Download D melanogaster - GEP Community Server

The Evolution of a Heterochromatic
Domain in Drosophila:
Investigating the Strange
Dot Chromosome
Sarah C R Elgin
January 2012
A collaborative investigation involving:
- former members of the Elgin Lab: Lee Silver, Carl Wu,
TC James, Joel Eissenberg, Lori Wallrath, Fang Lin Sun, Karmella
Haynes
- current members of the Elgin Lab: Nicole Riddle,
Tingting Gu, Chris Shaffer, Wilson Leung
- modENCODE: Gary Karpen, Mitzi Kuroda, Vincenzo Pirrotta,
Peter Park, and their colleagues
- Faculty and students of the Genomics Education
Partnership
Minimum Haploid DNA Content - the C Value Paradox
3
Britten and Davidson, 1969 Science 165:349
Larger genomes reflect high levels of repeats retroviral and DNA transposon remnants (TEs)
4
Allis et al: Epigenetics 2007
Considerations for Genome Sequencing
1. Satellite DNA, a sequence of tandem repeats, is very difficult to
sequence, as there are few markers to help order subclones;
hence centromeric regions of the chromosomes are usually left
unsequenced.
2. Other repetitious DNA, derived from transposable elements, also
causes difficulties; because one finds nearly identical sequences
located in different regions of the genome, mistakes can be made
in assembling sequence data. High quality discrepancies can
identify these.
3. Much of the repetitous DNA is packaged into heterochromatin,
which maintains these regions in a compact and transcriptionally
silent form.
4. However, in many higher organisms, protein-coding genes are
found embedded in repetitious DNA. Check out your favorite
human gene on the UCSC Browser by taking off RepeatMasker!
5
SCR Elgin
Eukaryotic genomes are very large – and most of that
DNA is non-coding!
Human
Genome
3 Gb
~2 m/cell !
Coding exons
1.5%
Conserved noncoding regulatory? 3.5%
Key Questions:
 Is it junk or garbage?
 How is DNA packaged into a nucleus?
 How is silencing maintained – while
allowing appropriate transcription ?
TEs –
retroviruses,
DNA transposons
What determines phenotypes?
It’s not just your DNA….
Environment (diet)
(grey bars = folate)
Phenotype
Genotype
Epigenetics
?
Development
(Waterland and Jirtle 2003)
Chromatin structure = epigenetics !
What sets and maintains tissue-specific gene
expression patterns? Differences are heritable through
mitosis, but independent of DNA sequence.
• DNA modification (mC)
• Chromatin structure
• Nuclear localization
It’s all about silencing!
How is chromatin assembled?
When, where and how does
gene silencing occur?
Incorrect silencing can lead
To genetic disability, as seen
In Fragile X syndrome
Zoghbi and Beaudet 2007
Fragile X Foundation
Chromatin formation:
DNA
First step - packaging in a
nucleosome array
Chromatin
Second - differential
packaging into
heterochromatin &
euchromatin
Histone
protein core
Chromosome
(metaphase)
Lodish et.al., Molecular Cell Biology, 4th Edition
Felsenfeld et al. Nature 2003, 421: 448
Electron Micrograph of Chromatin Fibers
(rat thymus nucleus)
0.1 mm
Olins et. al., 1975 J. Cell Biol, 64:528
10
“A eukaryotic chromosome made out of self-assembling
70A units, which could perhaps be made to crystallize,
would necessitate rewriting our basic textbooks on
cytology and genetics! I have never read such a naïve
paper purporting to be of such fundamental significance.
Definitely it should not be published anywhere!”
Anonymous review of paper submitted by C.F.L.
Woodcock, 1973, showing EM pictures of
nucleosome arrays.
Quoted
in “Chromatin” by K.D. van Holde, 1989
11
The Structure of the Nucleosome Core
Resolution: 2.8 Å
Half of the nucleosome structure is
shown
One turn of the DNA helix is visible
(73 bp)
View is down the superhelix axis
Protein - DNA contact: white hooks
Rhodes, 1997 Nature 389:231, after
12
Luger et. al., 1997 Nature 389:251
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
Heterochromatin formation – silencing counts!
1
2
How is heterochromatin organized and packaged to
promote silencing?
The fourth chromosome appears heterochromatic
but has ~80 genes:
-- do these genes have unusual characteristics?
-- how has the chromosome evolved?
-- how do these genes function?
Fruit Flies!
Short life cycle, easily
maintained: good genetic tools
Polytene chromosomes:
excellent cytology
Biochemical approaches
Simple genome, good
reference sequence
Mary Lou Pardue, MIT
euchromatin
expressed
heterochromatin
silenced
PEV – reporter for gene
silencing, heterochromatin
formation
Metazoan useful for behavioral,
developmental and human
disease research
Using a white transgene to sample chromatin
environments
inject
P[white+]
transposon
carrying
white gene
white67c23
mobilize P element
by crossing to stock
with transposase
insertion into
euchromatin
(99%)
insertion into
heterochromatin
( 1%)
16
Elgin Lab
Transposition of a P element reporter allows sampling
of euchromatic and 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 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
C
C
Phase
HP1
James & Elgin,1986; James et al 1989
Looking for heterochromatic proteins by immunofluorescent
staining of the polytene chromosomes: discovery of HP1a
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
HP1 interacts with both the modified histone H3K9me2/3
and the modifying enzyme
HP1:
Chromo
Histone 3
methyl-Lys9
Shadow
H3 K9 methyl
transferase
SU(VAR)3-9
[(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
Establishing silencing: a multi-step process
Loss of euchromatin marks
Gain of heterochromatin marks
wm4 reporter (screens by Reuter, Grigliatti, others)
Heterochromatin formation on the dot chromosome…
2
The fourth chromosome appears heterochromatic
but has ~80 genes:
-- do these genes have unusual characteristics?
-- how has the chromosome evolved?
- how do these genes function?
The Drosophila melanogaster fourth chromosome exhibits an amalgam of
euchromatic and heterochromatic properties (HP1a association)
But…
- the fourth has ~ 80 genes in distal 1.2 Mb
- these genes are transcriptionally active!
C
C
Phase
HP1
James & Elgin,1986; James et al 1989
Heterochromatic properties:
- late replication, lack of recombination
- high repeat density (30%)
- antibody staining of HP1, H3K9me2/3
Most hsp70-white reporters exhibit variegation
on insertion into the fourth chromosome
2-M1021
39C-12
2-M390
Sun et al 2004; Riddle et al 2007
39C-52
Our GEP Research Goal:
Use comparative genomics to learn more
about heterochromatic domains, analyzing the
dot chromosomes and a control euchromatic
region of Drosophila genomes
Status
Reference
Completed
Annotation
Sequence
Improvement
New Project
FlyBase: http://flybase.org
Genomics Education Partnership (GEP)
Partners are generally PUI schools; faculty join by
attending one-week workshop at WU. Shared
work organized on GEP website.
The D.melanogaster & D. virilis dot chromosomes
are 25% - 30% repetitious DNA
(typical – but up to 80% in D. ananassae)
D mel D vir
D mel
D vir
D vir
D mel
D vir
D vir
D vir
Leung et al 2010
Dot chromosome genes: introns are larger,
exons show less codon bias
% Introns This Size or Smaller
100%
Euchromatic
90%
80%
Leung et al. 2010 Genetics 185:1519-1534
70%
Dot
60%
Codon Bias
50%
40%
30%
20%
Heterochromatic
10%
0%
Intron Size
D. melanogaster Dot
D. virilis Dot
Intron Size (bin)
D. melanogaster Euch.
D. virilis Euch.
D. melanogaster Het.
Initial analysis of Drosophila virilis
dot chromosome fosmids
Almost all of the same genes are present (27/28), but
rearrangements within the chromosome are common!
Slawson et. al., 2006 Genome Biology, 7(2):R15.
Comparison of gene order and orientation
D. melanogaster
D. virilis
72 genes on both the D. virilis and D. melanogaster dots.
A minimum of 33 inversions are needed to convert order and orientation!
Leung et al 2010
“Wanderer” genes move between the dot chromosome and a
euchromatic site in the long arms; they adopt the
properties (gene size, codon bias) of their local environment
CG5262
CG9935
CG5367
rho-5
CG1732
dot
CG4038
dot: D. virilis
CG11077
CG11076
dot: D. melanogaster
Leung et al 2010
Some things to look for while annotating dot
chromosome genes….
•
•
•
•
•
•
•
Is there a homologous gene in D. melanogaster?
Is it on the dot chromosome?
Are all of the isoforms found in D melanogaster present?
How many exons?
Any unusual splice sites?
What is the order and orientation of genes compared to D. melanogaster?
Are there repetitious elements nearby?
Check out your gene on FlyBase – what is the pattern of expression in
D. melanogaster? Has a function been described?
Many dot chromosome genes are expressed at a high level - how can
this occur in a heterochromatic domain?
1. Crosslink proteins to DNA
Chromatin Immunoprecipitation - ChIP
(cells or nuclei)
2. Isolate chromatin and sonicate
qPCR
3. Incubate with antibody
ChIP-chip*
4. Isolate AB/chromatin complexes
5. Isolate DNA from complexes
ChIP-seq
Mapping chromatin marks by ChIP-chip:
Chromosome arm 3L shows a distinct shift between
heterochromatin and euchromatin
Centromere
Enrichment (log intensity ratio values)
S2 cells
HP1a
Su(var)3-9
H3K9me2
H3K9me3
genes
genes
Euchromatin
Heterochromatin
Euchromatin / heterochromatin transition point from Flybase
Pink boxes show significant enrichment (0.1% false discovery rate)
Chromosome 4 is largely heterochromatic, but shows distinct
peaks of H3K4me2/3, indicating transcription start sites
HP1a
H3K9me2
H3K9me3
H3K4me2
genes
Telomere
Enrichment (log intensity ratio values)
Centromere
A model of 9 chromatin states, based on clustering of
histone modification marks, identifies large-scale
genomic domains
 TSS (red; H3K4me3 rich)
 Polycomb (grey; H3K27me3 rich)
Heterochromatin (dark blue; H3K9me3)
An expanded view of the fourth chromosome reveals
TSS (state 1, red) and Pc (state 6, grey) domains
interspersed within heterochromatin (states 7 & 8, blue).
Pericentric heterochromatin
10 Mb
chr3L
chr4
Red
Variegating
chr4
500 kb
BG3 cells, chromatin states:
1 2 3 4 5 6 7 8 9
Might fourth chromosome genes function early, and be silenced
later? No, fourth chromosome genes show a variety of
expression patterns, including expression in the adult
S Celniker, modENCODE
Most 4th chromosome genes lie in heterochromatic space (blue),
but active genes achieve state 1 (red) at the TSS
1360
Active fourth chromosome genes show depletion of HP1a and
H3K9me3 at the TSS, but enrichment across the body of the gene
Average enrichment
chromosome 4
TSS-relative position
RNA pol II
H3K4me3
HP1 H3K9me2 H3K9me3
Transcription levels are similar to euchromatic genes!
The fourth chromosome: a repeat rich domain
with “heterochromatic” genes
1360
Future: try to determine what feature drives 4th chromosome
gene expression that is absent from euchromatic genes (hsp70).
Eight new genomes
Expanded dot
chromosomes?
44
Heterochromatin formation on the dot chromosome…
1
Heterochromatin formation changes chromatin at
the nucleosome level, eliminating HS sites at the TSS of
euchromatic genes; silencing is dependent on HP1a
2
Fourth chromosome genes are larger, have more
introns, and less codon bias than euchromatic genes
Fourth chromosome genes show high levels of HP1a
and H3K9 methylation over the body of the gene,
but maintain access at the TSS.
Next steps: what makes fourth chromosome
genes robust? Lets look for fourth chromosome motifs!
Question Slides
Are there chromosomal proteins unique to the fourth
chromosome? Yes – POF (Painting of Fourth) is uniquely
associated with active genes on the fourth chromosome
Green = HP1a Red = POF
A role for POF? A different configuration for HP1a?
(See J Larsson, PLoS Genet. 11:e209 for more on POF)
Drosophila melanogaster: 1/3 heterochromatin.
Pericentric heterochromatin is under-replicated in polytene
chromosomes; the arms fuse in the chromocenter
Drawing of polytene chromosomes modified from TS Painter, 1934, J. Hered 25: 465-476.
Clark and Elgin, 1992 Nucleic Acids Res. 20:6067
HP1 sequence from Drosophila, mouse, human and mealy bug
identifies chromo domain & chromo shadow domain
HP1 from mammals can rescue mutations in flies and yeast!
Position Effect Variegation in Drosophila:
an assay for heterochromatic packaging
white
Wild Type
Inversion
i
HP1 is a trans-acting modifier of PEV
Su(var)
(1 copy HP1)
E(var)
(3 copies HP1)
Eissenberg et al
The heterochromatic hsp26 transgene:
Cryderman et al 1999 (Wallrath & Gilmour labs)
- loss of accessibility at the TSS when in heterochromatin
- reversed in an HP1 mutant background
Chromosome 4 short arm is a unique heterochromatic domain
Haynes et al. 2007
Translocation away from the chromocenter results in loss
of silencing – spatial organization plays a role
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
The centroid of
each cluster is
closest mean.
the new mean.
Repeat the last
two steps.
Chromatin states are highly intersperseda folded view of the chromosome (Bg3 cells)
Polycomb
Pericentric
heterochromatin
Chromatin states reveal cell type specific patterns
(note facultative heterochromatin – light blue)
BG3
S2
Chromosome 3L
A folded view of the chromosome reveals
TSS and Pc states within chromosome 4
(Bg3 cells)
4th chromosome
Pericentric
heterochromatin
Chromosome 4 shows a distinct subset of Polycomb
sites in a cell-type specific pattern
H3K9me3
(S2 cells)
H3K9me3
(Bg3 cells)
Polycomb
(S2 cells)
Polycomb
(Bg3 cells)
genes
genes
A subset of 4th genes is associated with Polycomb;
these domains are permissive for reporter expression
(red eyed fly).
Heterochromatin formation on the dot chromosome…
3
How are specific domains targeted for
heterochomatin formation?
Repetitious elements such as 1360 are targeted by a
mechanism involving the RNAi system
Analysis of chromosome 4 identified 1360 as a target.
P[1360, hsp70-w] silencing depends on the
reporter’s position in the genome
3’ P
FRT
1360
FRT
hsp70-white
5’ P
Haynes et al 2006 Curr Biol 16: 2222
FLP-mediated removal of 1360 results in loss of silencing
3’P
FRT
1360 FRT
hsp70-white
3’P
5’P
+ FLP recombinase
FLP14
FLP16
FRT
hsp70-white
5’P
1360 excised
FLP4
FLP5
Haynes et al
2006 Curr Biol
16: 2222
Mutations in RNAi components impact PEV
spn-E (hls)
Dmp68/Lip
dFMR1
Results from Birchler, Elgin, Schedl, others;
note also esiRNA pathway, Siomi, Hannon & others
Mutations in RNAi components piwi, aubergine
and homeless suppress PEV
y w; 118E-10/+
+
+
piwi1
+
aubQC42
hls∆125
+
+
y w; 118E-10/+
y w; 39C-12/+
y w; 39C-12/+
B Leibovitch in Pal-Bhadra et al, 2004
piwi
aub
hls
A tentative model for heterochromatin targeting –
HP1a – PIWI interaction (piRNA)
Cytoplasm
Aub
piRNA
PIWI
Ago3
PIWI
HP1
Nucleus
Heterochromatin
PIWI (binds piRNA) is an argonaute family member
and nuclear protein which interacts with HP1a
transposon