1. Methods to read out regulatory functions
Regulomics I:
Methods to read out regulatory functions

2. Identifying regulatory functions in genomes
Merge into general discussion of regulatory space - regulomics
Noonan and McCallion, Ann Rev Genomics Hum Genet 11:1 (2010)

3. Genes are not just protein coding sequences
Expression of
gene A
gene A
limb
Limb TFs
gene A
forebrain
gene A
Brain TFs
Tissue specific TF
neural tube
gene A
Neural TFs

4. Regulatory mutations can cause
profound phenotypes
Lettice et al. Hum Mol Genet 12:1725 (2003)
Sagai et al. Development 132:797 (2005)

5. Three essential questions
Q1: Where are regulatory elements located in the genome?
Q2: What regulatory functions do they encode?
Q3: What genes do they control?
We will use promoters and enhancers as our examples, but there are other
regulatory functions

6. Q1: Mapping regulatory elements in genomes
Chr5: 133,876,119 – 134,876,119
Genes
Transcription
Regulatory elements are not easily detected by sequence analysis
Examine biochemical correlates of RE activity in cells/tissues:
Chromatin Immunoprecipitation (ChIP-seq)
DNase-seq and FAIRE
Methylated DNA immunoprecipitation (MeDIP)

7. Biochemical indicators of regulatory function
1. TF binding
2. Histone
modification
H3K27ac
H3K4me3
3. Chromatin
modifiers &
coactivators
p300
MLL
4. DNA looping
factors
cohesin

8. Methods ChIP-seq Chromatin accessibility TFs Histone mods DNase FAIRE
From Furey (2012) Nat Rev Genet 13:840

9. Method I: ChIP-seq ChIP Input Peak call Signal
Align reads to reference
Use peaks of mapped reads to
identify binding events
PCR

10. Calling peaks in ChIP-seq data
Input
Peak call
Enrichment
relative to control
Highlight the challenges for both ChIP and RNA-seq in both protocols
ChIP-seq is an enrichment method
Requires a statistical framework for determining the significance of enrichment
ChIP-seq ‘peaks’ are regions of enriched read density relative to an input control
Input = sonicated chromatin collected prior to immunoprecipitation

11. There are many ChIP-seq peak callers available
Wilbanks and Facciotti PLoS ONE 5:e11471 (2010)

12. Generating ChIP-seq peak profiles
Artifacts:
Repeats
PCR duplicates
From Park (2009) Nat Rev Genet 10:669

13. Assessing statistical significance
Assume read distribution follows a
Poisson distribution
Many sites in input data will have some
reads by chance
Some sites will have many reads
Poisson assumption + seq depth
# of reads at a site (S)
Empirical FDR: Call peaks in input (using ChIP as control)
FDR = ratio of # of peaks of given enrichment value called in input vs ChIP
From Pepke et al (2009) Nat Meth 6:S22

14. Assessing statistical significance
Sequencing depth matters:
Poisson assumption + seq depth
# of reads at a site (S)
From Park (2009) Nat Rev Genet 10:669

15. ChIP-seq signal profiles vary depending on factor
Transcription
factors
Pol II
Histone
mods
From Park (2009) Nat Rev Genet 10:669

16. Mapping chromatin accessibility
DNase I
FAIRE
From Furey (2012)
Nat Rev Genet
13:840

17. DNase I hypersensitivity identifies
regulatory elements…
DNase I hypersensitive sites
Case studies: TFs
Which?
Oct4?
CTCF?
Song et al., Genome Res 21:1757 (2011)

18. …but needs to be combined with other data to
determine what is actually bound – such as TF ChIP…
DHS signal in GM12878
RNA PolII ChIP
in GM12878

19. … or motif analysis DHS sites in human ES cells:
From Neph (2012) Nature 489:83

20. Q2: Making sense of regulatory functions
Compare multiple biological states
Integrate multiple data sources
TF function
Histone modification
Potential target genes
Existing genome annotations

21. Regulatory function is dependent on biological
context
forebrain
gene A
Brain TFs
neural tube
Neural TFs
limb
Limb TFs

22. Identifying tissue-specific regulatory function
Limb
Brain
Limb
Sites strongly
marked in Limb
Sites
strongly marked
in both
ChIP-seq
signal
Signal at 20,000 bound sites
Clustering
signal
Sites
strongly
marked
in Brain

23. Identifying tissue-specific regulatory function
Limb
Brain
Function?
Assign enhancers to genes based on proximity (not ideal)
GREAT: bejerano.stanford.edu/great/
Gene ontology annotation assigned to regulatory sequences

24. Q2: Making sense of regulatory functions
Compare multiple biological states
Integrate multiple data sources
TF function
Histone modification
Potential target genes
Existing genome annotations

25. Example from PS1: CTCF and RAD21 (cohesin)
Annotate (GREAT)

26. CTCF and cohesin co-occupy many sites
Promoters
Insulators
Enhancers
From Kagey et al (2010) Nature 467:430

27. CTCF: marks insulators and promoters
Enhancers?
Annotate (GREAT)
CTCF: marks insulators and promoters
RAD21 (cohesin): marks insulators, promoters and enhancers?
Include histone modification data (Wednesday’s lecture)

28. Identifying bound motifs from ChIP-seq data
CTCF
~20,000 binding sites identified by ChIP:
GREAT
MEME suite:
From Furey (2012) Nat Rev Genet 13:840

29. Single TF binding events often do not indicate regulatory function
Caveat:
Single TF binding events often do not indicate regulatory function
Enhancer-associated
histone modification
Many TFs are present at high concentrations
in the nucleus
TF motifs are abundant in the genome
Single TF binding events may be incidental
Combinations of marks/TF binding events

30. Q3: Identifying the target genes for
regulatory elements
forebrain
gene A
Brain TFs
neural tube
Neural TFs
limb
Limb TFs

31. Chromosome Conformation Capture ChIP for specific factors: ChIA-PET
Sequence: 5C
Sequence: Hi-C
Sequence: 4C

32. 3C evaluates specific interaction possibilities by qPCR
Dekker et al Nat Rev Genet 14:390 (2013)

33. 4C identifies genome-wide interactions for a single
“bait” sequence

34. ChIA-PET identifies interactions involving a particular factor
From Kieffer-Kwon et al. (2013) Cell 155:1507

35. In principle, Hi-C captures all interactions, but is
limited by sequencing depth
Dekker et al Nat Rev Genet 14:390 (2013)

36. Hierarchical organization of the genome
Cohesin-mediated interactions
Dekker et al Nat Rev Genet 14:390 (2013)
Gorkin et al Cell Stem Cell 14:762 (2014)

37. Summary Relevant overview papers on methodologies posted on class wiki
Wednesday: Epigenetics and the histone code