methods for chromatin analysis— precipitating protein, rna & dna from chromatin john m....
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Methods for Chromatin Analysis—Precipitating Protein, RNA & DNA from Chromatin
John M. Rosenfeld, Ph.D.
External Innovation Manager
EMD Millipore
Temecula, CA
Genomics & Pharmacogenomics 2015
Confidential and Property of EMD Millipore Corporation
Chromatin Biology has come a long way…
1st gen ChIP Kit ~1999Fully disclosed recipes
And protocol
4th gen ChIP KitEvery major supplier offers one
Major Consortium Efforts in Genetics & Epigenetics--The Age of Omics?
https://www.encodeproject.orghttp://www.roadmapepigenomics.org/http://cancergenome.nih.gov/
WGS 1000 tumor types11,000 samples
111 tissue & cell types
Multi-omic
To 2015
From 2003…
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Next Gen Sequencing Drives Discovery in Chromatin Analysis
Confidential and Property of EMD Millipore Corporation
Nextgenseq.com
http://stemcellthailand.org/chromosomes/
ChIP-seqDNAse-seq
Gro-seqRIP-seq
CLIP-seqMethyl-seq
…
Transcription and Chromatin
Shlueyva, Nat. Rev. Genetics, 2014
AcH3K27H3K4me1
H3K4me3AcH3/H4
H3K4me1H3K27me3
Access to chromatin influenced by PRC2, MLL/Compass, Swi/Snf (BAF)Large number of mutations in components of these pathways
20% of sequenced tumors have BAF subunit mutations
EnhancersPromoters
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A cryptic 3rd component of chromatin--ncRNAs
• RNA-seq (Gro-seq) reveals in eukaryotes, genomes are pervasively transcribed
Kaikkonen et al., Cardiovascular Research, 2011
Enhancer transcripts (H3K4me1) mRNA transcript (GRO-seq)
Antisense Transcripts (GRO-seq)
• A small percentage of these transcripts encode for proteins, whereas the majority are transcribed as non-coding RNAs (miRNA, piRNA, PAR, eRNA, lncRNA)
• Non-coding transcripts are marked by histone modifications characteristic of enhancers
and promoters
Confidential and Property of EMD Millipore Corporation
Interrogating Interactions in Chromatin
Confidential and Property of EMD Millipore Corporation
Protein:DNAChIP-seq
Histone Mods, Chromatin modifiers,Transcription Factors
General Txn Machinery
Protein:RNANuclear RIP-seq
CLIP-seq
Modified DNAMethyl-seq (BS mC)
MeDIP-seq (MBD-seq mC)Ox-BS (HMC)
TAB-seq (HMC)
Modified RNAMeRIP (m6A)BS-Seq (mC)
ncRNA:DNAChIRP-seqChart-seqRAP-seq
Many require high quality, high affinity & specificity antibodiesEsteller, Scientist, 2011
A Screen for Antibody Specificity for ChIP-seq
H3K9me1
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Results for Dot Blot and ChIP-seq Screen
Confidential and Property of EMD Millipore Corporation
52 Antibodies(30 modifications)
35 Passed15 no signal
2 non-specific
Dot Blot
NGS Library
Prep
21 LibrariesPassed
NGS13 Antibodies
ChIP-seq>90%
Top Quartile
Multiple antibodies to same modification passed, both polyclonal & monoclonalTracks published on website and antibodies offered as Trial Size
Used Magna ChIP HiSens Kit and commercial NGS Library KitUtilized embedded positive controls and replicate testing
Cutoffs for “ChIP-seq” claim ≥ 90% in top quartile
Histone Mod
Sample IDTotal Reads
Mapped reads
% Map Peaks% ENCODE total overlap
% ENCODE overlap top
quartile
ChIP-seq Valid
Justification
H3K4 05-1341 16870837 8523220 51 61584 N/A N/A yes Peak comparison to H3K4mod
H3K4me2 ABE250 16392094 12840005 78 50210 81% 90% yesH3K4me2 04-790 19560881 17130225 88 39590 84% 92% yesH3K4me2 05-1338 22477959 16466050 73 59383 79% 90% yesH3K4me3 07-473 22121742 20045414 91 20195 91% 99% yesH3K4me3 07-473 20170331 17968795 89 20572 91% replicate (1 pg spike in)
H3K4me3 05-745R 20788887 18939160 91 18841 92% 99% yesH3K4me3 05-745R 19944362 18157798 91 18847 92% replicate (10 pg spike in)
H3K4me3 05-1339 25415820 22539277 89 29407 89% 99% yesH3K9Ac ABE18 17220771 13625114 79 45284 70% 95% yesH3K9Ac 06-942 30861680 27715279 90 26797 83% 98% yes
H3K9me1 97202 15934423 9680715 61 36328 N/A N/A no low mapping, low overlap
H3K9me1 95301 29243959 22452146 77 495 63% (HUV) 69% (HUV) no low peaks
H3K9me2 85207 28796997 18185660 63 1217 N/A N/A no low mapping, low peaks
H3K14Ac 04-1044 24674468 18088959 73 20317 N/A N/A yes comparison to H3K9Ac, K18Ac
H3K18Ac 07-354 14928546 10170672 68 82404 N/A N/A yes comparison to H3K9Ac, K14Ac
H3K27Ac 07-360 21695249 14651365 68 25361 61% 96% yesH3K27me3 92784 11008397 6400089 58 38857 83% 85% no low depth, low overlap
H3K36me3 ABE435 16904103 11527539 68 85606 91% 96% yesH3K36me3 87402 107345215 71297489 66 40887 65% 73% no contamination
H4K20me1 95828 28223510 18551401 66 24980 52% 62% no index error
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A method to enhance low input ChIP-seq for Trans Factors
Patented technology outperforms Nextera& NEB in library complexity, duplicate reads, & input range
Confidential and Property of EMD Millipore Corporation
Tools For Exploring ncRNA & mRNA Function
Nuclear RIP—Like ChIP, but for discovering RNAs
ChIRP—Pull down lncRNAs from chromatin and discover genomic sites of action as well as protein interactors
meRIP—analysis of RNA methylation
Protein:RNA
Methylation of RNAs
RNA:DNA and RNA:Protein Interactions
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Magna Nuclear RIP—Two Options for Interrogation
Magna Nuclear RIP (Native) vs. Magna Nuclear RIP (Cross-Linked). While both native and cross linked approaches are used to analyze chromatin associated RNA, native RIP typically allows recovery of high affinity, more direct interactions. Conversely, the cross-linking method is designed to capture higher molecular weight complexes and more readily trap weaker interacting RNAs.
Confidential and Property of EMD Millipore Corporation
Nuclear RIP for PRC2 Components
HOTAIR HOXC11
Genome Wide RNA-seq or Transcript Specific Measurement (qRT-PCR)Confidential and Property of EMD Millipore Corporation
Nuclear RIP for PRC2 Components
Genome Wide RNA-seq or Transcript Specific Measurement (qRT-PCR)Confidential and Property of EMD Millipore Corporation Nat. Struct. Mol. Biol. 2013
Chromatin Isolation by RNA Purification
Split-probe’ strategy eliminates off-target hybridization artifacts
Given ncRNA of interest, designbiotinylated probes to tile and immunoprecipitate from glutaraldehydecrosslinked chromatin
• Verify RNA recovery via qRT-PCR• Discover genomic sites of actionvia DNA-seq• Discover protein partners via Mass Spec
Precipitation of ncRNA—chromatincomplexes
Confidential and Property of EMD Millipore Corporation
Neat1 interacts with intronic sequence from another ncRNA with motifs for various transcription factors
Analyzed similar to ChIP-seq, can infer interacting proteins by overlaying ChIP-seq data sets
CandidateChromatin RBPs
Odd Pool
Even Pool
Input
Confidential and Property of EMD Millipore Corporation
Positive Control Data from Neat1 ChIRP-seq
Positive Control Region
Neat1 autoregulates it’s own transcriptionRef. Simon MD, et al. Proc Natl Acad Sci U S A. 2011 108(51):20497-502
NEAT1 ChIRPOdd
Input
NEAT1 ChIRPEven
Odd & Even datasets allow verification of probeDesign to eliminate “false positives”
Confidential and Property of EMD Millipore Corporation
ChIRP-Mass Spec for Proteomic Discovery
Cell, 2015
m6A—Reversible Modification of RNAs
Confidential and Property of EMD Millipore Corporation
Found in many types of RNAMtase & demethylases knownInvolved in RNA stability, splicingchoices
meRIP of methylated RNA
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Summary
NGS has spawned many interesting sample prep methods for epigenomic profiling, allowing dissection of chromatin regulation from many angles
ChIP-seq for transcription factors and chromatin modifiers—Pure Genome Library Construction
Nuclear RIP-seq for discovery of RNAs associated with Chromatin
ChIRP-seq for characterization of long non coding RNA interactions (also utilizes Pure Genome NGS kit)
meRIP-seq for identification of m6a modified RNAs
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Acknowledgements
Zirong Li, Ph.D. (ChIP-seq)
Kan Saito, Ph.D. (RIP-seq, ChIRP-seq & meRIP)
Konstantin Taganov, Ph.D (ChIP-seq, ChIRP-seq)
Tracy Cooke (ChIP-seq)
Jeremy Simon, Ph.D. UNC Chapel Hill, NGS Analytics
Michael Sturges, Ph.D.
Nick Asbrock
James Hoberg