Intro to EpigeneticsJason Gardiner

MCDB 191Winter 2020

How do specialized cell types with the exact same genotype develop

Every cell has a distinct epigenetic pattern

Epigenetics

Genetics

These “genes” can be turned on

This “gene” cannot be turned on

Jansen A , and Verstrepen K J Microbiol. Mol. Biol. Rev. 2011;75:301-320

What is chromatin?

A complex of both DNA and proteins that organizes and compacts DNA. This allows a human cell's ~2 meter long DNA (stretched end-to-end) to fit inside a 6 um nucleus.

https://www.nature.com/scitable/topicpage/dna-packaging-nucleosomes-and-chromatin-310/

Jansen A , and Verstrepen K J Microbiol. Mol. Biol. Rev. 2011;75:301-320

https://www.nature.com/scitable/topicpage/dna-packaging-nucleosomes-and-chromatin-310/

Jansen A , and Verstrepen K J Microbiol. Mol. Biol. Rev. 2011;75:301-320

Histone tail modifications

Keppler and Archer. Expert Opin Ther Targets 2009; doi: 10.1517/14728222.12.10.1301

K = lysineR = arginineS = serineT = threonine

Me = methylationAc = acetylationP = phosphorylationUb = ubiquitination

H3K9me

H2BK28ac

H3K27acThese modifications have different effects on the DNA-histone interactions, and can open or compact chromatin. There are also proteins that recognize these modifications and can affect expression.

Tail modificationsrepresent epigenetic

information

Histone variants and modifications - associated regions & effect on expression

Li et al. Cell 2007; 128: 707-719 Lim et al. Epigenomics 2010; 2:775-795.

Histone tail modifications

histone modifications are reversiblesome residues (lysine) can support

multiple methyl groups

https://web.stanford.edu/group/gozani/cgi-bin/gozanilab/research/

http://www.web-books.com/MoBio/Free/Ch4G.htm e.g. H3K9me3

Histone Variants

Millar (2013) Biochem. Journal

H2A.B-containingnucleosomes wrap only 118 bp of DNA instead of 146 bp and areless stable

Nucleosome Remodeling

DNA methylation and gene expression

gene

TF

gene

TF

TE

HMTs

HDACs

MBD

TE

😈😈

DNA methylation and gene expression

constitutive expression = gene is always onectopic expression = gene is on where it's not supposed to be

Dolinoy et al., 2008

DNA methylation and gene expression

constitutive expression = gene is always onectopic expression = gene is on where it's not supposed to be

cryptic promoter(active)

PolII

normal expressionAvy

constitutive, ectopicexpression

PolII

Avy

cryptic promoter(inactive)

IAPIAP

Dolinoy et al., 2008

Putting it all together

Greco and Condorelli (2015). Nat Rev Cardio

Chromatin marks and dynamics: DNA topology in the cell:

Stevens et al., (2017). Nature.

Key Techniques Frequently Employed in Epigenetics Research

• ChIP-seq : Tells you WHERE a protein is BOUND on the genome, including histone variants, transcription factors, etc.

• Bisulfite Sequencing : Tells you the PROPORTION of METHYLATEDcytosines in your sample

ChIP-seq : Chromatin Immunoprecipitation [coupled to] High throughput Sequencing

TF

Step zero - Native state in the cell, transcription factor is bound to DNA

TF

ChIP-seq

TF

Step One – crosslink protein to DNA

X X X XTF

X X X X

ChIP-seq

TF

Step two – shear the DNA (aka chromatin)

X X X XTF

X X X X

ChIP-seq

TF

Step three – use antibody (aka immuno) to bind the protein.

X X X XTF

X X X X

ChIP-seq Step three – use antibody (aka immuno) to bind the protein, wash away unbound DNA.

TFX X X X

ChIP-seq

TF

Step four – de-crosslink

ChIP-seq

TF

Step five – purify DNA (precipitation) and sequence

High throughput sequencing

Example of ChIP seq data

Modified from Michael R. Tallack et al. Genome Res. 2010;20:1052-1063

Histone variants and modifications - associated regions & effect on expression

Li et al. Cell 2007; 128: 707-719 Lim et al. Epigenomics 2010; 2:775-795.

Bisulfite-sequencing

(read as T by transcriptional machinery)

Wikipedia commons

Bisulfite-sequencing

Example of bisulfite sequencing data

Wasson et al. (2016) eLIFE

mCpG

Example of ChIP-seq and BisulphiteSequencing Data

Harris et al., 2018. Science

Summary of topics in this course

• 9 topics (1/week), 2 papers each

What will you choose?

Today DNA methylation

Histone methylation / demethylation

Polycomb RNA-directed silencing

DNA demethylation

Enhancers Targeted epigenetics

3D chromatin architecture

Phase transition + epigenetics

Week 2 - DNA methylation

• UHRF1 Plays a Role in Maintaining DNA Methylation in Mammalian Cells. (2007) Magnolia Bostick, Jong Kyong Kim, Pierre-Olivier Estève, Amander Clark, Sriharsa Pradhan, Steven E. Jacobsen. Science,317:1760-1764.

• Looks at mechanism for DNA methylation maintenance after replication

• Highly Integrated Single-Base Resolution Maps of the Epigenome in Arabidopsis. (2008) Lister, R. et al. Cell, 133:523-536.

• One of the first papers published that did whole-genome bisulfite-sequencing to look at DNA methylation genome-wide

Week 3 - Histone methylation/demethylation

• Methylation of Histone H3 lysine 9 creates a binding site for HP1 proteins. (2001) Monika Lachner, Dónal O'Carroll, Stephen Rea, Karl Mechtler and Thomas Jenuwein. Nature, 410:116-120.

• Important early paper linking a histone modification (H3K9me) to heterochromatin maintenance and gene expression

• JHDM2A, a JmjC-Containing H3K9 Demethylase, Facilitates Transcription Activation by Androgen Receptor. (2006) Kenichi Yamane, Charalambos Toumazou, Yu-ichi Tsukada, Heiye Erdjument-Bromage, Paul Tempst, Jiemin Wong, Yi Zhang. Cell, 125(3):483-495.

• Identified one of the first histone demethylases, showing that histone modifications are reversible

Week 4 - Polycomb

• Role of Histone H3 Lysine 27 Methylation in Polycomb-Group Silencing. (2002) Ru Cao, Liangjun Wang, Hengbin Wang, Li Xia, Hediye Erdjument-Bromage, Paul Tempst, Richard S. Jones, and Yi Zhang. Science, 298:1039-1043.

• Showed that Polycomb group (PcG) proteins (PRC2) methylate H3K27 to silence target genes and establish a repressive chromatin state

• Chromatin Compaction by a Polycomb Group Protein Complex. (2004) Nicole J. Francis, Robert E. Kingston, and Christopher L. Woodcock. Science, 306:1574-1577.

• Showed that Polycomb Repressive Complex 1 (PRC1) creates compacted chromatin through interactions with nucleosomes independently of their histone tail.

Week 5 - RNA-directed silencing

• Discrete small RNA-generating loci as master regulators of transposon activity in Drosophila. (2007) Brennecke J, Aravin AA, Stark A, Dus M, Kellis M, Sachidanandam R, Hannon GJ. Cell, 128(6):1089-103.

• Role of piRNAs in maintaining silencing of TEs; piRNA loci help form an 'immune system' against TEs

• A nuclear Argonaute promotes multigenerational epigenetic inheritance and germline immortality. (2012) Buckley, B. A. et al. Nature, 489:447–451.

• Characterized a specialized Argonaute required for transgenerational maintenance of small-RNA mediated silencing signals, also required for fertility, in C. elegans

Week 6 - RNA-directed silencing

• Extensive Demethylation of Repetitive Elements During Seed Development Underlies Gene Imprinting

• Showed that DME (a DNA glycosylase) selectively demethylates certain loci in Arabidopsis endosperm, and association with genomic imprinting

• Dual functions of Tet1 in transcriptional regulation in mouse embryonic stem cells

• Linked Tet1 to maintenance of DNA hypomethylation, gene expression regulation

Week 7 - Enhancers

• Histone modifications at human enhancers reflect Global cell-type-specific gene expression. (2009) Nathaniel D Heintzman, Gary C Hon, R David Hawkins, Pouya Kheradpour, Alexander Stark, Lindsey F Harp, Zhen Ye, Leonard K Lee, Rhona K Stuart, Christina W Ching, Keith A Ching, Jessica E Antosiewicz-Bourget, Hui Liu, Xinmin Zhang, Roland D Green, Victor V Lobanenkov, Ron Stewart, James A Thomson, Gregory E Crawford, ManolisKellis, Bing Ren. Nature, 459:108-112.

• Early work identifying enhancers using whole-genome approaches, also showing that cell-type-specific histone modifications at enhancers correlate with expression

• Enhancer Divergence and cis-Regulatory Evolution in the Human and Chimp Neural Crest. (2015) Prescott SL, Srinivasan R, Marchetto MC, Grishina I, Narvaiza I, Selleri L, Gage FH, Swigut T, Wysocka J. Cell, 163(1):68-83.

• Systematically characterized enhancers associated with facial development & variation in humans

Week 8 - Targeted epigenetics

• Editing DNA Methylation in the Mammalian Genome. (2016) X. Shawn Liu, Hao Wu, Xiong Ji, Yonatan Stelzer, Xuebing Wu, SzymonCzauderna, Jian Shu, Daniel Dadon, Richard A. Young, Rudolf Jaenisch. Cell, 167: 233-247.

• Demonstrated that fusion of dCas9 to either Tet1 (removes 5mC) or Dnmt3a (adds 5mC) can be used to cause targeted DNA methylation/demethylation

• CRISPRi-mediated modular RNA-guided regulation of transcription in eukaryotes. (2013) Gilbert LA, Larson MH, Morsut L, Liu Z, Brar GA, Torres SE, Stern-Ginossar N, Brandman O, Whitehead EH, Doudna JA, Lim WA, Weissman JS, Qi LS. Cell, 154: 442-51.

• Optimizing CRISPR systems for targeted gene activation/repression via combinatorial manipulation of epigenetic marks & chromatin structure

Week 9 - 3D chromatin architecture

• Comprehensive mapping of long-range interactions reveals folding principles of the human genome. (2009) Lieberman-Aiden, E. et al. Science, 326:289–293.

• Initial publication of Hi-C, a method for mapping 3D chromatin architecture

• Targeted Degradation of CTCF Decouples Local Insulation of Chromosome Domains from Genomic Compartmentalization. (2017) Nora et al., Cell 169, 930–944.

• Demonstrates that CTCF sites are required for looping between CTCF sites and insulation of topologically associating domains through blocking enhancer binding.

Week 10 - phase transition & epigenetics

• Liquid droplet formation by HP1a suggests a role for phase separation in heterochromatin. (2017) Larson, A. G. et al. Nature, 547: 236-240.

• New potential mechanism for heterochromatin compaction & associated gene silencing, via liquid-liquid phase separation driven by HP1a

• Transcription Factors Activate Genes through the Phase-Separation Capacity of Their Activation Domains. (2018). Boija et al., 2018, Cell 175, 1842–1855.

• New model proposing that activation domains (ADs) can form phase-separated droplets via interaction with Mediator, and this phase-separation is associated w/ gene activation