Chromatin Immunoprecipitation - Chip • Chromatin Immunoprecipitation (Chip) Is the Only Method for Large-Scale Identification of Direct Transcriptional Targets

BioSci D145 Lecture #7

• Bruce Blumberg ([email protected]) – 4103 Nat Sci 2 - office hours Tu, Th 3:30-5:00 (or by appointment) – phone 824-8573

• TA – Angela Kuo ([email protected]) – 4311 Nat Sci 2– office hours W 10-12 – Phone 824-6873

• Updated lectures (with answers) will be posted after lecture – http://blumberg-lab.bio.uci.edu/biod145-w20120

• Midterms graded and key posted – Mean 24.8 ± 4.4 – High 31.5, low 16.5 – Those with grades < 20 should come talk with me

• Term paper outlines commented on and uploaded to Canvas

Which genes regulate what other genes? • The biggest defect of expression microarrays or RNA-seq transcript profiling is that neither can distinguish direct targets from indirect targets – Which genes are a primary response to the treatment vs. – Which ones have one or more intermediates? • How can we approach and solve this important problem? – Identify the genes to which a transcription factor binds – Identify to which genes RNA polymerase II is recruited • And from which it is dismissed • All eukaryotic DNA occurs as chromatin (DNA+histone and other proteins) – Chromatin conformation influences whether transcription can occur

— Open chromatin is accessible to transcriptional machinery • DNA is unmethylated • Histones are methylated and acetylated (acetylase activates) • Many transcriptional co-activators methylate and acetylate histones and other chromatin associated proteins − Opens the chromatin

BioSci D145 lecture 6 page 3 ©copyright Bruce Blumberg 2004-2016. All rights reserved Which genes regulate what other genes? — Closed chromatin is inaccessible to transcriptional machinery • DNA is methylated • Some histone tails are methylated, others not • Transcriptional co-repressors recruit histone deacetylases (HDACs) which lead to chromatin condenstation • Chromatin condensation leads to gene silencing

• Identification of chromatin-localized proteins is diagnostic for direct target genes of transcription factors − Most common application

• Identification of promoters to which RNA Pol II is recruited upon some treatment is diagnostic for genes directly upregulated by the treatment − Trickier but very useful

• Identification of promoters from which RNA Pol II is dismissed upon some treatment is diagnostic for genes downregulated by the treatment

BioSci D145 lecture 6 page 4 ©copyright Bruce Blumberg 2004-2016. All rights reserved Chromatin immunoprecipitation - ChIP • Chromatin ImmunoPrecipitation (ChIP) is the only method for large-scale identification of direct transcriptional targets

• General strategy – Crosslink proteins to nearby DNA with formaldehyde • Works for about 2 angstrom distances • What does this say about the specificity of the interaction? — Only specific interactions will be reflected in crosslinks

BioSci D145 lecture 6 page 5 ©copyright Bruce Blumberg 2004-2016. All rights reserved Chromatin immunoprecipitation - ChIP • ChIP - general strategy (contd) – Break chromatin into small chunks (sonicating is most common method) – Typically want ~500 bp fragments – Evaluate fragmentation quality and extent by gel electrophoresis to ensure that size range is obtained • Needs MUCH optimization

• ChIP - general strategy (contd) – Precipitate chromatin with antibody against protein of interest – Requires excellent, specific, well-characterized antibody!

• Bind antibody, then capture complex with protein G/protein A beads • Reverse crosslinks – and remove proteins with proteinase K digestion • Purify DNA away from proteins • Evaluate enrichment of individual candidate binding sites by PCR

BioSci D145 lecture 6 page 7 ©copyright Bruce Blumberg 2004-2016. All rights reserved Chromatin immunoprecipitation - ChIP • Flavors of ChIP commonly in use – Standard ChIP – one antibody, few targets analyzed • Most commonly used method – ChIP-chip – chromatin immunoprecipitation on chip • Recovered fragments are used to probe microarray of genomic DNA • Allows identification of novel binding sites • Requires good genomic microarrays — Whole genome requires MANY chips (at least 7 for human and mouse) = EXPENSIVE — may not be available for your target organism — Affymetrix, Agilent, Nimblegen are sources

• Flavors of ChIP commonly in use – ChIP-sequencing (ChIP-seq) – chromatin immunoprecipitation sequencing • Massively parallel sequencing of recovered fragments • Unbiased method to identify transcription factor binding sites • Price now much less than ChIP-chip

BioSci D145 lecture 6 page 9 ©copyright Bruce Blumberg 2004-2016. All rights reserved Chromatin immunoprecipitation – other applications • Various methods of probing chromatin structure with ChIP, etc – Chromatin conformation capture and related methods – useful to study spatial organization of chromosomes (mostly looping) and how this influences gene expression • 3C – chromatin conformation capture – Capture-C (most modern version) • 4C – circularized chromosome conformation capture • 5C – carbon-copy chromosome conformation capture • ChIP-loop • Hi-C (first truly genome wide 3C method)

– ChIA-PET – chromatin interaction analysis by paired-end tag sequencing • Useful to detect de novo long-range chromatin interactions

– Methods differ in how much you must sequence to get the information you need to evaluate structure.

BiSci D145 lecture 6 page 11 ©copyright Bruce Blumberg 2009. All rights reserved Chromatin immunoprecipitation – other applications • Various methods of probing chromatin structure with ChIP, etc – Chromatin conformation capture and related methods – useful to study spatial organization of chromosomes (mostly looping) and how this influences gene expression • 3C – chromatin conformation capture (Capture-C) • 4C – circularized chromosome conformation capture • 5C – carbon-copy chromosome conformation capture • ChIP-loop • Hi-C (first truly genome wide 3C method)

• Probe open chromatin – DNAse I-seq, Mnase-seq more sequences (higher counts) at promoters – FAIRE-seq – formaldehyde- assisted isolation of regulatory elements higher counts at non- promoters – ATAC-seq - Assay for Transposase-Accessible Chromatin with deep sequencing (easiest, most sensitive, least material needed)

• Probe open chromatin – DNAse I-seq, Mnase-seq more sequences (higher counts) at promoters – FAIRE-seq – formaldehyde- assisted isolation of regulatory elements higher counts at non-promoters – ATAC-seq - Assay for Transposase-Accessible Chromatin with deep sequencing (most sensitive, least material needed)

• Epigenetics involves changes in gene expression without changes in the DNA sequence – Heritable, maintained – Reversible – Encoded in chromatin – Cellular memory

• What are some examples of epigenetic phenomena ? – X inactivation – Genomic imprinting – Cancer – widespread silencing or overexpression of genes

• Methylation (inactivates)/demethylation of DNA, proteins • Acetylation (activates)/deacetylation of DNA-binding proteins • Changes can act at very long range, 100s of kb to mb – from other chromosomes!

• Chromatin conformation affects accessibility of DNA to transcriptional machinery • Widespread changes in DNA methylation can be associated with diseases, e.g., cancer

BioSci D145 lecture 7 page 20 ©copyright Bruce Blumberg 2004-2016. All rights reserved Genetics and epigenetics of disease • Some genetic diseases – Sickle cell anemia – Cystic fibrosis – Hemophilia – Marfan syndrome – Duchenne muscular dystrophy – Huntington’s disease

• Diseases with an epigenetic component (from twin studies) – Fragile X syndrome – Prader-Willi syndrome – Angelman’s syndrome – Autism – Schizophrenia – Inflammatory bowel disease – Various types of cancer

BioSci D145 lecture 7 page 21 ©copyright Bruce Blumberg 2004-2016. All rights reserved The DOHaD Hypothesis • Barker Hypothesis - gestational under-nutrition leads to a thrifty phenotype – reduced fetal growth is strongly associated with many cardiovascular disease and other related syndromes. – Increased susceptibility results from adaptations made by the fetus in an environment limited in its supply of nutrients

• Developmental Origins of Health and Disease (Mark Hanson, Peter Gluckman) more generally proposes that development is very sensitive to perturbations that lead to permanent changes in susceptibility to disease – Birth defects, low birth weight, premature birth – Functional changes – individual appears normal but has molecular abnormalities that persist and lead to increased disease sensitivity later in life

• Diseases with Developmental Origins (from animal models) – Cardiovascular, Pulmonary (asthma) – Neurological (ADHD, Neurodegenerative diseases), – Immune/autoimmune – Endocrine, reproductive/fertility, cancer – Obesity/diabetes

“How We're Already Killing Our Grandkids”

“Sins of the fathers, and their fathers”

“Men inherit hidden cost of Three generations from the dad's vices” Överkalix parish of Norrbotten

A single winter of overeating could lead to a 6 (actual) to 32 (adjusted for socioeconomic status) year decrease in longevity of paternal grandchildren

• Maternal care – licking studies in rats – (maternal) stress

• Diet - folic acid, vitamins B2, B6, B12 (influence DNA methylation)

• Other dietary factors – e.g., phytoestrogens

• Toxins - heavy metals, arsenic, tobacco

• Maternal caloric intake (paternal, too)

• Changes in DNA methylation responsible

• Epigenome – changes in chromatin states that may vary from cell to cell • Epigenetics – all processes that lead to heritable changes in gene expression (during development or across generations) – Without changes in DNA sequence – Usually involves changes in chromatin therefore epigenetic inheritance often involves epigenomics.

• What are some ways that epigenetic inheritance can be transmitted? – Changes in DNA methylation – Changes in histone methylation/retention – Changes in ncRNA partitioning – lncRNAs – Small RNAs – Modified tRNAs – Changes in chromatin accessibility – Changes in 3D-chromatin structure

BioSci D145 lecture 7 page 27 ©copyright Bruce Blumberg 2004-2016. All rights reserved Genome-wide de-methylation and re-methylation • Germ cell DNA methylation is erased and then re-established – Controversial whether some regions are resistant to demethylation – Consensus is that some regions are probably resistant (imprinted loci) • How is specific pattern of DNA methylation re-established? – Template use is unlikely based on Seisenberger paper. • How are changes in methylation propagated across generations?

• DNA methylation – Methylation sensitive restriction digestion (simple, fast, inexpensive) • DNA treated with bisulfite - converts C to U • Replication replaces U with T • Digest with restriction enzyme that does not cut methylated DNA • Evaluate products – Gel electrophoresis – QPCR

• DNA methylation – Methylation sensitive restriction digestion (simple, fast, inexpensive) • Genome wide variation – cut with frequent digesting enzyme e.g, MseI – Add linker sequences with PCR primer – Cut with methylation sensitive restriction enzyme – PCR with primers – only detects sequences with methylated DNA between primers – Analyze by microarray or nextgen sequencing

• DNA methylation – bisulfite sequencing (unmethylated C residues converted to U) • Directly sequence products of bisulfite treatment and infer degree of methylation • Mass spectrometric analysis of results • Massively parallel sequencing • 3rd generation sequencing (Pacific Biosciences and Nanopore) can (supposedly) directly detect methylated DNA – Whole genome bisulfite sequencing is often suggested as the way to identify altered methylation • Assumes (incorrectly) that all unmethylated cytosines are converted to uracil

BioSci D145 lecture 7 page 32 ©copyright Bruce Blumberg 2004-2016. All rights reserved Techniques to study epigenetics • DNA methylation – MeDIP – methylated DNA immunoprecipitation • Denature DNA, sonicate and immunoprecipitate with anti-methyl cytosine antibody • Identify sequences precipitated by microarray, nextgen sequencing – Methylminer-seq (MBD-seq) • Uses methylated DNA binding protein to capture meDNA

BioSci D145 lecture 7 page 33 ©copyright Bruce Blumberg 2004-2016. All rights reserved Techniques to study epigenetics • DNA methylation – MeDIP – methylated DNA immunoprecipitation – seq AND Methylminer-seq (MBD-seq) have a bias toward regions that are highly methylated and against sparsely methylated regions – Whole genome bisulfate sequencing is expensive and may not be necessary – One approach to reduce this bias is to use MBD/MeDIP-seq to identify regions of altered methylation then use bisulfite sequencing to get nucleotide-level resolution • Bisulfite treat DNA and cut with restriction enzyme • PCR amplify region of interest • Sequence using pyrosequencing to identify which cytosines have been converted to uracil

• Histone modification – Histones are considerably modified at many places and in a combinatorial fashion (many permutations) • Methylation • Phosphorylation • acetylation – Only methylation is heritable! – Methods of analysis • ChIP – precipitate chromatin with antibodies specific to particular methylation – Analyze by microarray – ChIP-CHIP – Or by high throughput sequencing – ChIP-seq

BioSci D145 lecture 7 page 35 ©copyright Bruce Blumberg 2004-2016. All rights reserved H3K4me3 – pro-transcription (open) H3K4me1 (enhancers) H3K9me3 – anti-transcription (alone) H3K27me3 – anti-transcription H3K27ac – pro-transcription meCpG – anti-transcription

BioSci D145 lecture 7 page 36 ©copyright Bruce Blumberg 2004-2016. All rights reserved “Open” chromatin takes multiple forms • Active promoters – H3K4me3 present – H3K4me1 at enhancers – H3K27ac at enhancer – DNAse I hypersensitive – eRNA transcribed at enhancers • Primed promoters (inactive, but receptive to activation) – H3K4me3 absent – H3K4me1 present at enhancers – 5hmC, 5mC present at enhancers – Pioneer factors may be present • Poised promoters (inactive and ready to be activated) – H3K4me3 at promoter, no tx – H3K4me1, H3K27me3 in enhancers – DNAse I hypersensitive enhancer – eRNA not transcribed