activator/repressor prediction using chromatin state dynamics
Motif disruption experiments in human enhancers
Selecting 5 activators and 2 repressors in two cell lines (x160)
Massively parallel reporter assay: 200,000+ expr measurements
Experimental results:
Activators: Disrupting, enhancing, and neutral motif changes
Context matters: sequence features of functional WT enhancers
Repressor disruption: aberrant enhancer activity in other cells

16. Massively parallel reporter assay (MPRA)
Multiplexed enhancer assay
10,000s of elements using unique tags
Normalize mRNA counts by Plasmid counts
Tarjei Mikkelsen
Melnikov, Murugan, Zhang, et al.

17. Experimental design Manipulations (*expected to disrupt enhancer):
Scramble of bases*
Least change in score
Complete removal of motif*
Greatest increase in score
Greatest decrease in score*
Random change (x2)
Activators
Repressors
HepG2
HNF1, HNF4, FOXA
ZFP161
K562
GATA, NRF2
GFI1
Same cell type
160 18
+ scramble
+ other manip (x7) 15
Opposite cell type
No. tested instances (x2 for ignoring/high conservation)

18. Dissecting motifs in 2000+ enhancers
Predict activator and repressor motifs in specific cell lines
Motif instances using comparative genomics
Activator/repressor prediction using chromatin state dynamics
Motif disruption experiments in human enhancers
Selecting 5 activators and 2 repressors in two cell lines (x160)
Massively parallel reporter assay: 200,000+ expr measurements
Experimental results:
Activators: Disrupting, enhancing, and neutral motif changes
Context matters: sequence features of functional WT enhancers
Repressor disruption: aberrant enhancer activity in other cells

19. Ex. activator: conserved HNF4 motif match
Found in enhancer state of target cell line (HepG2)
Coincident with “Dip” in H3K27ac chromatin signal, suggestive of nucleosome exclusion

20. Ex. activator: conserved HNF4 motif match
Motif match disruptions reduce expression to background
Non-disruptive modifications maintain expression
Random changes depend on effect to motif match
WT expression
specific to HepG2

21. All conserved HNF4 in HepG2 results
Most are tested sequences not highly expressed
Maybe not sufficient context?
Those that are almost always reduce in expression when scrambled
Max-increase and min-change in motif match score do not seem to reduce when scrambled

22. Scrambling has the same effect for all activators
Conserved instances consistently have higher expression than instances ignoring conservation

23. Additional modifications for 15 conserved instances per factor
The “disruptive” modifications (scrambling, removal, max 1-bp decrease) all reduce expression
The “neutral” modification does not
The max 1-bp increase significantly increases expression when looked in aggregate

24. Opposite cell line enrichments: Surprise functional factor
Conserved motif instances (18 per factor) found in the opposite cell type (K562 for HNF1, HNF4, FOXA; HepG2 for GATA, NRF2)
Do not generally show reduction on scrambling
Exception is NRF2 – shows signature of activity despite lack of expression or enrichment

25. Dissecting motifs in 2000+ enhancers
Predict activator and repressor motifs in specific cell lines
Motif instances using comparative genomics
Activator/repressor prediction using chromatin state dynamics
Motif disruption experiments in human enhancers
Selecting 5 activators and 2 repressors in two cell lines (x160)
Massively parallel reporter assay: 200,000+ expr measurements
Experimental results:
Activators: Disrupting, enhancing, and neutral motif changes
Context matters: sequence features of functional WT enhancers
Repressor disruption: aberrant enhancer activity in other cells

26. Estimating the number of functional enhancers tested
71% of conserved instances for activators drop expr in their target cell line when scrambled (n≈800)
We expect non-functional enhancers to increase in expression upon scrambling of motif instances with 50/50 probability
If we assume (conservatively) that only ‘fake’ enhancers will go up on scrambling, we estimate at most 2*(100 – 71) = 58% ‘fake’ enhancers, and at least 42% real enhancers

27. Expression of conserved
Context matters: identical motif matches considerably varied expression
Expression of conserved
NRF2 motif instances
Motif match score explains very little of variability in expression (r=0.15)
Even for these cell-type specific enhancers and conserved motif instances, context plays an important role

28. Features of functional enhancers
Instances ignoring conservation for all 5 activators in matched cell line
H3K27ac dip score (suggestive of nucleosome exclusion)
Motif conservation level (BLS)
No. different factors with matching motifs in 145-bp seq
From large database of known motifs
Strength of motif match

30. Dissecting motifs in 2000+ enhancers
Predict activator and repressor motifs in specific cell lines
Motif instances using comparative genomics
Activator/repressor prediction using chromatin state dynamics
Motif disruption experiments in human enhancers
Selecting 5 activators and 2 repressors in two cell lines (x160)
Massively parallel reporter assay: 200,000+ expr measurements
Experimental results:
Activators: Disrupting, enhancing, and neutral motif changes
Context matters: sequence features of functional WT enhancers
Repressor disruption: aberrant enhancer activity in other cells

31. Model of GFI1 activity in HepG2 / K562 enhancers

32. Ex. GFI1 match in HepG2 enhancer
Disruption leads to increase in K562 expression

33. Disruption of repressor motifs leads to aberrant expression
Much more subtle effect than for activators
Not surprising, we don’t expect every enhancer in one cell type to be “primed” for the other
Significant effect seen for GFI1, but not for ZFP161

34. Dissecting motifs in 2000+ enhancers
Predict activator and repressor motifs in specific cell lines
Motif instances using comparative genomics
Activator/repressor prediction using chromatin state dynamics
Motif disruption experiments in human enhancers
Selecting 5 activators and 2 repressors in two cell lines (x160)
Massively parallel reporter assay: 200,000+ expr measurements
Experimental results:
Activators: Disrupting, enhancing, and neutral motif changes
Context matters: sequence features of functional WT enhancers
Repressor disruption: aberrant enhancer activity in other cells

35. Contributions
Hundreds of experimentally validated enhancers with necessary motif instance.
Resource for data mining enhancer sequence elements
General principles of activator/repressor motifs:
Motif matters: Scrambling, removing, or disrupting predicted activator motifs abolishes enhancer activity
PWM matters: Silent/positive changes maintain activity
Context matters: (1) Conservation, (2) nucleosome exclusion, (3) binding of other TFs, (4) motif strength
Repressors matter: help activators maintain specificity by repressing enhancer activity in aberrant cell types
General methodology for enhancer motif dissection

36. Acknowledgements Jason Ernst Alexandre Melnikov Peter Rogov Li Wang
Xiaolan Zhang
Jessica Alston
Brad Bernstein
Tarjei S. Mikkelsen
Manolis Kellis