Marc Bhler

(Epi)genome regulation
by RNA turnover pathways

INTRODUCTION
Data from a variety of organisms have shown that
the assembly of silent chromatin coincides with
the presence or absence of non-protein-coding
RNAs (ncRNAs). These range from long ncRNAs,
which have been classically implicated in the reg-
ulation of dosage compensation and genomic
imprinting, to small ncRNAs involved in hetero-
chromatin assembly via the RNA interference
(RNAi) pathway. This raises the question of how
common ncRNAs control gene expression at the
GROUP LEADER
level of chromatin.
Marc Bhler
Research in our group is focused on the mecha-
marc.buehler@fmi.ch
nisms and conservation of epigenetic processes
that are under the control of ncRNAs and the
TECHNICAL / RESEARCH
processing thereof, in the conviction that a better
ASSOCIATES
understanding of the role of RNA in cellular
Nathalie Laschet
mechanisms will yield promising new drug targets
Yukiko Shimada
and tools for the treatment of human disease. Our
approach is first to study fundamental processes
POSTDOCTORAL
in simple model organisms, taking advantage of
FELLOWS
the power of fission yeast. The insights gained
Julien Bethune
from our work with yeast we then expand in
Philip Knuckles
higher eukaryotes.
Raghavendran Kulasegaran Shylini
Currently, we are investigating the role of yeast
Daniele Oberti
and vertebrate RNAi pathways in genome regula-
Tanel Punga*
tion. RNAi pathways are prevalent throughout the
eukaryotic kingdom and are well known as regula-
PhD STUDENTS
tors of gene expression post-transcriptionally in
Stephan Emmerth*
the cytoplasm. However, the nuclear activity of
Claudia Keller
RNAi is less understood. Recently, we discovered
Katarzyna Kowalik
that the fission yeast RNAi pathway represses
Veronika Ostapcuk
genome activity by a chromatin silencing mecha-
Rieka Stunnenberg
nism we refer to as co-transcriptional gene silenc-
Rodrigo Villasenor Molina
ing (CTGS). Employing a wide range of ap-
Katrina Woolcock
proaches, our lab is unraveling the mechanism,
physiological relevance, and evolutionary conser-
vation of CTGS and related pathways.
*left the group

FMI Report 2011/2012 49

Epigenetics

CO-TRANSCRIPTIONAL GENOME REGULATION sociated proteins in the respective complexes by

BY RNAi direct epifluorescence in living cells.
This work identified the S. pombe RNAi protein
K.Woolcock, in collaboration with D.Gaidatzis
Dicer (Dcr1) as a predominantly nuclear protein
and H.-R.Hotz
enriched at nuclear pores (5). We have found that
In the fission yeast Schizosaccharomyces pombe, this subcellular localization pattern is regulated
the RNA interference (RNAi) pathway generates through its double-stranded RNA-binding do-
small interfering RNAs (siRNAs), which mediate main (dsRBD). The fold of this domain is rather
heterochromatic silencing of centromeric repeats. unusual and embeds a novel zinc-binding motif
Recently, we demonstrated that RNAi also re- (4). We have demonstrated that nuclear retention
presses genomic elements other than constitutive of Dcr1 is lost once the coordination of zinc is
heterochromatin. Using DNA adenine methyl- disrupted. As a result, centromeric heterochroma-
transferase identification (DamID), we discov- tin is lost and the tight repression of stress-
ered that RNAi proteins physically associate with response genes via CTGS is disrupted.
some euchromatic genes, noncoding RNA genes, Interestingly, similar to disrupting zinc-coordi-
and retrotransposon long terminal repeats (3). nation genetically, Dcr1 is deported to the cyto-

Fig.1. Model for RNAi-me-

diated co-transcriptional A B C
gene silencing (CTGS) of
(e.g. transient heat shock)

Atf1-bound genes (BANCs)

at nuclear pores. A. RNAi

chronic heat stress

normal conditions

stressful condition

tightly represses BANCs

NC

NC
under normal conditions.

BA
BA

NC
BA

B. Strong transcriptional nucleus

nucleus Atf1 Dcr1
Atf1 Dcr1

activation of BANCs over- P Atf1 Dcr1 nucleus

rides CTGS. C. Dcr1 leaves cytoplasm
cytoplasm
the nucleus at prolonged cytoplasm
heat stress, abrogating
CTGS

This physical association of RNAi with chromatin plasm under chronic heat-shock conditions. Our
is sufficient to trigger a silencing response but not latest results support a model in which the Dcr1
to assemble heterochromatin. The mode of silenc- dsRBD acts as a thermoswitch that deports Dcr1
ing at the newly identified RNAi targets is consist- to the cytoplasm under chronic heat-stress condi-
ent with a co-transcriptional gene silencing model tions (2). This disrupts RNAi-mediated CTGS and
(CTGS) and functions with trace amounts of siR- may enable the expression of genes implicated in
NAs. Our most recent work has highlighted a role the acquisition of thermotolerance (Fig.1).
for nuclear pores and the stress response tran-
scription factor Atf1 in coordinating RNAi-medi- IDENTIFICATION OF A POTENTIAL ANTIFUNGAL
ated CTGS (2). Our observation that RNAi com- DRUG TARGET
ponents interact with chromatin at nuclear pores,
S.Emmerth and Y.Shimada, in collaboration with
keeping stress response genes tightly repressed,
H.-R.Hotz and H.Gut (FMI), and P.Barraud and
provided first insights into the physiological rel-
F.Allain (ETH Zurich)
evance of RNAi-mediated CTGS (Fig.1).
Dicer is a key enzyme in RNAi pathways that is
RNAi REGULATION IN RESPONSE TO AN conserved from yeast to humans. It is involved in
ENVIRONMENTAL CUE the biogenesis of siRNAs and microRNAs from
double-stranded RNA precursors. Dicer is a large
R.Stunnenberg, S.Emmerth, Y.Shimada
multidomain protein containing RNA helicase/
Biochemical and genetic analyses over the last ATPase, DUF, and PAZ domains, two RNAseIII
years have greatly improved our mechanistic un- domains and a C-terminal dsRBD domain. Coun-
derstanding of how RNAi pathways function. ter-intuitively, we found that RNA binding of S.
However, little has been examined at a cell bio- pombe Dcr1s dsRBD is dispensable for the gen-
logical level. Important questions such as the sub- eration of siRNAs in S. pombe. Instead, the dsRBD
cellular localization of the RNAi pathway and the controls the nucleo-cytoplasmic distribution of
regulation thereof remain largely unanswered. Dcr1. Although adopting a dsRBD fold, the dsRBD
For a more comprehensive understanding of the domain of S. pombe Dcr1 harbors additional
spatial organization of the fission yeast RNAi structural elements such as a novel zinc-binding
pathway, we analyze RNAi factors and their as- motif. The coordination of zinc by this motif is

50 FMI Report 2011/2012


A B
100
50
relative SPR response (RU)
0 heterochromatin occurs,
0.01 0.1
-50
-100
HP1 wild type
-150 HP1 RNA binding mutant
crucial for the proper folding of the dsRBD of
Dcr1, which in turn is a prerequisite for the reten-
tion of Dcr1 in the nucleus. This zinc-binding
motif is present in more than 30 proteins of the
dicer family. Strikingly, many of the Dicers con-
taining zinc-binding ligands are found in human
and plant pathogens, such as species of Aspergillus.
This is particularly interesting because a S. pombe
mutant strain expressing Dcr1 that fails to coor-
dinate zinc grows more slowly and is less viable
than wild-type cells or cells completely lacking
Dcr1 (4). Importantly, the zinc-binding motif is
not found in Dicers of plant and animal cells.
Thus, its high degree of conservation among dif-
ferent yeast species and its mutational intolerance
make the zinc-binding motif a prime antifungal
target. Compounds reacting with the Dicer dsRBD
zinc-binding motif may offer new antifungal
strategies to cope with the increasing incidence of
invasive mycoses.
LINKING TRANSCRIPTION WITHIN HETERO-
CHROMATIN TO RNA DEGRADATION
C.Keller, K.Woolcock and R.Stunnenberg,
in collaboration with R.Adaixo and S.Hiller
(Biozentrum, University of Basel)
While studying the mechanisms leading to the as-
sembly of higher order chromatin structures such
as heterochromatin, we are also investigating how
such chromatin structures impact on the expres-
sion of genetic information. Although generally
perceived as an inert and transcriptionally inactive
chromatin structure, our studies in S. pombe have
demonstrated that heterochromatin is not always
refractory to transcription. Clearly, some promot-
ers can be transcribed within heterochromatic
domains and we have demonstrated that robust
silencing of heterochromatic genes can involve
RNA processing events.
Recently, we obtained novel insights into the
mechanism linking transcription within hetero-
chromatin to downstream RNA degradation. We
made the rather surprising observation that the
Fig.2. A. SPR responses
for competitive binding of
H3K9me3 and RNA to
either wild-type (black) or
mutant HP1Swi6 (red).
[RNA] / [HP1]
HP1 B. If transcription within
exchange
1 10
HP1Swi6 binds newly syn-
eviction
thesized RNA (red) and
dissociates from nucleo-
me
me
me
me
K9
K9
K9
K9
somes (grey) as a result of
H3
H3
H3
H3
competition between RNA
and the histone tail for
HP1Swi6 binding (blue)
RNA degradation
highly conserved HP1 protein, one of the major
components of heterochromatin, plays a crucial
role in this process. By a combination of in vivo
and in vitro experiments, we found that one of the
two HP1 proteins in S. pombe, HP1Swi6, guarantees
tight repression of heterochromatic genes through
RNA sequestration and degradation. Stimulated
by positively charged residues in the hinge region,
RNA competes with methylated histone H3K9 for
binding to the chromodomain of HP1Swi6 (Fig.2).
Hence, HP1Swi6 binding to RNA is incompatible
with stable heterochromatin association. We pro-
pose a model in which HP1Swi6 acts as an hetero-
chromatin-specific checkpoint capturing and
priming heterochromatic RNAs for the RNA
degradation machinery. Sustaining a functional
checkpoint requires continuous exchange of
HP1Swi6 within heterochromatin, which explains
the dynamic localization of HP1 proteins on het-
erochromatin. p
Selected publications
1. Keller C, Adaixo R, Stunnenberg R, 4. Barraud P, Emmerth S, Shimada Y,
Woolcock K, Hiller S, Bhler M (2012) Hotz HR, Allain FH, Bhler M (2011)
HP1Swi6 mediates the recognition and An extended dsRBD with a novel
destruction of heterochromatic RNA zinc-binding motif mediates nuclear
transcripts. Mol Cell (in press) retention of fission yeast Dicer.
EMBO J 30:4223-4235
2. Woolcock KJ, Stunnenberg R,
Gaidatzis D, Hotz HR, Emmerth S, 5. Emmerth S, Schober H, Gaidatzis D,
Barraud P, Bhler M (2012) RNAi Roloff T, Jacobeit K, Bhler M (2010)
keeps Atf1-bound stress response Nuclear retention of fission yeast Dicer
genes in check at nuclear pores. is a prerequisite for RNAi-mediated
Genes Dev 26:683-692 heterochromatin assembly. Dev Cell
18:102-113
3. Woolcock KJ, Gaidatzis D, Punga T,
Bhler M (2011) Dicer associates with
chromatin to repress genome activity
in Schizosaccharomyces pombe. Nature
Struct Mol Biol 18:94-99
FMI Report 2011/2012 51