Trends in Biomedical Science 2015 Epigenetics – Editing the Epigenome

adapted from Opinion: Engineering the Epigenome Stephan Beck, Stefan H adapted from Opinion: Engineering the Epigenome Stephan Beck, Stefan H. Stricker and Anna Köferle | August 26, 2015 http://www.the-scientist.com//?articles.view/articleNo/43837/title/Opinion--Engineering-the-Epigenome/

Cells can add or remove epigenetic marks. this is called modifying the chromatin. proteins which add or remove epigenetic marks are chromatin modifiers. there are ways to send chromatin modifiers to particular regions of the chromosome the modifiers are targeted to the chromosome region

Epigenetic processes influence how cells use genetic information. the cell adds chemical tags onto the genome, labels features such as genes or regulatory elements millions of these tags—chromatin marks—have been profiled different tissues cell types

It is difficult to say how a single mark affects gene activity It is difficult to say how a single mark affects gene activity. If we alter chromatin marks globally through mutational approaches or pharmacological inhibition.

Epigenome engineering makes it possible to investigate the function of individual chromatin marks by adding them to, or removing them from single locations of interest in the genome.

Targeted modification fuse an existing chromatin modifying enzyme (or a functional part of such an enzyme) a programmable DNA binding domain.

Example chromatin modifying enzymes.

Demethylases are enzymes that remove methyl (CH3-) groups from nucleic acids, proteins (in particular histones), and other molecules. Several families of histone demethylases act on different substrates and do different things in cellular function.

The DNA methyltransferase (DNA MTase) family of enzymes catalyze the transfer of a methyl group to DNA. All the known DNA methyltransferases use S-adenosyl methionine (SAM) as the methyl donor.

Other enzymes include those which attach or remove actyl groups, ubiquitin, phosphoryl groups, and so on.

Programmable DNA binding domains There are several types of DNA binding domains.

TALE TAL effectors (Transcription Activator-Like Effectors ) are proteins that are injected into plant cells by Xanthomonas bacterial. They enter the nucleus, bind to effector-specific promoter sequences, and activate the expression of individual plant genes, which can either benefit the bacterium or trigger plant defenses.

TALE In each TAL effector, a variable number of tandem amino acid repeats (which are usually 34 residues in length), terminated by a truncated “half repeat,” recognize DNA sequences. Each of the repeats associates with one of the four nucleotides in the target site.

TALE The repeats are located centrally in the protein between N-terminal sequences required for bacterial type III secretion and C-terminal sequences required for nuclear localization and activation of transcription. From “The Crystal Structure of TAL Effector PthXo1 Bound to Its DNA Target.” Amanda Nga-Sze Mak et al. (2012) 10 FEBRUARY 2012 VOL 335 SCIENCE

Transcription Activator-Like Effectors can be used for genome editing and gene modulation

Transcription Activator-Like Effector Nucleases (TALENs) TALENs are engineered restriction enzymes generated by fusing the TAL effector DNA binding domain to a DNA cleavage domain (eg. FokI).

Transcription Activator-Like Effector Activators or Repressors Created by fusing the TAL effector DNA binding domain that targets a specific promoter region to a transcription activation or repression protein domain.

TAL effector–based sequence-specific mutagens or chromatin-modifying proteins created by fusing TAL effectors to domains such as cytidine deaminases, histone acetyltransferases or deacetylases, or DNA methyltransferases may be possible.

Fig. 2 Genomic control enabled by engineered TAL effector proteins. Genomic control enabled by engineered TAL effector proteins. Fusion of TAL effector proteins to FokI creates sequence-specific nucleases that enable targeted DNA cleavage for gene knockouts and genome editing. TAL effector proteins fused to transcriptional activation domains (AD) and putatively to repression domains (RD) provide artificial switches for gene regulation in vivo. TAL effector–based sequence-specific mutagens or chromatin-modifying proteins created by fusing TAL effectors to domains such as cytidine deaminases, histone acetyltransferases or deacetylases, or DNA methyltransferases can also be envisioned. Adam J. Bogdanove, and Daniel F. Voytas Science 2011;333:1843-1846 Published by AAAS

A Brief History of Zinc Fingers 1985 Discovery of the zinc finger protein Jonathon Miller, A. D. McLachlan, and Sir Aaron Klug first identify the repeated binding motif in Transcription Factor IIIA and are the first to use the term ‘zinc finger.'

1991 First crystal structure of a zinc finger Carl Pabo and Nikola Pavletich of Johns Hopkins University solve the crystal structure of zif268, now the most-commonly studied zinc finger. This paved the way for construction of binding models to describe how zinc fingers bind to DNA, setting the foundation for future custom engineering of zinc finger proteins.

1996 Srinivasan Chandrasegaran publishes work on fusing the Fok I nuclease to zinc fingers By attaching nuclease proteins to zinc fingers, a new genome editing tool was created.

2008 Rapid open source production of zinc finger nucleases becomes available Researcher Keith Joung shows how to make zinc finger nuclease proteins that bind to custom target sequences, using a bacterial two-hybrid screening system to identify specific zinc finger binders to a DNA sequence of interest.

2009 Zinc finger nuclease enters clinical trials Sangamo begins clinical trials with a zinc finger nuclease designed to target the CCR5 gene and inhibit HIV. Success of this therapeutic could prove a significant advance for gene therapy.

2011 Context-dependency improves open-source zinc finger engineering Keith Joung publishes tables of zinc finger binding sites that account for context-dependent effects and can be rearranged to form custom zinc finger proteins that bind to a variety of DNA sequences. This greatly increases the ease of engineering novel zinc fingers based on the structures of previously characterized zinc fingers.

Clustered, regularly interspaced, short palindromic repeat (CRISPR) technology

Fig. 1 Timeline of CRISPR-Cas and genome engineering research fields Fig. 1 Timeline of CRISPR-Cas and genome engineering research fields.Key developments in both fields are shown. Timeline of CRISPR-Cas and genome engineering research fields.Key developments in both fields are shown. These two fields merged in 2012 with the discovery that Cas9 is an RNA-programmable DNA endonuclease, leading to the explosion of papers beginning in 2013 in which Cas9 has been used to modify genes in human cells as well as many other cell types and organisms. Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:1258096 Published by AAAS

Fig. 2 Biology of the type II-A CRISPR-Cas system Fig. 2 Biology of the type II-A CRISPR-Cas system.The type II-A system from S. pyogenes is shown as an example. Biology of the type II-A CRISPR-Cas system.The type II-A system from S. pyogenes is shown as an example. (A) The cas gene operon with tracrRNA and the CRISPR array. (B) The natural pathway of antiviral defense involves association of Cas9 with the antirepeat-repeat RNA (tracrRNA:crRNA) duplexes, RNA co-processing by ribonuclease III, further trimming, R-loop formation, and target DNA cleavage. (C) Details of the natural DNA cleavage with the duplex tracrRNA:crRNA. Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:1258096 Published by AAAS

Fig. 4 CRISPR-Cas9 as a genome engineering tool. CRISPR-Cas9 as a genome engineering tool.(A) Different strategies for introducing blunt double-stranded DNA breaks into genomic loci, which become substrates for endogenous cellular DNA repair machinery that catalyze nonhomologous end joining (NHEJ) or homology-directed repair (HDR). (B) Cas9 can function as a nickase (nCas9) when engineered to contain an inactivating mutation in either the HNH domain or RuvC domain active sites. When nCas9 is used with two sgRNAs that recognize offset target sites in DNA, a staggered double-strand break is created. (C) Cas9 functions as an RNA-guided DNA binding protein when engineered to contain inactivating mutations in both of its active sites. This catalytically inactive or dead Cas9 (dCas9) can mediate transcriptional down-regulation or activation, particularly when fused to activator or repressor domains. In addition, dCas9 can be fused to fluorescent domains, such as green fluorescent protein (GFP), for live-cell imaging of chromosomal loci. Other dCas9 fusions, such as those including chromatin or DNA modification domains, may enable targeted epigenetic changes to genomic DNA. Jennifer A. Doudna, and Emmanuelle Charpentier Science 2014;346:1258096 Published by AAAS

Cas9 functions as an RNA-guided DNA binding protein when engineered to contain inactivating mutations in both of its active sites. Dead Cas9 (dCas9) can be fused to: activator or repressor domains for transcriptional down-regulation or activation . fused to fluorescent domains, (eg. GFP), for live-cell imaging of chromosomal loci. fused chromatin or DNA modification domains, to target epigenetic changes to genomic DNA.

Make fusion proteins which have a DNA binding domain fused to a chromatin modifying domain.

Zinc fingers, TALEs, and CRISPR/Cas9 proteins can be used to target changes in gene expression, "epigenome editing." Eg. fuse the gene-activating VP64 or p65 domains to an engineered DNA binding domain targeted to an endogenous gene promoter to achieve gene activation.

Zinc finger, TALE, and Cas9-gRNA platforms for editing regulatory states. Zinc finger, TALE, and Cas9-gRNA platforms for editing genomic sequence and regulatory states. Individual zinc finger domains (A) and TALE repeats (B) that recognize unique triplets or single base pairs, respectively, can be arrayed in engineered proteins to target specific genomic sequences. (C) Cas9 in complex with a chimeric guide RNA (gRNA) can recognize a specific genomic address through complementarity between the protospacer segment of the gRNA and target DNA. The formation of this complex is dependent upon the presence of a protospacer adjacent motif (PAM). The RuvC and HNH nuclease domains of Cas9 cleave genomic DNA that matches the protospacer (i.e., the noncomplementary strand) and genomic DNA with complementarity to the protospacer (i.e., the complementary strand), respectively (indicated by black triangles). (D) Zinc fingers and TALEs fused to nuclease domains or Cas9 in complex with a gRNA can cleave targeted sequences to generate double-strand breaks (DSBs). DSB resolution through nonhomologous end joining (NHEJ) or homology-directed repair (HDR) can lead to various alterations in genomic sequence. (E) Zinc finger, TALE, or deactivated nuclease-null Cas9 (dCas9) platforms can also be fused to diverse effector domains to modify endogenous gene regulation and epigenetic states: (TSS) transcription start site; (GOI) gene of interest. Isaac B. Hilton, and Charles A. Gersbach Genome Res. 2015;25:1442-1455 © 2015 Hilton and Gersbach; Published by Cold Spring Harbor Laboratory Press

Editing Epigenetic States. Zinc finger, TALE, and Cas9-gRNA platforms for editing genomic sequence and regulatory states. Individual zinc finger domains (A) and TALE repeats (B) that recognize unique triplets or single base pairs, respectively, can be arrayed in engineered proteins to target specific genomic sequences. (C) Cas9 in complex with a chimeric guide RNA (gRNA) can recognize a specific genomic address through complementarity between the protospacer segment of the gRNA and target DNA. The formation of this complex is dependent upon the presence of a protospacer adjacent motif (PAM). The RuvC and HNH nuclease domains of Cas9 cleave genomic DNA that matches the protospacer (i.e., the noncomplementary strand) and genomic DNA with complementarity to the protospacer (i.e., the complementary strand), respectively (indicated by black triangles). (D) Zinc fingers and TALEs fused to nuclease domains or Cas9 in complex with a gRNA can cleave targeted sequences to generate double-strand breaks (DSBs). DSB resolution through nonhomologous end joining (NHEJ) or homology-directed repair (HDR) can lead to various alterations in genomic sequence. (E) Zinc finger, TALE, or deactivated nuclease-null Cas9 (dCas9) platforms can also be fused to diverse effector domains to modify endogenous gene regulation and epigenetic states: (TSS) transcription start site; (GOI) gene of interest. Isaac B. Hilton, and Charles A. Gersbach Genome Res. 2015;25:1442-1455 © 2015 Hilton and Gersbach; Published by Cold Spring Harbor Laboratory Press

What do you want to change What do you want to change? Think of the problem then look it up in ENCODE.

Figure 4. ENCODE chromatin annotations in the HLA locus. The ENCODE Project Consortium (2011) A User's Guide to the Encyclopedia of DNA Elements (ENCODE). PLoS Biol 9(4): e1001046. doi:10.1371/journal.pbio.1001046 http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.1001046

Epigenomic enrichments of genetic variants associated with diverse traits. Roadmap Epigenomics Consortium et al. Nature 518, 317-330 (2015) doi:10.1038/nature14248

Epigenomic enrichments of genetic variants associated with diverse traits. Roadmap Epigenomics Consortium et al. Nature 518, 317-330 (2015) doi:10.1038/nature14248

Epigenomic information across tissues and marks. Roadmap Epigenomics Consortium et al. Nature 518, 317-330 (2015) doi:10.1038/nature14248

Which of the large number of catalogued chromatin marks possess real gene-regulatory capabilities? GWAS and ENCODE involves statistical correlation of chromatin marks with expression levels of associated genes.

By directing chromatin modifiers to a range of sites at different genomic loci and measuring resulting changes in transcription of associated candidate genes, a number of functional chromatin marks have now been identified. Eg. removal of methylation from lysine4 of histone H3 at enhancers and promoters with dCas9-LSD1 results in downregulation of proximal genes , while adding histone acetylation using dCas9-p300 gives upregulation.

Changing individual chromatin marks at relevant sites can significantly alter levels of transcription. This effect depends both on the enzymatic activity of the chromatin modifier and its guided binding to the target site. The biological relevance of these engineering efforts must still be established.

Measuring changes in protein levels orphenotypic changes in addition to changes in mRNA levels,   or comparing engineered gene expression to physiological levels of activity should show that changing specific chromatin marks can influence cellular behavior.

It will soon be possible to see the effect of altering combinations of chromatin marks by using different targeting platforms that can act independently in the same cell.

The ease of targeting chromatin modifiers through an RNA-based DNA binding mechanism will help us discover functional marks using screening approaches. Some of the findings may be translated into therapeutic use by using epigenetic engineering technologies in vivo.

Eg in living mice, Heller et al (2014), engineered Zinc Finger Proteins (ZFPs), which were targeted to the transcription factor, ΔFosB, which plays important roles, acting in the nucleus accumbens (Nac). The ZFPs were fused to enzymes which  acetylate histones.

The DNA coding for the ZFP-acetyltransferase was puut into a virus The DNA coding for the ZFP-acetyltransferase was puut into a virus. The virus was injected into the nucleus accumbens. The nucleus accumbens is part of the brain which is a key brain reward region, in drug and stress action.

The results displayed that once ZFPs bound to the gene, histones were modified by the FosB-ZFPs in the area of the FosB gene, which either activated or repressed expression. Histone methylation or histone acetylation “was sufficient to control drug- and stress-evoked transcriptional and behavioral responses via interactions with the endogenous transcriptional machinery.”