supplementary information for - nature research · 2013-10-04 · calculated (est2 n = 48, pd 30;...
TRANSCRIPT
Supplementary Information
for
Telomeric RNA-DNA hybrids affect telomere length dynamics and senescence
Bettina Balk1,2, André Maicher1,2, Martina Dees1, Julia Klermund1, Sarah Luke-
Glaser1, Katharina Bender1 and Brian Luke1,3
1Zentrum für Molekulare Biologie der Universität Heidelberg (ZMBH), DKFZ-
ZMBH Alliance, Heidelberg, Germany
2These authors contributed equally
List of Supplementary Information
Supplementary Figures (5)
Supplementary Tables (3)
Nature Structural and Molecular Biology: doi:10.1038/nsmb.2662
Supplementary Figure 1. Telomeric RNA-DNA hybrids are regulated by
RNase H enzymes
(a) RNA-DNA hybrids exist at telomeres and accumulate in RNase H mutants. The
identical ChIP as described in Fig. 1a, however quantified showing % input on the Y-
axis. Values are represented as % input of telomeric DNA recovered and are the
means of seven (+ab) and five (-ab) biological replicates, error bars depict ± s.e.m.
P values were derived from two-tailed Student’s t-tests (NS = not significant). (b) The
RNA-DNA hybrid signal is RNase H sensitive. Following overnight
immunoprecipitation with the S9.6 antibodies, one wild type sample was treated with
recombinant RNase H before the washing steps (see online methods). (c) TERRA
levels are not increased in rnh1 rnh201 mutants. qRT-PCR was performed for the
6Y’, 1L and 15L telomeres using the indicated mutants (at approx. PD 15 following
tetrad dissection). sir2 cells served as positive control where TERRA is upregulated.
Average values were derived from 3 biological replicates where error bars depict ±
s.e.m.
ba
c
WT
rnh1 rnh201
0
0.2
0.4
0.6
0.8
1.0
ab
RNase H
6Y´15L rDNA
+ +
+
1L
0
0.05
0.10
0.60
0.80
1.00
P = 0.46
ab + + +
6Y´15L rDNA
+
1L
NS
P = 0.61
P = 0.001
***
P = 0.03
*P = 0.003
**
NS
P = 0.09
**P =0.005
NS
P = 0.02
*
% I
np
ut
% I
np
ut
Fo
ld e
nrich
me
nt
co
mp
are
d t
o W
T
0
100
200
300
1
2
3
4
5
1L 15L
WT
rnh1 rnh201est2est2 rnh1 rnh201sir2
– – – –
–
– –
+ +
+
–
– –
+ +
+
–
– –
+ +
+
–
– –
Nature Structural and Molecular Biology: doi:10.1038/nsmb.2662
Supplementary Figure 2. RNA-DNA hybrids accumulation promotes
homologous recombination
(a) Raw data from Fig. 1b. The individual pairs within a graph were derived from the
same tetrad for direct comparisons. (b) The second and third biological replicates
from Fig. 2a. (c) Premature survivor formation does not account for the increased rate
of HR in rnh1 rnh201 cells. Genomic DNA derived from tetrad 2 of the curve shown
in Fig. 1b was digested with Xho1. TRFs of Y´ telomeres can be seen between 1164
and 992 bps. Recombination rates for Fig. 2a were determined at around PD 35, when
type II survivors had clearly not yet formed.
Day 1 3 5 7 9 11 1 3 5 7 9 11
est2 est2 rnh1 rnh201
19531882
15151482
1164
992
710
2799
3639
4899
bp
PD 9 27 43 55 73 92 9 26 44 59 71 85
Type IIsurvivors
Type IIsurvivors
8
6
4
2
0
est2 est2 rnh1 rnh201 est2 est2 rnh1 rnh201
Cel
l den
sity 8
6
4
2
0
8
6
4
2
0150100500 150100500
PDs PDs
a
b
Tetrad 2
Undiverged region Diverged region
050
100150200250300350
Telomeres from est2 rnh1 rnh201 tetrad 2 – PD 36 Telomeres from est2 tetrad 2 – PD 36
11 % Divergence 18 % Divergence
Tetrad 1 Tetrad 2
Tetrad 3 Tetrad 4
Tetrad 5 Tetrad 6
cTe
lom
ere
leng
th (
bp)
12% Divergence 19% Divergence
Telomeres from est2 tetrad 3 – PD 36 Telomeres from est2 rnh1 rnh201 tetrad 3 – PD 36
est2 est2 rnh1 rnh201
050
100150200250300350
Telo
mer
e le
ngth
(bp
)
Nature Structural and Molecular Biology: doi:10.1038/nsmb.2662
Supplementary Figure 3. Rnh1 and Rnh201 act redundantly to remove telomeric
RNA-DNA hybrids.
(a) The increased rate of senescence shown in Fig. 2c only occurs in absence of
telomerase. Senescence curves were performed as previously described (Fig. 1b). The
loss of RNase H activity does not result in decreased cell viability in rad52 cells, but
accelerates senescence only when both EST2 and RAD52 are co-deleted. Curve
averages were derived from 6 biological replicates for each genotype depicted, error
bars depict ± s.e.m. est2 rad52 and est2 rad52 rnh1 rnh201 curves are taken form
Fig. 2c. (b, c) Senescence curves in an est2 rad52 background were performed with
only one of the RNase H genes deleted (rnh1 or rnh201, respectively). Rates of
senescence were not affected upon deletion of one RNase H gene only. Curves
represent the means of 6 biological replicates ± s.e.m. (d) Raw data from Fig. 2c.
Genotypes are indicated.
0 10 20 30 40 50
0
50
100
150
est2 rad52est2 rad52 rnh1 rnh201 rad52 rnh1 rnh201
rad52R
ela
tive
ce
ll d
en
sity
Average PDs
est2 rad52
est2 rad52 rnh1
Re
lative
ce
ll d
en
sity
est2 rad52est2 rad52 rnh201
Average PDs
Re
lative
ce
ll d
en
sity
0 10 20 30 400
50
100
150
0 10 20 30 40 500
50
100
150
Average PDs
ba
c
PDs
0 10 20 30 40 50
6
4
2
0
Ce
ll d
en
sity (
OD
600)
est2 rad52 1
est2 rad52 rnh1 rnh201 1est2 rad52 rnh1 rnh201 exo1 1est2 rad52 2
est2 rad52 rnh1 rnh201 2est2 rad52 rnh1 rnh201 exo1 2est2 rad52 3
est2 rad52 rnh1 rnh201 3est2 rad52 rnh1 rnh201 exo1 3
est2 rad52 6
est2 rad52 rnh1 rnh201 6est2 rad52 rnh1 rnh201 exo1 6
est2 rad52 4
est2 rad52 rnh1 rnh201 4est2 rad52 rnh1 rnh201 exo1 4est2 rad52 5
est2 rad52 rnh1 rnh201 5est2 rad52 rnh1 rnh201 exo1 5
d
Nature Structural and Molecular Biology: doi:10.1038/nsmb.2662
Supplementary Figure 4. The THO mutant hpr1 shows increased telomeric
RNA-DNA hybrids and increased rates of telomeric recombination.
(a) hpr1 shows increased telomeric RNA-DNA hybrids. ChIP was carried out as
described in Fig. 1a. The values are represented as % input of telomeric DNA
recovered relative to wild type (set to 1) and are the means of 5 biological replicates,
error bars depict ± s.e.m. P values were derived from a two-tailed one-sample
Student’s t-test. (b) Deletion of HPR1 accelerates senescence in HR deficient
telomerase mutants. In the presence of RAD52 the viability loss was alleviated,
suggesting HR-mediated compensation. Liquid senescence assays were performed on
the indicated strains. Curves represent the average value for 6 biological
replicates ± s.e.m. (c) hpr1 shows increased rates of telomeric recombination. 1L
Telomere-PCR was performed on genomic DNA extracted from the two indicated
clones, which were derived form the same tetrad. 1L Telomere-PCR products were
cloned and sequenced as described in Fig. 2a and % of diverged telomeres was
calculated (est2 n = 48, PD 30; est2 hpr1 n = 39; PD 26).
a b
Rel
ativ
e ce
ll de
nsity
est2 rad52est2 rad52 hpr1
Average PDs
est2 est2 hpr1
c
Div
erge
d te
lom
eres
(%)
0
5
10
15
20
est2 est2 hpr1
0.0
0.5
1.0
1.5
2.0P = 0.05
*
6Y´15L rDNA
Rel
ativ
e to
WT
(% In
put)
NSP = 0.06 P = 0.17
NS
WT
hpr1
0 20 40 60 80 1000
50
100
150
Nature Structural and Molecular Biology: doi:10.1038/nsmb.2662
Supplementary Figure 5. Overexpression of RNH1 increases senescence in
recombination competent cells.
(a) Raw data for Fig. 5a for the indicated genotypes. (b) Raw data for Fig. 5e for the
indicated genotypes.
est2 rad52 + RNH1 3est2 rad52 + VC 3
Cell d
ensit
y (OD
600) 6
4
2
00 10 20 30 40
est2 rad52 + VC 1est2 rad52 + RNH1 1
6
4
2
00 10 20 30 40
est2 rad52 + VC 2est2 rad52 + RNH1 2
6
4
2
0 10 20 30 40
6
4
2
0 10 20 30 40
est2 rad52 + VC 5est2 rad52 + RNH1 5
Cell d
ensit
y (OD
600) 6
4
2
0 10 20 30 40
est2 rad52 + VC 4est2 rad52 + RNH1 4
6
4
2
0 10 20 30 40
est2 rad52 + VC 6est2 rad52 + RNH1 6
0
0 0 0
PD
6
4
2
0 10 20 30 40
est2 rad52 + VC 8est2 rad52 + RNH1 8
0
PD
6
4
2
0 10 20 30 40
est2 rad52 + VC 9est2 rad52 + RNH1 9
0
PD
Cell d
ensit
y (OD
600)
6
4
2
0 10 20 30 40
est2 rad52 + VC 7est2 rad52 + RNH1 7
0
PD
0 20 40 60 80 1000
2
4
6
8 est2 +VC 1est2 +RNH1 1
Cell d
ensit
y (OD
600) est2 +VC 2
est2 +RNH1 2
PD
20 40 60 80 1000
2
4
6
8
PD
20 40 60 80 1000
2
4
6
8
Cell d
ensit
y (OD
600)
PD
20 40 60 80 1000
2
4
6
8
PD
20 40 60 80 1000
2
4
6
8
Cell d
ensit
y (OD
600)
PD
20 40 60 80 1000
2
4
6
8
est2 +VC 3est2 +RNH1 3
est2 +VC 4est2 +RNH1 4
est2 +VC 5est2 +RNH1 5
est2 +VC 6est2 +RNH1 6
a
b
Nature Structural and Molecular Biology: doi:10.1038/nsmb.2662
Supplementary Table 1. Yeast strains used in this study
Yeast strains used in this study were derived from BY4741 background (his3-1, leu2-
0, ura3-0, met15-0)
Code Name Genotype
YBL7 wild type MATa
YBL588 rnh1 rnh201 MATa rnh1::KAN rnh2::NAT
YAM76 hpr1 MATa hpr1::KAN
YBB 236 est2 rnh1 rnh201 MATa/MATalpha EST2/est2::HYG RNH1/rnh1::KAN
RNH201/rnh201::NAT
YBB 261 est2 rad52 +VC MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::KAN +
pRS426 GPD
YBB 261 est2 rad52 +RNH1 MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::KAN +
pRS426 GPD RNH1
YAM184 est2 rad52 rnh1 MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::NAT
RNH1/rnh1::KAN
YAM186 est2 rad52 rnh201 MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::NAT
RNH201/rnh201::HYG
YBB238 est2 rad52 rnh1 rnh201 exoI MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::NAT
RNH1/rnh1::KAN RNH201/rnh201::HYG EXOI/exoI::URA3
YAM188
YSLG432
est2 rad52 rnh1 rnh201
7L*
MATa/ MATalpha EST2/est2::HIS3 RAD52/rad52::NAT
RNH1/rnh1::KAN RNH201/rnh201::HYG
7L::URA3
YAM156 est2 rad52 hpr1 MATa/MATalpha EST2/est2::HIS3 RAD52/rad52::NAT
HPR1/hpr1::KAN
Supplementary Table 2. Plasmids Code Name Genotype
pBL189 VC pRS426 GPD
pBB39 RNH1 pRS426 GPD RNH1
Nature Structural and Molecular Biology: doi:10.1038/nsmb.2662
Supplementary Table 3. Oligos Code Name Genotype Experiment
oBL358 1L GCGGTACCAGGGTTAGATTAGGGCTG Telomere-PCR
oBL360 6R AAATGAGGACTGGGTCATGG Telomere-PCR
oBL361 6Y` TTAGGGCTATGTAGAAGTGCTG Telomere-PCR
oAM26 7L CGGATCCCAGAGTAGAGGTAG Telomere-PCR
oBL359 oligo-dG CGGGATCCGGGGGGGGGGGGGGGGGG Telomere-PCR
oBL295 1L-fwd CGGTGGGTGAGTGGTAGTAAGTAGA ChIP
oBL296 1L-rev ACCCTGTCCCATTCAACCATAC ChIP
oLK49 6Y’-fwd GGCTTGGAGGAGACGTACATG ChIP
oLK50 6Y’-rev CTCGCTGTCACTCCTTACCCG ChIP
oLK57 15L-fwd GGGTAACGAGTGGGGAGGTAA ChIP
oLK58 15L-rev CAACACTACCCTAATCTAACCCTGT ChIP
oBL292 actin-fwd CCCAGGTATTGCCGAAAGAATGC ChIP
oBL293 actin-rev TTTGTTGGAAGGTAGTCAAAGAAGCC ChIP
oAM47 rDNA-fwd TCCAATTGTTCCTCGTTAAG ChIP
oAM48 rDNA-rev ATTCAGGGAGGTAGTGACAA ChIP
Nature Structural and Molecular Biology: doi:10.1038/nsmb.2662