Transcript ppt

Trends in Biomedical Science
2015 Epigenetics – Editing the
Epigenome
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adapted from Opinion: Engineering the
Epigenome Stephan Beck, Stefan H.
Stricker and Anna Köferle | August 26, 2015
http://www.thescientist.com//?articles.view/articleNo/43837/ti
tle/Opinion--Engineering-the-Epigenome/
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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
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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
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It is difficult to say how a single
mark affects gene activity.
If we alter chromatin marks
globally through mutational
approaches or pharmacological
inhibition.
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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.
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Targeted modification
• fuse
–an existing chromatin modifying
enzyme (or a functional part of
such an enzyme)
–a programmable DNA binding
domain.
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Example chromatin
modifying enzymes.
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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.
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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.
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Other enzymes include those
which attach or remove actyl
groups, ubiquitin, phosphoryl
groups, and so on.
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Programmable DNA binding
domains
There are several types of DNA
binding domains.
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TALE
TAL effectors (Transcription Activator-Like
Effectors ) are proteins that are injected
into plant cells by Xanthomonas bacterial.
They enter the nucleus, bind to effectorspecific promoter sequences, and activate
the expression of individual plant genes,
which can either benefit the bacterium or
trigger plant defenses.
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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.
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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
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Transcription Activator-Like
Effectors can be used for genome
editing and gene modulation
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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).
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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.
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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.
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Fig. 2 Genomic control enabled by engineered TAL effector proteins.
Adam J. Bogdanove, and Daniel F. Voytas Science
2011;333:1843-1846
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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.'
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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.
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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.
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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.
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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.
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2011
Context-dependency improves open-source
zinc finger engineering
Keith Joung publishes tables of zinc finger
binding sites that account for contextdependent 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.
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Clustered, regularly
interspaced, short palindromic
repeat (CRISPR) technology
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Fig. 1 Timeline of CRISPR-Cas and genome engineering research fields.Key developments in
both fields are shown.
Jennifer A. Doudna, and Emmanuelle Charpentier Science
2014;346:1258096
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Published by AAAS
Fig. 2 Biology of the type II-A CRISPR-Cas system.The type II-A system from S. pyogenes is
shown as an example.
Jennifer A. Doudna, and Emmanuelle Charpentier Science
2014;346:1258096
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Published by AAAS
Fig. 4 CRISPR-Cas9 as a genome engineering tool.
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 livecell imaging of chromosomal loci.
•fused chromatin or DNA modification domains, to
target epigenetic changes to genomic DNA.
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Make fusion proteins which have
a DNA binding domain fused to a
chromatin modifying domain.
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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.
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Zinc finger, TALE, and Cas9-gRNA
platforms for editing regulatory states.
Isaac B. Hilton, and Charles A. Gersbach Genome Res.
2015;25:1442-1455
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© 2015 Hilton and Gersbach; Published by Cold Spring Harbor Laboratory Press
Editing Epigenetic States.
Isaac B. Hilton, and Charles A. Gersbach Genome Res.
2015;25:1442-1455
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© 2015 Hilton and Gersbach; Published by Cold Spring Harbor Laboratory Press
What do you want to
change?
Think of the problem then
look it up in ENCODE.
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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
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Epigenomic enrichments of genetic variants associated with
diverse traits.
Roadmap Epigenomics Consortium et al. Nature 518, 317-330 (2015)
doi:10.1038/nature14248
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Epigenomic enrichments of genetic variants associated with
diverse traits.
Roadmap Epigenomics Consortium et al. Nature 518, 317-330 (2015)
doi:10.1038/nature14248
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Epigenomic information across tissues and marks.
Roadmap Epigenomics Consortium et al. Nature 518, 317-330 (2015)
doi:10.1038/nature14248
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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.
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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.
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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.
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• 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.
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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.
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• 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.
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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.
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The DNA coding for the ZFPacetyltransferase 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.
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The results displayed that once ZFPs bound to
the gene, histones were modified by the FosBZFPs 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.”
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