Transcript Heterochromatin Formation - Washington University in St. Louis

Heterochromatin Formation It’s all about silencing!
Shhh!
Sarah C. R. Elgin
Bio 4342/434W
Copyright 2017, Washington University in St. Louis
What determines phenotypes?
It is not just your DNA….
Environment (diet)
(black bars = folate)
Phenotype
Genotype
Epigenetics
?
Development
From N. Riddle
(Waterland and Jirtle 2003)
A tale of two mice – littermates
(inbred line)
Yellow mouse
• High risk of cancer,
diabetes, obesity;
• Reduced lifespan
Maternal
supplements with
zinc, methionine,
betaine, choline,
folate, B12
Pseudo-agouti mouse
• Low risk of cancer,
diabetes, obesity;
• Prolonged lifespan
Slide prepared by T Wang
Methylation and diet: impact on the agouti locus
•
Mothers fed BPA (soaks up
methyl)  coats shift toward
yellow (higher gene expression)
•
Mothers fed folate (methyl donor)
 shift toward pseudoagouti
•
Trait stable at least to the
following generation (your
grandparents diet could effect
your epigenetics!)
What is epigenetics?
Study of gene expression states that are
heritable, through mitosis and/or meiosis, but
that are independent of DNA sequence
changes.
How is this accomplished?
from N. Riddle
(Brickner et al 2007)
• DNA modification – 5mC
• Nuclear localization
• Chromatin structure
Packaging large genomes:
DNA
First step - packaging in a
nucleosome array
Chromatin
Histone
protein
core
Second - differential
packaging into
heterochromatin &
euchromatin
Chromosome
(metaphase)
Lodish et.al., Molecular Cell Biology, 4th Edition
Felsenfeld et al. Nature 2003, 421: 448
DNA packaging domains
• Euchromatin
– Less condensed
– Chromosome arms
– Unique sequences;
gene rich
– Replicated throughout S
– Recombination during meiosis
• Heterochromatin
– Highly condensed
– Centromeres and telomeres
– Repetitious sequences;
gene poor
– Replicated in late S
– No meiotic recombination
Transcriptional activators
Heterochromatin Protein 1 complex
Hyper-acetylated histone tail
Hypo-acetylated histone tail; methylated H3/K9
The histone N-terminal tails are unstructured and
accessible for modification
The complete histone octamer in
the absence of DNA.
The view is down the superhelix
axis.
Color code:
H2A
H2B
H3
H4
Rhodes, Nature (1997) 389, 231-233
The core histone tails are extensively modified
Histone modifications are reversibleExamples: HAT / HDAC
HMT / KDM
Allis et al 2007 Epigenetics
Role of acetylation of histone tails in yeast transcription controlGcn5 is a histone acetyltransferase!
David Allis
Lodish et.al., Molecular Cell Biology, 4th Ed
Specific mutations in the core histones have specific
impacts on gene regulation
Mike Grunstein
Silencing at HML, HMR & telo
Repression of basal transcription
Inter-nucleosomal contact
Silencing at HML, HMR & telo
Durrin et al 1991 Cell 65:1023
Repression of basal transctiption
Ragunathan et al 2015 Science 348: 90.
Histone modification patterns can be inherited
Model system – fruit flies!
Inexpensive and easy to culture,
short life cycle, easily visible
phenotypes: good genetic
approaches
Biochemical approaches
Polytene chromosomes:
excellent cytology
Simple genome, good reference
sequence
Mary Lou Pardue, MIT
euchromatin
heterochromatin
PEV – reporter for gene silencing,
heterochromatin formation
Metazoan useful for behavioral,
developmental. and
human disease research
expressed
silenced
Distribution of Heterochromatin Protein 1
C
James et al, 1989, Europ J Cell Bio 50: 170
Identified by screening monoclonal antibodies against tight-binding nuclear proteins
C
Phase
HP1
Position Effect Variegation in Drosophila
provides a functional assay for putative chromatin proteins
white
Wild Type
Inversion
Trans-acting modifiers of PEV:
Su(var):
E(var):
Elgin lab
Heterochromatin-associated gene silencing is
dependent on HP1
Mutations in
gene for HP1a
Mutations recovered by T Grigliatti as suppressors of PEV.
Dosage dependent response.
Eissenberg et al, 1990, PNAS 87: 9923
Using a white transgene to sample
chromatin environments
inject
transposon
white67c23
insertion into
euchromatin
(99%)
P[white+]
mobilize P element
by crossing to stock
with transposase
insertion into
heterochromatin
( 1%)
Wallrath & Elgin, 1995
Transposition of a P element reporter allows sampling
of heterochromatin domains
X
Silenced
1%
2L
2R
3L
3R
4
And the Y chromosome
Active
99%
Wallrath and Elgin, 1995
Assessing chromatin structuresame gene, different environments
Analysis: MNase cuts between nucleosomes;
DNase I and restriction enzymes cut hypersensitive sites
What happens to the same gene in heterochromatin?
Elgin lab
The nucleosome array associated with the silenced
transgene in heterochromatin shows more regular
spacing and sharper MNase cleavage sites
Sun et al, 2001, Mol Cell Biol 21: 2867
Cryderman et al 1999, Nucleic Acids Res 27: 3364
Loss of accessibility in the heterochromatic hsp26
transgene is reversed in an HP1 mutant background
Assessing chromatin structuresame gene, different environments
Analysis based on nuclease digestion of chromatin
The euchromatic hsp26 transgene:
- DH sites: accessibility at the TSS, upstream regulatory region
- irregular nucleosome array
The heterochromatic hsp26 transgene:
- loss of DH sites
- regular nucleosome array
Clark and Elgin, 1992 Nucleic Acids Res. 20:6067
HP1 sequence from Drosophila, mouse, human and mealy
bug identifies conserved chromo & chromo shadow domains
HP1 from mammals can rescue mutations in flies and yeast!
HP1 interacts with both the modified histone H3K9me2/3
and the modifying enzyme
HP1:
Chromo
Histone 3
methyl-Lys9
Shadow
H3 K9
methylation
SU(VAR)3-9
HMT
[(SU(VAR)3-9 identified in screen by Reuter;
H3 interaction first shown from work in mammals – Jenuwein, Kouzarides;
demonstrated in flies by Imhof.]
Model for spreading of heterochromatin
(and epigenetic inheritance):
create a system that can recognize a histone modification mark
and can generate that same mark.
Elgin summary
PEV transition: loss of euchromatin marks
Heterochromatin
Reuter & Elgin, 2006, Epigenetics
Euchromatin
-and probably others!
Establishing silencing: gain of heterochromatin marks
Heterochromatin
Reuter & Elgin, 2006, Epigenetics
Euchromatin
And no doubt, additional proteins are required!
Three different H3K9 methyltransferases!
Mutations in these have different impacts on different
heterochromatin compartments
118E-10
39C-12
B Brower-Toland et al, 2008
Feedback loops to maintain silencing
Richards & Elgin, 2002
1. Crosslink proteins to DNA
Chromatin Immunoprecipitation – ChIP 
mapping chromatin structure
2. Isolate chromatin and sonicate
qPCR
3. Incubate with antibody
ChIP-chip
4. Isolate AB/chromatin complexes
ChIP-seq
5. Isolate DNA from complexes
N Riddle
ChIP-chip mapping of chromosomal proteins shows distinct
differences between heterochromatin and euchromatin
Centromere
S2 cells
HP1
Su(var)3-9
H3K9me2
H3K9me3
genes
genes
Euchromatin
Heterochromatin
Euchromatin / heterochromatin transition point from Flybase
Riddle et al, 2011, Genome Research 21: 147-63.
H3K4me3 
Define chromatin states by K-means clustering
(using enrichment values for 1 kb chromatin fragments)
H3K9me3 
Select k means
as starting
points.
Assign each
data point to
closest mean.
The centroid of
each cluster is
the new mean.
Repeat the last
two steps.
Slide by N Riddle
Karchenko et al, 2011, Nature 471: 480-5.
H3K9me2
H3K9me3
Clustering chromatin fragments according to histone
modification marks suggests nine major chromatin states
TSS
X chr
Polycomb
HeteroSimilar to
chromatin
heterochromatin
S2 and BG3 cells
Histones modifications are critical for setting levels
of gene expression.
•
H3/H4 acetylation associated with the active state
•
HAT’s are activators; HDAC’s are silencers
•
H4K16 acetylation associated with dosage compensation
•
H3K9 di- and tri-methylation are found in constitutive silent
domains (centromeres, telomeres)
•
H3K27 tri-methylation is associated with developmentally
regulated silencing (Pc complex)
•
H3K4 di- and tri-methylation occurs at the 5’ end of active
genes, and H3K36me3 over the gene body
•
Chromatin modification states switch to achieve gene
regulation, controlling frequency of transcription.
SCR Elgin
Chromatin states identify large-scale
genomic domains
1Mb
Mapping the nine chromatin
states onto the Drosophila
genome (Bg3 cells).
Karchenko et al 2011 Nature 471: 480
Summing up…..
Much of the genome is packaged in heterochromatin.
Heterochromatic structure differs- in a loss of accessibility, loss of 5’ HS sites;
- in having increased nucleosome stability;
- in having a more ordered nucleosome array.
Heterochromatin is marked by- hypoacetylated H4, H3K9me2/3, (methylated DNA)
Epigenetic inheritance and spreading are achieved by
- a protein (HP1) or protein complex that interacts with a
modified histone and with the modifying enzyme.
The genome can be subdivided into different states based
on histone modifications –
- specific patterns associated with transcription start
sites, enhancers, transcript elongation;
- two types of silencing domains: H3K27me3 (Polycomb)
and H3K9me2/3 (HP1a)
- and more!
SCR Elgin