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ChIP-seq and related applications. I dentifying regulatory functions 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:

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ChIP-seq and related applications


Identifying regulatory functions 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)


Identifying regulatory functions in genomes

Noonan and McCallion, Ann Rev Genomics Hum Genet 11:1 (2010)


Biochemical indicators of regulatory function

1. TF binding

2. Histone

modification

  • H3K27ac

  • H3K4me3

3. Chromatin

modifiers &

coactivators

p300

MLL

4. DNA looping

factors

cohesin


Regulatory functions are tissue/cell type/time point-specific

From Visel et al. (2009) Nature 461:199


I point-specificdentifying regulatory functions in genomes

Chr5: 133,876,119 – 134,876,119

Genes

Transcription

Histone mods

TF binding


Methods point-specific

ChIP-seq

Chromatin accessibility

TFs

Histone mods

DNase

FAIRE

From Furey (2012) Nat Rev Genet 13:840


ChIP-seq point-specific

ChIP

Peak call

Signal

Input

Align reads to reference

Use peaks of mapped reads to

identify binding events

PCR


Calling peaks in point-specificChIP-seq data

ChIP

Peak call

Enrichment

relative to control

Input

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


There are many point-specificChIP-seq peak callers available

Wilbanks and Facciotti PLoS ONE 5:e11471 (2010)


Generating point-specificChIP-seq peak profiles

  • Artifacts:

  • Repeats

  • PCR duplicates

From Park (2009) Nat Rev Genet 10:669


Assessing statistical significance point-specific

Assume read distribution follows a

Poissondistribution

Many sites in input data will have some

reads by chance

Some sites will have many reads

# 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 vsChIP

From Pepke et al (2009) Nat Meth 6:S22


Assessing statistical significance point-specific

Sequencing depth matters:

# of reads at a site (S)

From Park (2009) Nat Rev Genet 10:669


ChIP-seq point-specific signal profiles vary depending on factor

Transcription

factors

Pol II

Histone

mods

From Park (2009) Nat Rev Genet 10:669


Quantitative analysis of point-specificChIP-seq signal profiles

HeLa

K562

HeLa

Sites strongly

marked in HeLa

Sites

strongly marked

in both

Clustering

Signal at 20,000 bound sites

ChIP-seq

signal

Sites

strongly

marked

in K562


ChIP-seq point-specific analysis workflow

From Park (2009) Nat Rev Genet 10:669


Interpreting point-specificChIP-seq datasets

  • Requires some prior knowledge

  • TF function

  • Histone modification

  • Potential target genes

  • Exploit existing annotation

  • Promoter locations

  • Known binding sites

  • Known histone modification maps



CTCF and cohesin co-occupy many sites point-specific

Promoters

Insulators

Enhancers

From Kagey et al (2010) Nature 467:430


P point-specificromoter

Enhancers?

CTCF: marks insulators and promoters

RAD21 (cohesin): marks insulators, promoters and enhancers


Discovering regulatory functions specific to a biological state

Limb

Brain

Function?

Assign enhancers to genes based on proximity (not ideal)

GREAT: bejerano.stanford.edu/great/

Gene ontology annotation assigned to regulatory sequences


TF motif elicitation from stateChIP-seq data

CTCF

~20,000 binding sites identified by ChIP:

MEME suite:

http://meme.nbcr.net/meme/

From Furey (2012) Nat Rev Genet 13:840


Single TF binding events may not indicate regulatory function

  • Many TFs are present at high concentrations

  • in the nucleus

  • TF motifs are abundant in the genome

  • Single TF binding events may be incidental

Enhancer-associated

histone modification


Mapping chromatin accessibility function

DNase I

FAIRE

From Furey (2012)

Nat Rev Genet

13:840


DNase function I hypersensitivity identifies TF binding events

From Furey (2012) Nat Rev Genet 13:840


DNase function I hypersensitivity identifies regulatory elements

DNase I hypersensitive sites

Song et al., Genome Res 21:1757 (2011)


De novo functionTF motif discovery by DNase I hypersensitivity mapping

In human ES cells:

From Neph (2012) Nature 489:83


De novo TF motif discovery by functionDNase I hypersensitivity mapping

Across tissue types:

From Neph (2012) Nature 489:83


Summary function

  • Relevant overview papers on ChIP-seq and DNase-seq posted on class wiki

  • Monday: Epigenetics and the histone code

  • Wednesday: Regulatory architecture of the genome


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