genome widetargets of dna binding molecules: from seq to
TRANSCRIPT
![Page 1: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/1.jpg)
Genome‐wide targets of DNA‐binding molecules: from ChIP‐Seq to Chem‐Seq
Mario Nuvolone
Technical Journal Club
May 05th 2015
![Page 2: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/2.jpg)
DNA‐binding proteins
DNA‐binding small molecules
ChIPChIP‐on‐chipChIP‐seq
Chem‐seq
![Page 3: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/3.jpg)
DNA‐binding proteins
‐ Include histones, transcription factors, DNA polymerases, RNA polymerases, DNA nucleases etc.
‐ Play crucial roles in many cellular processes (transcription, splicing, replication, DNA repair etc.)
Furey Nat Rev Genet 2012Schones Nat Rev Genet 2008
![Page 4: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/4.jpg)
Mapping DNA‐binding proteins
‐ Bound genomic locations in a particular cell type cannot be predicted using DNA sequence features alone
‐ Functional assays are necessary
‐ Mapping binding sites is vital to study epigenome and regulatory networks underlying different biological processes
Furey Nat Rev Genet 2012Park Nat Rev Genet 2009
![Page 5: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/5.jpg)
![Page 6: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/6.jpg)
Chromatin immunopurification (ChIP)
Solomon et al. Cell 1988
![Page 7: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/7.jpg)
Collas Methods Mol Biol 2009
Evolution of ChIP: ChIP‐on‐chip
"Chromatin immunoprecipitation"
Year# Pub
Med
Entrie
s
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
0
500
1000
1500
2000
ChIP‐on‐chip
![Page 8: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/8.jpg)
![Page 9: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/9.jpg)
ChIP‐on‐chip
Park Nat Rev Genet 2009Schones Nat Rev Genet 2008
![Page 10: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/10.jpg)
Collas Methods Mol Biol 2009
Evolution of ChIP: ChIP‐seq
"Chromatin immunoprecipitation"
Year# Pub
Med
Entrie
s
1998
1999
2000
2001
2002
2003
2004
2005
2006
2007
2008
2009
2010
2011
2012
2013
2014
2015
0
500
1000
1500
2000
ChIP‐seq
![Page 11: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/11.jpg)
ChIP‐seq
Furey Nat Rev Genet 2012
Landt et al. Genome Res 2012
![Page 12: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/12.jpg)
Park Nat Rev Genet 2009
ChIP‐seq: peak identification
‐ Fragments are sequenced at the 5′ end
‐ Locations of reads form two distributions (+ and – strand)
‐ Combined profiles used to identify peak
![Page 13: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/13.jpg)
Park Nat Rev Genet 2009
ChIP‐seq: peak identification
![Page 14: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/14.jpg)
Park Nat Rev Genet 2009
ChIP‐seq: types of peaks
Sharp binding sites
Mixed peaks
Medium size broad peaks
Large size broad peaks
Insulators
Transcribed regions
Transcription elongation
Transcription repression
![Page 15: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/15.jpg)
ChIP‐on‐chip vs ChIP‐seq
Park Nat Rev Genet 2009
*
* Now reduced→ ChIP‐seq:higher resolution fewer artefactsgreater coverage larger dynamic range
![Page 16: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/16.jpg)
ChIP‐seq: issues in experimental design
Park Nat Rev Genet 2009
‐ Antibody quality
‐ Sample quantity
‐ Control experiment
‐ Sequencing
‐ Data analysis
‐ Downstream analyses
![Page 17: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/17.jpg)
ChIP‐seq: Antibody quality
Park Nat Rev Genet 2009
‐ Crucial determinant of any ChIP experiment
‐ Sensitivity and specificity of antibody influence enrichment and peak detection
‐ Batches differences in commercial antibody
‐ Need for rigorous validation
![Page 18: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/18.jpg)
![Page 19: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/19.jpg)
Landt et al. Genome Res 2012
ChIP‐seq: Antibody quality
![Page 20: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/20.jpg)
ChIP‐seq: Sample quantity
Park Nat Rev Genet 2009
‐ Lower amount of DNA required(ChIP‐on‐chip: > 2μg; ChIP‐seq: 10‐50 ng)
‐ Fewer rounds of amplifica on → Less PCR bias
‐ Required amount depends on:
‐ Abundance of target protein
‐ Quality of antibody
![Page 21: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/21.jpg)
ChIP‐seq: Control experiments I
Park Nat Rev Genet 2009
‐ Potential sources of artefacts:‐ Euchromatin vs heterochromatin‐ Repetitive regions
‐ Controls are required
‐ Commonly used controls:‐ Input DNA‐ Mock IP DNA‐ DNA from non‐specific IP
![Page 22: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/22.jpg)
ChIP‐seq: Control experiments II
Furey Nat Rev Genet 2012Landt et al. Genome Res 2012
‐ Experimental replication:
‐ Minimum of two experimental replicates
‐ Biological rather than technical replicates
‐ For duplicates:‐ 80% of the top 40% of identified targets in one replicate must be replicated
OR‐ 75% of targets lists must be in common between both replicates
![Page 23: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/23.jpg)
ChIP‐seq: Sequencing I
Park Nat Rev Genet 2009
‐ Higher sequence depth allows detection of more sites with lower level of enrichment over background
![Page 24: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/24.jpg)
ChIP‐seq: Sequencing II
Park Nat Rev Genet 2009
‐ Multiplexing at the sequencing stage is possible
‐ Paired‐end sequencing for particular applications
![Page 25: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/25.jpg)
ChIP‐seq: Data analysis I
Furey Nat Rev Genet 2012Park Nat Rev Genet 2009
‐ Sequence alignment (some mismatches allowed)
‐ Identification of enriched reagions (peaks)
‐ Data quality assessment
‐ Fractions of reads in peaks (FRiP) >1%
‐ Correlations of sequence reads from + and ‐ strands
![Page 26: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/26.jpg)
Furey Nat Rev Genet 2012
ChIP‐seq: Data analysis II
![Page 27: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/27.jpg)
ChIP‐seq: Downstream analyses
Park Nat Rev Genet 2009
![Page 28: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/28.jpg)
→ Introduc on of ChIP‐seq
![Page 29: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/29.jpg)
Johnson et al. Science 2007
‐ Jurkat human T lymphoblast cell line
‐ IP with anti‐human neuron‐restrictive silencer factor (NRSF/REST)
‐ Validated target genes
‐ Known DNA motif bound (NRSE)
‐ Additional predicted target genes
‐ High‐quality monoclonal antibody validated for ChIP
‐ Two replicate IPs (one with and one without PCR amplification)
‐ Illumina sequencing on two IPs and two DNA input controls
Study design
![Page 30: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/30.jpg)
Johnson et al. Science 2007
‐ 2‐5 x106 25bp sequences per sample
‐ Removal of reads with multiple sites
‐ Alignment allowing up to 2 mismatches
‐ Generation of ChIPSeq peak locator for peaks identification
‐ Peaks required to have ≥5 fold enrichment vs input DNA control and ≥ 13 reads
![Page 31: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/31.jpg)
Johnson et al. Science 2007
‐ #Reads/peak: from 13 to 6718
‐ Most peaks contains one canonical NRSE binding motif
‐ 83 NRSF‐binding sites previously identified (true positives)
‐ 130 known negatives (true negatives)
‐ 87% sensitivity‐ 98% specificity
Sensitivity
1‐Specificity
![Page 32: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/32.jpg)
Johnson et al. Science 2007
‐ Based on 771 computationally identified NRSF binding sites which were positive also at ChIP‐seq
‐ Distance <50 bp in 94% cases
‐ Based on all computationally identified NRSF binding sites
‐ Virtually all strong canonical NRSF binding sites were detected
![Page 33: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/33.jpg)
![Page 34: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/34.jpg)
![Page 35: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/35.jpg)
→ Introduc on of HiC to study 3D architectures of whole genomes
![Page 36: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/36.jpg)
Lieberman‐Aiden et al. Science 2009
Study design
‐ Karyotypically normal lymphoblastoid cell line (GM06990)
‐ Illumina sequencing with (paired end)
![Page 37: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/37.jpg)
Lieberman‐Aiden et al. Science 2009
‐ 8.4 x 106 read pairs mapped to human genome
‐ 6.7 x 106 read pairs correspond to long‐range contacts (>20 kb apart or interchromosomal)
‐ Genome‐wide contact matrix M:‐ 1 Mb regions (loci)‐ Matrix entry mij: #ligation products between locus i and locus j
![Page 38: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/38.jpg)
Lieberman‐Aiden et al. Science 2009
‐ Contact probability:intrachrom >> interchrom(also at high distances)
‐ Small, gene‐rich chromosomes preferentially interact with each other
→ Chromosome territories
![Page 39: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/39.jpg)
Lieberman‐Aiden et al. Science 2009
![Page 40: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/40.jpg)
→ Introduc on of Chem‐seq
![Page 41: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/41.jpg)
Anders et al. Nat Biotechnol 2014
Study design
‐ MM1.S multiple myeloma cell line
‐ Bromodomain inhibitor JQ1, known to bind the BET bromodomainfamily members BRD2, BRD3 and BRD4
![Page 42: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/42.jpg)
Anders et al. Nat Biotechnol 2014
Bio‐JQ1: biotinylated derivative of JQ1
Only slightly reduced bioactivity on MM1.S cells
![Page 43: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/43.jpg)
Anders et al. Nat Biotechnol 2014
‐ Similar results with Chem‐Seqwith bio‐JQ1 in vivo and in vitro
‐ Similar profile with ChIP‐seqwith BDR2, BDR3 and BDR4
![Page 44: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/44.jpg)
Anders et al. Nat Biotechnol 2014
No significant binding with bio‐JQ1R
![Page 45: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/45.jpg)
Anders et al. Nat Biotechnol 2014
JQ1 occupancy of chromatin is most strongly correlated with that of BRD4 in MM1.S cells
![Page 46: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/46.jpg)
![Page 47: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/47.jpg)
![Page 48: Genome widetargets of DNA binding molecules: from Seq to](https://reader033.vdocuments.site/reader033/viewer/2022042614/6263d9ff007a253c307a988b/html5/thumbnails/48.jpg)
DNA‐binding proteins
DNA‐binding small molecules
ChIPChIP‐on‐chipChIP‐seq
Chem‐seq