Dissection of regulatory motifs in 2,000 human enhancers using a massively parallel reporter assay

Pouya Kheradpour Kellis Lab In collaboration with Mikkelsen Lab

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 2000+ 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

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 2000+ 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

Identifying conserved motif instances

mutations movement missing short branches CTCF

BLS = 2.23

sps (78%)

Allows for:

1. Mutations permitted by motif degeneracy 2. Misalignment/movement of motifs within window (up to hundreds of nucleotides) 3. Missing motif in dense species tree

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

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 2000+ 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

Combinations of chromatin marks define Chromatin States

Ernst, Kheradpour, et al. Nature 2011

Coordinated activity reveals activators/repressors 1. Oct4 predicted activator of embryonic stem cells 2. Gfi1 predicted repressor K562/GM12878 cells Ernst, Kheradpour, et al. Nature 2011

Coordinated activity reveals activators/repressors

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

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 2000+ 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

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

Motifs predicted to be functional in HepG2 and K562 cells selected

Model for cell line specific motifs

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 2000+ 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

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.

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)

HepG2 K562 Same cell type + scramble + other manip (x7) Opposite cell type + scramble + other manip (x7)

15 18 18 0

Activators

HNF1, HNF4, FOXA GATA, NRF2 160 160

Repressors

ZFP161 GFI1 18 18 0 160 160 15 No. tested instances (x2 for ignoring/high conservation)

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 2000+ 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

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

Ex. activator: conserved HNF4 motif match WT expression specific to HepG2 to motif match expression to background Non-disruptive modifications maintain expression

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

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

• • • • 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

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

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 2000+ 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

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

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

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

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 2000+ 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

Model of GFI1 activity in HepG2 / K562 enhancers

Ex. GFI1 match in HepG2 enhancer

Disruption leads to increase in K562 expression

• • 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

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 2000+ 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

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

Jason Ernst

Acknowledgements

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