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 ID: 811092
Download The PPT/PDF document "The Evolution of a Heterochromatic" is the property of its rightful owner. Permission is granted to download and print the materials on this web site for personal, non-commercial use only, and to display it on your personal computer provided you do not modify the materials and that you retain all copyright notices contained in the materials. By downloading content from our website, you accept the terms of this agreement.
Slide1
The Evolution of a Heterochromatic
Domain in
Drosophila
:
Investigating the Strange
Dot Chromosome
Sarah C R Elgin
January 2012
Slide2A 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
Goal: to understand the organization and functioning of the dot
chromosome in Drosophila, an unusual heterochromatic domain.
Funding: HHMI Professors Program
NIH General Medical Sciences, National Human Genome Research Institute
3
Minimum Haploid DNA Content - the C Value Paradox
Britten and Davidson, 1969 Science 165:349
Slide44
Allis et al:
Epigenetics
2007
Larger genomes reflect high levels of repeats -
retroviral and DNA transposon remnants (TEs)
Slide55
Considerations for Genome Sequencing
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
.
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.
Much of the
repetitous
DNA is packaged into heterochromatin, which maintains these regions in a compact and
transcriptionally
silent form.
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!
SCR Elgin
Slide6Coding exons
1.5%
Conserved noncoding - regulatory? 3.5%
Human Genome 3 Gb
~2 m/cell !
Key Questions
:
Is it junk or garbage?
How is DNA packaged into a nucleus?
How is silencing maintained – while
allowing appropriate transcription ?
Eukaryotic genomes are very large – and most of that DNA is non-coding!
TEs –
retroviruses,
DNA transposons
Slide7What determines phenotypes?
It
’
s not just your DNA….
Phenotype
Development
Environment (diet)
(grey bars = folate)
Epigenetics
?
Genotype
(Waterland and Jirtle 2003)
Slide8Chromatin 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 structureNuclear 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 seenIn Fragile X syndrome
Zoghbi and Beaudet 2007
Fragile X Foundation
Slide9Felsenfeld
et al. Nature 2003, 421: 448
Chromatin formation:
First step - packaging in a nucleosome array
Second - differential packaging into heterochromatin & euchromatin
DNA
Chromatin
Lodish
et.al
., Molecular Cell Biology, 4th Edition
Chromosome
(metaphase)
Histone
protein core
Slide1010
Electron Micrograph of Chromatin Fibers
(rat thymus nucleus)
Olins
et. al., 1975 J. Cell
Biol
, 64:528
0.1
m
m
Slide1111
“
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
Slide1212
The Structure of the Nucleosome Core
Rhodes, 1997 Nature 389:231, after
Luger et. al., 1997 Nature 389:251
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
Slide13DNA packaging domains
EuchromatinLess condensedChromosome armsUnique sequences;
gene rich
Replicated throughout SRecombination during meiosis
Heterochromatin
Highly condensed
Centromeres and telomeres
Repetitious sequences; gene poorReplicated in late S
No meiotic recombination
Transcriptional activators
Hyper-acetylated histone tail
Heterochromatin Protein 1 complex
Hypo-acetylated histone tail; methylated H3/K9
Slide14Heterochromatin formation – silencing counts!
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?
1
2
Slide15Short life cycle, easily maintained: good
genetic tools Polytene chromosomes: excellent cytologyBiochemical approaches
Simple genome, good
reference sequence PEV – reporter for gene silencing, heterochromatin formation Metazoan useful for behavioral, developmental and human disease research
expressed silenced
euchromatin heterochromatin
Fruit Flies!
Mary Lou
Pardue
, MIT
Slide1616
Using a
white
transgene to sample chromatin environments
mobilize P element
by crossing to stock
with
transposase
inject
transposon
carrying
white
gene
P[
white
+
]
white
67c23
insertion into
euchromatin
(99%)
insertion into
heterochromatin
( 1%)
Elgin Lab
Slide17Transposition of a P element reporter allows sampling
of euchromatic and heterochromatin domains
X
2L
3L
2R
3R
4
Silenced
1%
Active
99%
Wallrath and Elgin, 1995
And the Y chromosome
Slide18Assessing chromatin structure-
same gene, different environmentsAnalysis 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
Slide19Looking for heterochromatic proteins by immunofluorescent staining of the polytene chromosomes: discovery of HP1a
C
C
HP1
Phase
James & Elgin,1986; James et al 1989
Slide20Heterochromatin-associated gene silencing is dependent on HP1
Eissenberg et al, 1990, PNAS 87: 9923
Mutations in
gene for HP1a
Mutations recovered by T Grigliatti as suppressors of PEV.
Dosage dependent response.
Slide21HP1 interacts with both the modified histone H3K9me2/3
and the modifying enzyme
HP1:
Shadow
Chromo
SU(VAR)3-9
Histone 3
methyl-Lys9
H3 K9 methyl
transferase
[(SU(VAR)3-9 identified in screen by Reuter;
H3 interaction first shown from work in mammals – Jenuwein, Kouzarides;
demonstrated in flies by Imhof.]
Slide22Model for spreading of heterochromatin
Slide23Establishing silencing: a multi-step process
w
m4
reporter (screens by Reuter, Grigliatti, others)
Loss of euchromatin marks
Gain of heterochromatin marks
Slide24Heterochromatin formation on the dot chromosome…
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?
2
Slide25The Drosophila melanogaster
fourth chromosome exhibits an amalgam of euchromatic and heterochromatic properties (HP1a association)
C
C
HP1
Phase
James & Elgin,1986; James et al 1989
Heterochromatic properties:
late replication, lack of recombination
high repeat density (30%)
antibody staining of HP1, H3K9me2/3
But…
the fourth has ~ 80 genes in distal 1.2 Mb
these genes are transcriptionally active!
Slide26Most
hsp70-white
reporters exhibit variegation
on insertion into the fourth chromosome
2-M1021
39C-12
2-M390
39C-52
Sun et al 2004; Riddle et al 2007
Slide27Use comparative genomics to learn more
about heterochromatic domains, analyzing the
dot chromosomes and a control euchromatic
region of Drosophila genomesOur GEP Research Goal:
FlyBase
:
http://flybase.org
Reference
Status
Completed
Annotation
Sequence Improvement
New Project
Slide28Genomics Education Partnership (GEP)
Partners are generally PUI schools; faculty join by
attending one-week workshop at WU. Shared
work organized on GEP website.
Slide29The
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
Slide30Dot chromosome genes: introns are larger, exons show less codon bias
Heterochromatic
Dot
Euchromatic
D. melanogaster
Het.
D. melanogaster
Euch.
D. virilis
Euch.
D. melanogaster
Dot
D. virilis
Dot
Intron
Size
Codon
Bias
Leung
et al
.
2010 Genetics 185:1519-1534
Slide31Almost all of the same genes are present (27/28), but rearrangements within the chromosome are common!
Initial analysis of Drosophila virilis
dot chromosome fosmids
Slawson
et. al., 2006 Genome Biology, 7(2):R15.
Slide32Comparison of gene order and orientation
72 genes on both the
D.
virilis and D. melanogaster
dots.
A minimum of 33 inversions are needed to convert order and orientation!
D. virilis
D. melanogaster
Leung et al 2010
Slide33dot:
D. virilis
dot:
D. melanogaster
CG9935
CG5367
rho-5
CG4038
CG11076
CG1732
CG5262
CG11077
dot
“
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
Leung et al 2010
Slide34Is 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?
Some things to look for while annotating dot chromosome genes….
Slide35Ch
romatin
I
mmuno-
p
recipitation - ChIP
(cells or nuclei)
qPCR
ChIP-chip*
ChIP-seq
1. Crosslink proteins to DNA
2. Isolate chromatin and sonicate
3. Incubate with antibody
4. Isolate AB/chromatin complexes
5. Isolate DNA from complexes
Slide36Mapping chromatin marks by ChIP-chip:
Chromosome arm 3L shows a distinct shift between heterochromatin and euchromatin
HP1a
Su(var)3-9
H3K9me2
H3K9me3
genes
genes
Euchromatin
Heterochromatin
Centromere
S2 cells
Euchromatin / heterochromatin transition point from Flybase
Pink boxes show significant enrichment (0.1% false discovery rate)
Enrichment (log intensity ratio values)
Slide37Chromosome 4 is largely heterochromatic, but shows distinct
peaks of H3K4me2/3, indicating transcription start sites
HP1a
H3K9me2
H3K9me3
H3K4me2
genes
Centromere
Telomere
Enrichment (log intensity ratio values)
Slide38A model of 9 chromatin states, based on clustering of histone modification marks, identifies large-scale genomic domains
Heterochromatin (dark blue; H3K9me3)
TSS (red; H3K4me3 rich)
Polycomb (grey; H3K27me3 rich)
Slide3910 Mb
chr3L
BG3 cells, chromatin states:
Pericentric heterochromatin
1
2
3
4
5
6
7
8
9
chr4
500 kb
chr4
Red
Variegating
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).
Slide40Might 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
Slide41Most 4
th
chromosome genes lie in heterochromatic space (blue),
but active genes achieve state 1 (red) at the TSS
1360
Slide42Active fourth chromosome genes show depletion of HP1a and
H3K9me3 at the TSS, but enrichment across the body of the gene
Transcription levels are similar to
euchromatic
genes!
Average enrichment
TSS-relative position
RNA pol II
H3K4me3
HP1
H3K9me2
H3K9me3
chromosome 4
Slide43The fourth chromosome: a repeat rich domain with
“heterochromatic” genes
1360
Future: try to determine what feature drives 4
th
chromosome
gene expression that is absent from euchromatic genes (hsp70).
Slide44Eight new genomes
Expanded dot
chromosomes?
44
Slide45Heterochromatin formation on the dot chromosome…
Heterochromatin formation changes chromatin at
the nucleosome level, eliminating HS sites at the TSS of euchromatic genes; silencing is dependent on HP1a
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!
1
2
Slide46Question Slides
Slide47A role for POF? A different configuration for HP1a?
Are there chromosomal proteins unique to the fourth chromosome? Yes – POF (Painting of Fourth) is uniquely associated with active genes on the fourth chromosome
(
See J Larsson,
PLoS
Genet. 11:e209 for more on POF)
Green = HP1a Red = POF
Slide48Drosophila 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.
Slide49HP1 sequence from Drosophila, mouse, human and mealy bug identifies chromo domain & chromo shadow domain
Clark and Elgin, 1992 Nucleic Acids Res. 20:6067
HP1 from mammals can rescue mutations in flies and yeast!
Slide50Position Effect Variegation in Drosophila:
an assay for heterochromatic packaging
white
Wild Type
Inversion
HP1 is a trans-acting modifier of PEV
E(var)
(3 copies HP1)
Su(var)
(1 copy HP1)
i
Eissenberg et al
Slide51The heterochromatic hsp26
transgene: - loss of accessibility at the TSS when in heterochromatin
- reversed in an HP1 mutant background
Cryderman et al 1999 (Wallrath & Gilmour labs)
Slide52Chromosome 4 short arm is a unique heterochromatic domain
Haynes et al. 2007
Slide53Translocation away from the chromocenter results in loss of silencing – spatial organization plays a role
Slide54Define chromatin states by K-means clustering
(using enrichment values for 1 kb chromatin fragments)
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.
H3K9me3
H3K4me3
Slide55Chromatin states are highly interspersed-a folded view of the chromosome (Bg3 cells)
Polycomb
Pericentric heterochro-matin
Slide56Chromatin states reveal cell type specific patterns
(note facultative heterochromatin – light blue)
Chromosome 3L
BG3
S2
Slide57A folded view of the chromosome reveals TSS and Pc states within chromosome 4
(Bg3 cells)
Pericentric heterochromatin
4th chromosome
Slide58Chromosome 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
Slide59A subset of 4
th
genes is associated with Polycomb;
these domains are permissive for reporter expression
(red eyed fly).
Slide60Heterochromatin formation on the dot chromosome…
How are specific domains targeted for
heterochomatin formation?
Repetitious elements such as
1360
are targeted by a mechanism involving the RNAi system
3
Slide61hsp70-white
5
’
P
3
’
P
FRT
FRT
1360
Analysis of chromosome 4 identified
1360
as a target.
P
[
1360, hsp70-w
] silencing depends on the
reporter
’
s position in the genome
Haynes et al 2006 Curr Biol 16: 2222
Slide62FLP-mediated removal of
1360
results in loss of silencing
+ FLP recombinase
hsp70-white
5
’
P
3
’
P
FRT
FRT
1360
hsp70-white
5
’
P
3
’
P
FRT
1360
excised
FLP14
FLP16
FLP4
FLP5
Haynes et al 2006 Curr Biol 16: 2222
Slide63Mutations in RNAi components impact PEV
spn-E (hls)
Dmp68/Lip
dFMR1
Results from Birchler, Elgin, Schedl, others;
note also esiRNA pathway, Siomi, Hannon & others
Slide64y w
; 118E-10/+
y w
; 39C-12/+
piwi
aub
hls
Mutations in RNAi components
piwi, aubergine
and
homeless
suppress PEV
y w
; 118E-10/+
y w
; 39C-12/+
piwi
1
+
aub
QC42
+
hls
∆125
+
+
+
B Leibovitch in Pal-Bhadra et al, 2004
Slide65A tentative model for heterochromatin targeting –
HP1a – PIWI interaction (piRNA)
PIWI
transposon
HP1
Aub
piRNA
Ago3
PIWI
Heterochromatin
PIWI (binds piRNA) is an argonaute family member
and nuclear protein which interacts with HP1a
Cytoplasm
Nucleus