Epigenetic genome control by RNAi and transposonderived proteins

1
Epigenetic genome control by RNAiand
transposon-derived proteins

• Shiv Grewal, Ph.D.
• Center for Cancer Research
• National Cancer Institute

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Distinct levels of chromatin organization
Nuclear membrane
Chromatin fiber
Chromatin fiber(30 nm dia.)
Variant histones (H2A.Z etc)
H1
Nucleosomes
DNA
Nuclear pore
Nuclear matrix
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Chromatin modifiers and RNA processing factors
suppress transcriptional noise across genome
Repeats
ORF
Accumulation of aberrant RNAs can lead to genomic
instability
4
RNAi and heterochromatin factors cooperate with a
variant histone H2A.Z to suppress antisense RNAs
Chromatin modifiers and RNA processing activities
Readthrough transcript
Repeats
ORF
Accumulation of aberrant RNAs can lead to genomic
instability
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Topics

• Silencing of retrotransposons and repeat elements
  by RNAi and chromatin-modifying factors
• Genome-wide suppression of antisense RNAs by a
  variant histone and the RNAi machinery

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S. pombe genome contains several classes of
repeat elements that are assembled in repressive
chromatin
All retroelements are bound by transposase-derived
CENP-B proteins (Cam et al Nature 2008)
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CENP-Bs localize to retrotransposons and their
remnants in the S. pombe genome
Abp1
Cbh1
Cbh2
Chromosome III
Chromosome II
Chromosome I
cen3
tel3L
tel3R
cen2
mat
tel2L
tel2R
cen1
tel1L
tel1R
rDNA
rDNA
Abp1
Fold enrichment
500
1,000
1,500
2,000
Cbh1
30
25
20
Fold enrichment
15
Tf2-5
10
5
1,000
2,000
3,000
4,000
1,000
2,000
3,000
4,000
5,000
Chromosome Position (Kb)
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CENP-Bs recruit Clr3 and Clr6 histone
deacetylases to repress Tf2 retroelements
IP Western
Clr3 SHREC
Relative fold enrichment
Chromosome III Position (Kb)
RT-PCR
Clr6 HDAC
Expression Mutant/ WT
abp1? cbh1?
abp1? cbh2?
abp1?
cbh1?
cbh2?
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CENP-Bs recruit SHREC and Clr6 histone
deacetylases to repress Tf2 retroelements
HDACs
Clr3/SHREC
Clr6
CENP-B
LTR
Transcriptional and recombinational suppression
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CENP-Bs and their associated HDACs protect
integrity of the genome
Tf1-neo expression
neo
Tf1
LTR
abp1?
cbh1?
WT
neoR colonies
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SHREC activities facilitate positioning of
nucleosomes to suppress transcriptional noise
Silencing and recombination suppression
Micrococcal nuclease digestion patterns
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S. pombe genome contains several classes of
repeat elements that are assembled in repressive
chromatin
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Heterochromatin a versatile recruiting platform
S-phase transcription Nucleation
Effectors
H3K9me
HP
HP
HP
Repeat elements
HP
Pol II
HP
Ac
HP
HP
RDRC
Chp1
Clr4
Ago1
Tas3
HP
TFIIIC
RITS
Dcr1
siRNA
Boundary element
HP Swi6, Chp2
Cis-PTGS
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HP1 proteins and their associated HDACs
collaborate to enforce heterochromatic
transcriptional silencing
Transcriptional silencing
HDACs
Clr3/ SHREC
Clr6
cen2
HP1
Swi6
Chp2
H3K9me
Clr4 HMTase
repeat
Pol2 txn RNAi
cen2
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Topics

• Silencing of retrotransposons and repeat elements
  by RNAi and chromatin-modifying factors
• Genome-wide suppression of aberrant RNAs by a
  variant histone and the RNAi machinery

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Large proportion (gt90) of the S. pombe genome
including the intergenic regions are transcribed
in both directions
Dutrow et al 2008 (Cairns lab) Wilhelm et al 2008
(Bahler lab) Nicolas et al 2007 (Our lab)
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RNAi and heterochromatin factors cooperate with a
variant histone H2A.Z to suppress antisense RNAs
Chromatin modifiers and RNA processing activities
Readthrough transcript
Repeats
ORF
Accumulation of aberrant RNAs can lead to genomic
instability
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H2A.Z is enriched at 5 ends of genes
ChIP profiling of H2A.Z

• H2AZ is a histone H2.A variant that is deposited
  onto chromatin by the SWR-C
• Loss of H2A.Z affects various chromosomal
  processes but its exact function is not known

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Loss of H2A.Z (pht1) causes disproportionate
increase in antisense transcripts at convergent
gene loci
Strand-specific RT-PCR
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H2A.Z acts synergistically with Clr4 and Ago1 to
suppress antisense transcripts
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Antisense RNAs correspond to read-through
transcripts rather than new initiation events
cyp7
c16h5.04
Strand-specific probe 1
probe 2
probe 3
5'
3'
read-through transcript
5'
3'
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PolII
PolII
PolII
CONVERGENT GENE
CONVERGENT GENE
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Readthrough antisense RNAs accumulate in
exosome (rrp6) mutant
cyp7
c16h5.04
probe 1
probe 2
probe 3
5'
3'
read-through transcript
5'
3'
Exosome
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Loss of exosome causes upregulation of antisense
transcripts in a pattern identical to H2A.Z clr4
mutant
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H2A.Z and Pol II-associated Ago1 are components
of RNA quality control mechanism involved in
antisense suppression
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Summary

• Transposon-derived CENP-B proteins and RNAi
  target chromatin modifying activities which in
  turn facilitate nucleosome positioning to
  suppress transcriptional noise at repeat elements
• H2A.Z is a component of genome indexing mechanism
  that cooperates with RNAi and heterochromatin
  factors to suppress antisense RNAs

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Acknowledgments
Grewal Lab
Martin Zofall Tamas Fischer Ke Zhang Bowen
Cui Francisca Reyes-Turcu Natalia
Kommissarova Ken-ichi Yamane Chanan Rubin Takeshi
Mizuguchi Nazanin Ashourian
Former Lab Members
Tomoyasu Sugiyama (Tsukuba Univ) Estelle Nicolas
(CNRS, Toulouse) Ee Sin Chen (National Univ.
Singapore) Ken-ichi Noma (Wistar
Institute) Songtao Jia (Columbia, NY) Ira Hall
(Univ Virginia) Jun-ichi Nakayama (Riken,
Kobe) Takatomi Yamada (Univ Tokyo) Hugh Cam
(Boston)
Collaborators
Peter FitzGerald (NCI) Tim Veenstra and Ming
Zhou (NCI)