Methods for Chromatin Analysis— Precipitating Protein, RNA & DNA from Chromatin John M. Rosenfeld,...

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…


https://www.encodeproject.org/

Next Gen Sequencing Drives Discovery in Chromatin Analysis


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

Confidential & Property of EMD Millipore Corporation

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


Interrogating Interactions in Chromatin


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


Results for Dot Blot and ChIP-seq Screen


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


A method to enhance low input ChIP-seq for Trans Factors

Patented technology outperforms Nextera& NEB in library complexity, duplicate reads, & input range


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


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.


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


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


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”


ChIRP-Mass Spec for Proteomic Discovery

Cell, 2015

m6A—Reversible Modification of RNAs


Found in many types of RNAMtase & demethylases knownInvolved in RNA stability, splicingchoices

meRIP of methylated RNA


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


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

Methods for Chromatin Analysis— Precipitating Protein, RNA & DNA from Chromatin John M. Rosenfeld,...

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