Song et al. Page 1
Supplementary Table S1. Telomeric silencing associated with histone ubiquitylation and
methylation, related to supplementary Fig. S3 and S4.
TEL05R
TEL07L
Gene Deletion
Phenotype
Reference
Phenotypea
Phenotypeb
Reference
RAD6
++
[1,2]
+++++
+++++++
[2,3]
BRE1
++
+++++
+++++++
[3]
LGE1
++
+++++
++++++
UBP8
NC
NC
NC
[4]
UBP10
NC
+++
NC
[4,5,6]
SET1
++
+++++
++++++
[8,9,10]
SET2
−−−
−
−−
[9]
DOT1
++
[11]
+++++
++++
[5,9,11,12]
SIR2
++
[1]
+++++
+++++++
[13,14]
RPD3
−−−−−−
[1]
−−−
−−−−
[15]
[7]
A summary of the silencing phenotypes at the telomere-proximal regions in various deletion
lines. Rad6 is an E2 ubiquitin-conjugating enzyme, and Bre1 is an E3 ubiquitin ligase; Ubp8
and Ubp10 are histone H2B deubiquitylases. Lge1 is a Bre1-associated protein that promotes
H2B ubiquitylation [16]. Set1, Set2, and Dot1 are histone H3 lysine methyltransferases that
methylate lysine residues at lysine 4, lysine 36, and lysine 79, respectively. Strains lacking
Sir2 or Rpd3 were used as controls. TEL05R, the right-end telomere of chromosome V;
TEL07L, the left-end telomere of chromosome VII; +, increase in URA3 expression caused
by a disruption of silencing; −, decrease in URA3 expression caused by enhanced silencing;
NC, no change. aUCC1111 background. bUCC1001 background.
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 2
Supplementary Table S2. rDNA silencing associated with histone ubiquitylation and
methylation, related to supplementary Fig. S3.
rDNA
Gene Deletion
Phenotype
Reference
RAD6
+++
[17]
BRE1
++
LGE1
++
UBP8
−−−−
UBP10
++
[5]
SET1
++
[7]
SET2
−−−−
DOT1
NC
[5]
SIR2
++
[1,13,18]
RPD3
−−−−
[1,18]
A summary of the silencing phenotypes at the rDNA locus in various deletion lines. Strains
lacking Sir2 or Rpd3 were used as controls for this assay. See supplementary Table S1 for
details.
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 3
Supplementary Table S3. HM silencing associated with histone ubiquitylation and
methylation, related to supplementary Fig. S3 and S4.
HMR
HML
Gene Deletion
Phenotype
Reference
Phenotype
Reference
RAD6
+++++
[2]
+++++
[2]
BRE1
+++++
+++++++
LGE1
+++++
+++++++
UBP8
NC
NC
UBP10
+
[5]
NC
[5]
SET1
+++++
[7]
++++
[7]
SET2
−−−
[19]
NC
DOT1
+++++
[5]
+
[5]
SIR2
+++++
[20]
+++++++
[13]
RPD3
−−−−
[21]
−−−−−
A summary of the silencing phenotypes at the HM loci in various deletion lines. Strains
lacking Sir2 or Rpd3 were used as controls for this assay. See supplementary Table S1for
details.
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 4
Supplementary Table S4. Strains used in this study.
Figure
Strain
Genotype
Fig. 1
YKH045
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1
htb1-1 htb2-1 pRS314 [FLAG-HTB1-CEN-TRP1]
pRG145 [GAPDHprom-3HA-UBI4-URA3 Integrative]
Fig. 2A
BY4741
FY124
MATa his3Δ1 leu2Δ0 met15Δ0 ura3Δ0
MATa
his3Δ1
leu2Δ0
met15Δ0
rad6Δ::KanMX4
MATa
his3Δ1
leu2Δ0
met15Δ0
bre1Δ::KanMX4
MATa
his3Δ1
leu2Δ0
met15Δ0
lge1Δ::KanMX4
FY127
FY167
Fig. 2B
Y131
Y133
YZS276
YZS277
Fig. 2C
YKH045
YKH046
YKH047
Fig. 2D
BY4741
FY231
SY387a
Fig. 2E
YKH045
SY390
SY394
Source
ura3Δ0
Euroscarf
ura3Δ0
Euroscarf
ura3Δ0
Euroscarf
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1
can1-100 hta1Δ-htb1Δ::LEU2 hta2Δ-htb2Δ::URA3
(non-functional) pRS426-HTA1-HTB1
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1
can1-100 hta1Δ-htb1Δ::LEU2 hta2Δ-htb2Δ::URA3
(non-functional) pRS426-HTA1-htb1-K123R
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1
can1-100 hta1Δ-htb1Δ::LEU2 hta2Δ-htb2Δ::URA3
(non-functional) pRS413-HTA1-FLAG-HTB1
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1
can1-100 hta1Δ-htb1Δ::LEU2 hta2Δ-htb2Δ::URA3
(non-functional) pRS413-HTA1-FLAG-htb1-K123R
See Fig. 1
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1
htb1-1 htb2-1 pRS314 [FLAG-htb1-K123R-CENTRP1] pRG145 [GAPDHprom-3HA-UBI4-URA3
Integrative]
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1
htb1-1 htb2-1 ubp8Δ::KanMX4 pRS314 [FLAG-HTB1CEN-TRP1] pRG145 [GAPDHprom-3HA-UBI4-URA3
Integrative]
See Fig. 2A
MATa
his3Δ1
ubp8Δ::KanMX4
MATa
his3Δ1
ubp10Δ::KanMX6
Mary Ann Osley
[24]
Mary Ann Osley
[24]
Mary Ann Osley
& C. David Allis
[25,26]
Mary Ann Osley
& C. David Allis
[25,26]
Jerry L. Workman
& Shelley L.
Berger
[22,23]
Jerry L. Workman
& Shelley L.
Berger
[22,23]
met15Δ0
ura3Δ0
Euroscarf
leu2Δ0
met15Δ0
ura3Δ0
This study
From BY4741
This study
From
YKH045
This study
From
See Fig. 1
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1
htb1-1 htb2-1 sir2Δ::KanMX4 pRS314 [FLAG-HTB1CEN-TRP1] pRG145 [GAPDHprom-3HA-UBI4-URA3
Integrative]
MATa ura3-1 leu2-3,112 his3-11,15 trp1-1 ade2-1
htb1-1 htb2-1 sir2Δ::KanMX4 pRS314 [FLAG-htb1K123R-CEN-TRP1] pRG145 [GAPDHprom-3HAUBI4-URA3 Integrative]
BY4741
FY124
FY127
FY231
SY387
See Fig. 2A
See Fig. 2A
See Fig. 2A
See Fig. 2D
See Fig. 2D
Fig.
S1
and S2
YKH045
See Fig. 1
Fig. S3A
UCC1111
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15 hhf1-hht1::LEU2 pRS412 (ADE2 CEN
ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2-
Histone Ubiquitylation Regulates Lifespan
[22,23]
leu2Δ0
Fig. 3
SY420
Jerry L. Workman
& Shelley L.
Berger
Note
(Reference)
YKH046
Kevin Struhl &
Mark R. Parthun
[11,27]
This study
From
UCC1111
Song et al. Page 5
SY421
SY422
SY423
SY424
SY238
SY365
SY189
SY190
SY191
Fig. S3B
UCC1188
SY469
SY470
SY471
SY472
SY473
SY345
SY368
hht2::MET15
hhf1-hht1::LEU2
rad6Δ::KanMX4
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15
hhf1-hht1::LEU2
bre1Δ::KanMX4
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15
hhf1-hht1::LEU2
lge1Δ::KanMX4
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15
hhf1-hht1::LEU2
ubp8Δ::KanMX4
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15
hhf1-hht1::LEU2
ubp10Δ::KanMX6
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15
hhf1-hht1::LEU2
sir2Δ::KanMX4
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15
hhf1-hht1::LEU2
rpd3Δ::KanMX6
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15
hhf1-hht1::LEU2
set1Δ::KanMX4
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15
hhf1-hht1::LEU2
set2Δ::KanMX4
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3Δ0 leu2Δ0 trp1Δ63 his3Δ200 ade2Δ::hisG
lys2Δ0 met15Δ0 adh4::URA3-TEL (VII-L) hhf2hht2::MET15
hhf1-hht1::LEU2
dot1Δ::KanMX4
pRS412 (ADE2 CEN ARS) - HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 rad6Δ::KanMX4 pMP9 (LYS2 CEN ARS) HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 bre1Δ::KanMX4 pMP9 (LYS2 CEN ARS) HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 lge1Δ::KanMX4 pMP9 (LYS2 CEN ARS) HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 ubp8Δ::KanMX4 pMP9 (LYS2 CEN ARS) HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 ubp10Δ::KanMX6 pMP9 (LYS2 CEN ARS)
- HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 sir2Δ::KanMX4 pMP9 (LYS2 CEN ARS) HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
Histone Ubiquitylation Regulates Lifespan
This study
From
UCC1111
This study
From
UCC1111
This study
From
UCC1111
This study
From
UCC1111
This study
From
UCC1111
This study
From
UCC1111
This study
From
UCC1111
This study
From
UCC1111
This study
From
UCC1111
Daniel E.
Gottschling
[12]
This study
From
UCC1188
This study
From
UCC1188
This study
From
UCC1188
This study
From
UCC1188
This study
From
UCC1188
This study
From
UCC1188
This study
From
Song et al. Page 6
SY474
SY475
SY476
Fig. S3C
UCC7262
SY445
SY446
SY447
SY448
SY449
SY346
SY369
SY450
SY451
SY452
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 rpd3Δ::KanMX6 pMP9 (LYS2 CEN ARS) HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 set1Δ::KanMX4 pMP9 (LYS2 CEN ARS) HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 set2Δ::KanMX4 pMP9 (LYS2 CEN ARS) HHF2-HHT2
MATa ura3(-52 or -167) leu2Δ1 trp1(Δ901 or ::HIS3)
his3Δ200 lys2-801 RDN1::URA3 hhf2-hht2::HIS3 hhf1hht1::LEU2 dot1Δ::KanMX4 pMP9 (LYS2 CEN ARS) HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 pMP9 (LYS2 CEN ARS)
- HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 rad6Δ::KanMX4 pMP9
(LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 bre1Δ::KanMX4 pMP9
(LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 lge1Δ::KanMX4 pMP9
(LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 ubp8Δ::KanMX4 pMP9
(LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15
hhf1-hht1::LEU2
ubp10Δ::KanMX6
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 sir2Δ::KanMX4 pMP9
(LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 rpd3Δ::KanMX6 pMP9
(LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 set1Δ::KanMX4 pMP9
(LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 set2Δ::KanMX4 pMP9
(LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
Histone Ubiquitylation Regulates Lifespan
UCC1188
This study
From
UCC1188
This study
From
UCC1188
This study
From
UCC1188
Daniel E.
Gottschling
[12]
This study
From
UCC7262
This study
From
UCC7262
This study
From
UCC7262
This study
From
UCC7262
This study
From
UCC7262
This study
From
UCC7262
This study
From
UCC7262
This study
From
UCC7262
This study
From
UCC7262
This study
From
Song et al. Page 7
his3Δ200 ade2(::hisG or -101) met15Δ0 or MET15
lys2(Δ0 or -801) ADE2-TEL-VR hmr::URA3 hhf2hht2::MET15 hhf1-hht1::LEU2 dot1Δ::KanMX4 pMP9
(LYS2 CEN ARS) - HHF2-HHT2
Fig. S4A
UCC1001
SY425
SY426
SY427
SY428
SY429
SY049
SY432
SY430
SY431
SY067
Fig. S4B
UCC506
SY433
SY434
SY435
SY436
SY437
SY344
SY367
SY438
SY439
SY440
Fig. S4C
UCC7266
SY457
SY458
SY459
SY460
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 rad6Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 bre1Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 lge1Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 ubp8Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 ubp10Δ::KanMX6
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 sir2Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 rpd3Δ::KanMX6
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 set1Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 set2Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 TELadh4::URA3 dot1Δ::KanMX4
lys2-801
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR rad6Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR bre1Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR lge1Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR ubp8Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR ubp10Δ::KanMX6
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR sir2Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR rpd3Δ::KanMX6
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR set1Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR set2Δ::KanMX4
MATa ura3-52 leu2Δ1 trp1Δ1 his3Δ200
ade2-101 URA3-TEL-VR dot1Δ::KanMX4
UCC7262
lys2-801
E.J. Cho & Daniel
E. Gottschling
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
lys2-801
Daniel E.
Gottschling
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
lys2-801
This study
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 pMP9 (LYS2 CEN
ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 rad6Δ::KanMX4
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 bre1Δ::KanMX4
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 lge1Δ::KanMX4
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
Daniel E.
Gottschling
[12]
This study
From
UCC7266
This study
From
UCC7266
This study
From
UCC7266
This study
From
Histone Ubiquitylation Regulates Lifespan
[28,29]
From
UCC1001
From
UCC1001
From
UCC1001
From
UCC1001
From
UCC1001
From
UCC1001
From
UCC1001
From
UCC1001
From
UCC1001
From
UCC1001
[30]
From
UCC506
From
UCC506
From
UCC506
From
UCC506
From
UCC506
From
UCC506
From
UCC506
From
UCC506
From
UCC506
From
UCC506
Song et al. Page 8
SY461
SY347
SY370
SY462
SY463
SY464
Fig. S5
a
UCC1111
SY420
SY421
SY423
SY424
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 ubp8Δ::KanMX4
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 ubp10Δ::KanMX6
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 sir2Δ::KanMX4
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 rpd3Δ::KanMX6
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 set1Δ::KanMX4
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 set2Δ::KanMX4
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
MATa ura3(Δ0 or -52) leu2(Δ0 or Δ1) trp1Δ63
his3Δ200 lys2(Δ0 or -801) ADE2-TEL-VR hml::URA3
hhf2-hht2::MET15 hhf1-hht1::LEU2 dot1Δ::KanMX4
pMP9 (LYS2 CEN ARS) - HHF2-HHT2
UCC7266
This study
From
UCC7266
This study
From
UCC7266
This study
From
UCC7266
This study
From
UCC7266
This study
From
UCC7266
This study
From
UCC7266
See Fig. S3A
See Fig. S3A
See Fig. S3A
See Fig. S3A
See Fig. S3A
The deletion strain UBP10 (SY387) was generated by replacing the open reading frame of
UBP10 with the KanMX6 marker using the pFA6-KanMX6 plasmid [31]. For the other gene
deletion strains, each indicated gene was deleted by high efficiency transformation using
PCR products amplified from genomic DNA in which the target gene of interest had already
been replaced by KanMX4 (Euroscarf, Frankfurt, Germany). All deletion strains were
confirmed by PCR using gene-specific and deletion cassette-specific primers.
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 9
Supplementary Table S5. oligonucleotide sequences used in ChIP.
Fig.
Name
Sequence
Source
Fig. 1B
TEL05R-1-for (5385-5407)
TEL05R-1-rev (5432-5454)
TEL05R-2-for (6648-6671)
TEL05R-2-rev (6703-6725)
TEL05R-3-for (6867-6888)
TEL05R-3-rev (6947-6970)
TEL06R-1-for (74-93)
TEL06R-1-rev (323-344)
TEL06R-2-for (496-516)
TEL06R-2-rev (768-787)
TEL06R-3-for (7388-7411)
TEL06R-3-rev (7632-7651)
TEL07L-1-for (148-177)
TEL07L-1-rev (263-289)
TEL07L-2-for (492-515)
TEL07L-2-rev (542-564)
TGTAGACCATCACGTGGTTTGTT
CGTTTGTTGAAGACGAACCAGAT
CATCCATCCCTCTACTTCCTACCA
TGGAGTTGGATATGGGTAATTGG
TGCCATACTCACCCTCACTTGT
CCATGGAGTGGAATGTGAGAGTAG
ACCCAGTCCTCATTTCCATC
TTTTTACGTTTAGCTGAGTTTA
ATCCTTAAGTAAAACACATTC
AGGATTTTAGCAACGACTTC
CATTGTGGCACTATTAACGAGAGG
ATGCGAAATAAGAACACGAT
AACCACCATCCATCTCTCTACTTACTACTA
AGAACAACAGTACAGTGAGTAGGACATG
TCAGTACTAAATGCACCCACATCA
TGGGTAAATGGCACAGGGTATAG
[32]
[32]
[32]
[32]
[32]
[32]
[33]
[33]
[33]
[33]
[33]
[33]
[32]
[32]
[32]
[32]
Fig. 3
See Fig. 1B
Fig. S1C
and S1D
See Fig. 1B
rDNA-1-for
(ChXII 463118-463137)
rDNA-1-rev
(ChXII 463344-463363)
rDNA-2-for
(ChXII 460408-460427)
rDNA-2-rev
(ChXII 460632-460651)
rDNA-3-for
(ChXII 459359-459378)
rDNA-3-rev
(ChXII 459590-459609)
rDNA-4-for
(ChXII 458177-458196)
rDNA-4-rev
(ChXII 458425-458444)
HMR-1-for
(ChIII 293848-293872)
HMR-1-rev
(ChIII 294338-294362)
GTGGTGTCTGATGAGCGTGT
[34]
TCCCTCAGGATAGCAGAAGC
[34]
TCCCCACTGTTCACTGTTCA
[34]
AGGGCTTTCACAAAGCTTCC
[34]
GCCACCATCCATTTGTCTTT
[34]
GAGGTGTTATGGGTGGAGGA
[34]
AAGATGCCCACGATGAGACT
[34]
GGGAGGTACTTCATGCGAAA
[34]
GTAGTATGGCGGAAAACATAAACAG
[35]
AGAAAAACCCGACTATGCTATTTTA
[35]
Fig. S2
See Fig. S1
Fig. S5
TEL07L-URA3-1-for
(123- 144)a
TEL07L-URA3-1-rev
(306-329)a
TEL07L-URA3-2-for
(732- 756)a
TEL07L-URA3-2-rev
(1003-1024)a
TEL07L-URA3-3-for
(4912- 4933)a
TEL07L-URA3-3-rev
(5141-5164)a
TEL07L-URA3-4-for
(15987-16009)a
TEL07L-URA3-4-rev
(16261-16281)a
CCCAGCCTGCTTTTCTGTAACG
This study
TGGGTGGAAGAGATGAAGGTTACG
This study
TGGGACCTAATGCTTCAACTAACTC
This study
GGAAGAACGAAGGAAGGAGCAC
This study
ATTTCCTCTCCCTGCCATCCTC
This study
GCTCTCCTCCCACAAAATAATCTT
This study
GAATAATCGGCTGTAATCGGACC
This study
ACCAACCGGATCAGGCAAGAC
This study
ChrV NO-ORF-forb
ChrV NO-ORF-revb
GGCTGTCAGAATATGGGGCCGTAGTA
CACCCCGAAGCTGCTTTCACAATAC
[36,37]
[36,37]
Fig. 1B,
3, S1, S2,
and S5
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 10
The primer locations denote the distance from the TG1-3 repeats at the end of each
chromosome (+1), unless otherwise indicated.
a
The nucleotide numbers are relative to the first nucleotide of the URA3 initiation codon (+1).
b
Primers used to amplify the non-transcribed regions of chromosome V.
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 11
Supplementary Table S6. Antibodies used in this study.
Specificity
Supplier
Catalog #
Applications
Anti-H3
Abcam
ab1791
ChIP
Anti-H3K4me3
Abcam
ab8580
ChIP
Anti-H3K36me3
Abcam
ab9050
ChIP
Anti-H3K79me3
Abcam
ab2621
ChIP
Anti-H4
Abcam
ab31827
ChIP, WB
Anti-H4K16ac
Millipore
07-329
ChIP, WB
Anti-Sir2
Santa Cruz Biotechnology
sc-6666
ChIP, WB
Anti-HA
Roche
12CA5
ChIP
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 12
Supplementary Table S7. Mean lifespans and p-values for lifespan analysis, related to Fig. 2.
Strain A
Strain B
Figure
a
p-values
Name
Meana
nb
Name
Meana
nb
Fig. 2A
WT (BY4741)
WT (BY4741)
WT (BY4741)
24.5
24.5
24.5
50
50
50
rad6∆
bre1∆
lge1∆
12.1
12.4
18.5
48
48
48
1.5×10-11
5.5×10-12
6.1×10-3
Fig. 2B
HTA1-HTB1,CEN
(YZS276)
HTA1-HTB1,CEN
( YZS276)
HTA1-HTB1,CEN
( YZS276)
21.4
50
13.3
50
6.9×10-7
21.4
50
13.6
38
6.2×10-5
21.4
50
HTA1-htb1-K123R,CEN
( YZS277)
HTA1-HTB1, 2μ
(Y131)
HTA1-htb1-K123R, 2μ
(Y133)
12.0
39
2.3×10-7
Fig. 2C
WT (YKH045)
WT (YKH045)
15.9
15.9
49
49
htb1-K123R (YKH046)
ubp8∆ (YKH047)
10.2
19.0
50
49
1.7×10-3
0.12
Fig. 2D
WT (BY4741)
WT (BY4741)
36.3
36.3
50
50
ubp8∆
ubp10∆
40.8
26.3
46
49
0.27
1.5×10-3
Fig. 2E
WT (YKH045)
WT (YKH045)
sir2∆
18.4
18.4
11.7
50
50
46
sir2∆
sir2∆+ htb1-K123R
sir2∆+ htb1-K123R
11.7
10.0
10.0
46
50
50
6.1×10-5
2.2×10-6
0.14
Mean lifespan; bnumber of cells analyzed in each experiment.
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 13
Supplementary legends
Supplementary Fig. S1. Histone levels at the telomere, rDNA and HMR Loci were decreased
in aged Cells, related to Fig. 1. (A) Western blot analysis of extracts from young and aged
cells (strain YKH045) using the indicated antibodies. The membrane was stained with
Ponceau S as a loading control (bottom). Average bud scar numbers for both types of cells
are shown in parentheses. (B) Schematic diagrams of the TEL05R, TEL06R, TEL07L, HMR,
and rDNA loci. The bars and numbers below each locus show the positions of the PCR
products used in ChIP analysis. (C) ChIP analysis of histones H3, H4, and H2B at the
TEL06R, rDNA, and HMR loci as described in Fig. 1B. Quantitative histone levels were
used to normalize the ChIP results for acetylated H4 and methylated H3 in Fig. 1B and
supplementary Fig. S1D. (D) ChIP analysis of telomere-proximal regions in young and aged
cells
(strain
YKH045).
Antibodies
against
Sir2
and
H4K16ac
were
used
to
immunoprecipitate chromatin. The results for acetylated H4 were further normalized to the
total H4 signals.
Supplementary Fig. S2. Changes in Sir2 levels and histone modifications at the rDNA and
HMR Loci in aged Cells, related to Fig. 1. (A-B) ChIP analysis of rDNA (A) and HMR (B)
regions in young and aged cells (strain YKH045). Antibodies against Sir2, H4K16ac,
H3K4me3, H3K36me3, and H3K79me3 were used to immunoprecipitate chromatin. To
analyze the levels of ubiquitylated H2B, a ChDIP assay was carried out as described in Fig.
1B. All methods and procedures were performed as described in Fig. 1.
Supplementary Fig. S3. Histone H2B ubiquitylation and H3 lysine methylation regulate Sir2mediated heterochromatin silencing. (A-C) To examine silencing at the telomeric, rDNA, and
HMR regions, the indicated genes were deleted in strains UCC1111, UCC1188, and
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 14
UCC7262, all of which contain a URA3 reporter gene, as shown in the diagram.
Exponentially growing cells were spotted onto SC, SC-Ura, and 5-FOA plates at 5-fold
dilutions and further incubated at 30°C for 1-3 days. Strains lacking SIR2 or RPD3 were used
as controls.
Supplementary Fig. S4. Histone H2B ubiquitylation and H3 lysine methylation regulate Sir2mediated heterochromatic silencing. (A-C) To determine silencing at telomeric and HML
regions, the indicated genes were deleted in strains UCC1001, UCC506, and UCC7266, all of
which contain a URA3 reporter gene, as shown in the diagram. All methods and procedures
were performed as described in supplementary Fig. S3.
Supplementary Fig. S5. Histone H2B ubiquitylation regulates Sir2 association at the URA3based TEL07L region, related to Fig. 3. (A) A schematic diagram of the URA3 reporter at
TEL07L is shown in the top panel. The bars and numbers below the subtelomeric region
show the positions of the PCR products used for the ChIP analysis and their distances from
the telomeric DNA sequence (TG1-3). Sir2 recruitment and histone H4K16ac were analyzed
by ChIP in WT, rad6Δ, bre1Δ, ubp8Δ, and ubp10Δ cells. (B) Quantitation of the ChIP
experiments in (A). The PCR signals were quantitated and normalized as described in Fig.
1B. The error bars indicate the standard deviation (S.D.) of three PCRs from two independent
chromatin preparations.
Histone Ubiquitylation Regulates Lifespan
Song et al. Page 15
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