DDK/Hsk1 phosphorylates and targets fission yeast histone deacetylase Hst4 for degradation to stabilize stalled DNA replication forks
- Laboratory of Chromatin Biology and Epigenetics, Centre for DNA Fingerprinting and Diagnostics, India
- Graduate Studies, Manipal Academy of Higher Education, India
Figures
Figure 1 with 1 supplement
Replication stress leads to downregulation of Hst4.
(A) Wild-type (wt) strain (ROP238) containing TAP-tagged Hst4 was grown to 0.5 O.D., treated with 0.015 % MMS and 10 mM HU for 2 hr, respectively, whole cell extracts were prepared and immunoblotted …
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Figure 1—source data 1
Uncropped western blot images for Figure 1A– E and G.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig1-data1-v2.zip
Figure 1—figure supplement 1
S-phase dependent downregulation of Hst4 upon MMS treatment.
(A) The cdc25-22 cells (DHP38) were first synchronized at G2 phase and then released into the S phase in the presence of 0.015 % MMS, time points were collected and western blot was performed. (B) …
Figure 2 with 1 supplement
The C terminus of Hst4 is required for proteasome dependent degradation of Hst4 upon replication stress.
(A) Half-life of Hst4 was measured by growing wt (ROP238) cells to O.D 0.5 and treating with cycloheximide (CHX) at100 µg/ml in the presence and absence of 0.015 % MMS. Cells were collected at …
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Figure 2—source data 1
Uncropped western blot images for Figure 2A–E.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig2-data1-v2.zip
Figure 2—figure supplement 1
Hst4 is post-translationally regulated and minimum region required for Hst4 degradation lies at the C-terminus.
(A) Wild-type strain of Hst4 (ROP238) was grown in absence and presence of 0.015 % MMS and RNA was extracted and RT-qPCR was performed. The qPCR data are shown as expression fold changes after …
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Figure 2—figure supplement 1—source data 1
Uncropped western blot images for Figure 2—figure supplement 1D.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig2-figsupp1-data1-v2.pdf
Figure 3 with 1 supplement
Replication-dependent regulation of Hst4 via phosphorylation by DDK/Hsk1.
(A) wt strain (ROP238) containing TAP-tagged Hst4 was grown to 0.5 O.D., treated with 0.015 % MMS for 1 hr and samples were immunoprecipitated using IgG-sepharose beads and western blot was …
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Figure 3—source data 1
Uncropped western blot images for Figure 3B–J.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig3-data1-v2.zip
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Figure 3—source data 2
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig3-data2-v2.zip
Figure 3—figure supplement 1
Gsk3-beta and Mcm4 are not required for downregulation of Hst4.
(A) The wt (ROP238) and gsk3∆ strain (DHP77) were grown to mid-log phase in YES medium, treated with 0.015 % MMS for 2 hr, whole cell extracts were prepared and western blot was performed. …
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Figure 3—figure supplement 1—source data 1
Uncropped western blot images for Figure 3—figure supplement 1A, B, C.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig3-figsupp1-data1-v2.pdf
Figure 4 with 1 supplement
Phosphorylation of Hst4 at serine residues 354, 355, 362, and 363 is required for cell survival upon replication stress.
(A) The C-terminal residues 352–370 required for degradation of Hst4 are shown. The serine residues marked with star, which are putative consensus DDK sites were mutated to alanines by site-directed …
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Figure 4—source data 1
Uncropped western blot images for Figure 4C–E.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig4-data1-v2.zip
Figure 4—figure supplement 1
Stability and sensitivity of Hst4 C-terminal domain deletion and serine to alanine mutants in replication stress.
(A) The hst4∆ strain (ROP57) transformed with 4SA-Hst4-pSLF273 (354,355,362,363), 354-355SA-Hst4-pSLF273, and 362-363SA-Hst4-pSLF273 were grown to mid-log phase in EMM-Ura medium, treated with 0.015 …
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Figure 4—figure supplement 1—source data 1
Uncropped western blot images for Figure 4—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig4-figsupp1-data1-v2.pdf
Figure 5 with 1 supplement
SCFPof3-mediated degradation of Hst4 on replication stress.
(A) wt strain (ROP238) and F box mutant strain pof3Δ (DHP35) containing TAP-tagged Hst4 were grown to 0.5 O.D., treated with 0.015 % MMS for 2 hr and whole cell extracts were prepared and …
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Figure 5—source data 1
Uncropped western blot images for Figure 5A–C and E.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig5-data1-v2.zip
Figure 5—figure supplement 1
Pop1 is not required for downregulation of Hst4.
(A) The wt (ROP238) and pop1∆ strain transformed with FL-Hst4-pSGP573 were grown to mid-log phase in EMM-Ura medium, treated with 0.015 % MMS for 2 hr, whole cell extracts were prepared and western …
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Figure 5—figure supplement 1—source data 1
Uncropped western blot images for Figure 5—figure supplement 1C.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig5-figsupp1-data1-v2.pdf
Figure 6 with 1 supplement
Dynamic regulation of Hst4 is required for stable fork stalling and recovery from replication stress.
(A) Wild-type strain (ROP238) and 4SA-hst4 (DHP146) were grown to OD 0.5 and treated with 12 mM HU for 6 hr and DNA was stained with Hoechst and life cell microscopy was performed. Scale- 5 µM. (B) …
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Figure 6—source data 1
Uncropped western blot images for Figure 6C.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig6-data1-v2.zip
Figure 6—figure supplement 1
4SA-hst4 is required for DNA replication fork restart post replication stress.
(A) The cdc25-22 (FY4225) and cdc25-22 4SA-hst4 (DHP148) strains engineered to incorporate halogenated nucleotides were grown to midlog phase in YES medium at 25 °C, were shifted to 36°C for 4 hr to …
Figure 7 with 1 supplement
The degradation of Hst4 is required for the stable association of Fork Protection Complex at the replication fork.
(A) Tetrads each from the hst4∆ strain (ROP58) crossed with the swi1∆ (EN3182) and swi3∆ (EN3366) are shown, photographed after 4 days of growth on YES plates. The genotypes were determined by …
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Figure 7—source data 1
Uncropped western blot images for Figure 7B–D and F.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig7-data1-v2.zip
Figure 7—figure supplement 1
Regulation of replisome components in 4SA-hst4 mutant.
(A) The wt Swi1 (EN3381) or Swi3 (EN3382) FLAG-tagged strains were grown and immunoprecipitation was performed using FLAG M2 beads, followed by western blot and probed with anti-FLAG and ant-acetyl …
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Figure 7—figure supplement 1—source data 1
Uncropped western blot images for Figure 7—figure supplement 1D.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig7-figsupp1-data1-v2.pdf
Figure 8 with 1 supplement
H3K56 acetylation-dependent stabilization of Fork Protection Complex at the chromatin regulates genomic stability.
(A) wt strain (ROP238) and 4SA-hst4 strain (DHP146) were grown to 0.5 O.D., treated with 0.015 % MMS for 2 hr, whole cell extracts were prepared and immunoblotted with indicated antibodies. (B) wt …
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Figure 8—source data 1
Uncropped western blot images for Figure 8A–E.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig8-data1-v2.zip
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Figure 8—source data 2
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig8-data2-v2.zip
Figure 8—figure supplement 1
Hypoacetylation of histone H3K56 in hsk1-89 and pof3 mutant cells.
(A) The wt (ROP238), hsk1-89 (DHP78) and H3K56R strain (ROP253) were grown to mid-log phase, whole cell extracts were prepared and western blot was performed. (B) The wt (ROP238) and pof3∆ (DHP35) …
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Figure 8—figure supplement 1—source data 1
Uncropped western blot images for Figure 8—figure supplement 1A and B.
- https://cdn.elifesciences.org/articles/70787/elife-70787-fig8-figsupp1-data1-v2.pdf
Tables
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Antibody | Anti-PAP(Peroxidase Peroxidase Soluble Complex antibody produced in rabbit) | Sigma-Aldrich | Cat#P1291RRID:AB_1079562 | WB (1:5,000) |
| Antibody | Anti-HA(rabbit polyclonal antibody) | Bethyl laboratories | Cat#A190-208ARRID:AB_67466 | WB (1:10,000) |
| Antibody | Anti-HA(rabbit polyclonal antibody) | Abcam | Cat#ab9110RRID:AB_307019 | WB (1:5000)IP- (4 µg) |
| Antibody | Anti-H3K56ac(rabbit polyclonal antibody) | Abcam | Cat#ab71956RRID:AB_10861799 | WB (1:2,000) |
| Antibody | Anti-H3(rabbit polyclonal antibody) | Abcam | Cat#ab1791RRID:AB_302613 | WB (1:5,000) |
| Antibody | Anti-FLAG(mouse monoclonal antibody) | Sigma-Aldrich | Cat#F1804RRID:AB_262044 | WB (1:2,000) |
| Antibody | Anti-myc(mouse monoclonal antibody) | Sigma-Aldrich | Cat#M4439RRID:AB_439694 | WB (1:5,000) |
| Antibody | Anti-And-1 (Mcl1)(mouse monoclonal antibody) | Biolegend | Cat#630,301RRID:AB_2215084 | WB (1:5,000)IP- (2 µg) |
| Antibody | anti-GFP(mouse monoclonal antibody) | Cell Signalling | Cat#2,955RRID:AB_1196614 | WB (1:2,000)IF- (1:250) |
| Antibody | Anti-His(mouse monoclonal antibody) | BD biosciences | Cat#552,565RRID:AB_394432 | WB (1:5,000) |
| Antibody | Anti-Phosphoserine(rabbit polyclonal antibody) | Abcam | Cat#ab9332RRID:AB_307184 | WB (1:3,00) |
| Antibody | H2A-P(rabbit polyclonal antibody) | Abcam | Cat#ab15083RRID:AB_301630 | WB (1:5,000) |
| Antibody | Anti-BrdU(mouse monoclonal antibody) | BD biosciences | Cat#347,580RRID:AB_400326 | IF (1:1,00) |
| Antibody | Anti-CldU(rat monoclonal antibody) | Abcam | Cat#ab6326RRID:AB_305426 | IF (1:1,00) |
| Antibody | Anti-Timeless(rabbit monoclonal antibody) | Abcam | Cat#ab109512RRID:AB_10863023 | WB (1:2000) |
| Antibody | Anti-Tipin | Abcam | Cat#ab229329 | WB (1:2000) |
| Antibody | Anti-Sirt2(rabbit monoclonal antibody) | Cell Signalling | Cat#D4O5ORRID:AB_2716762 | WB (1:2500) |
| Antibody | Anti-GAPDH(rabbit monoclonal antibody) | Cell Signalling | Cat#14C10RRID:AB_561053 | WB (1:5000) |
| Strain, strain background (Escherichia coli) | BL21(DE3) pLysS | Lab stock | Chemically Competent cells | |
| Cell line (Homo-sapiens) | U2OS(Osteosarcoma), cells | ATCC | ATCC ID: HTB-96RRID:CVCL_0042 | Gift from Dr. Rashna Bhandari’s lab |
| Transfected construct (human) | siRNA toAsf1a | Thermo Fisher Scientific | Cat#4392420Assayid- S226044 | |
| Recombinant DNA reagent | pSLF273 | Forsburg Lab | Cloning of FL-Hst4, T1Δ-Hst4, T2Δ-Hst4 and point mutations. | |
| Recombinant DNA reagent | pSLF272 | Forsburg Lab | Cloning of FL-Hsk1, KD-Hsk1 and Pof3 | |
| Recombinant DNA reagent | pET28a | Lab stock | Cloning of FL-Hst4 and 4SA-Hst4 | |
| Recombinant DNA reagent | pSGP573 | Forsburg Lab | Cloning of FL-Hst4 and CΔ-Hst4 | |
| Recombinant DNA reagent | pREP1-His6-ubiquitin | Takashi Toda | ||
| Chemical compound, drug | Methyl methane sulphonate (MMS) | Sigma-Aldrich | Cat#129,925 | |
| Chemical compound, drug | Hydroxyurea (HU) | Sigma-Aldrich | Cat#H8627 | |
| Other | Bisbenzimide Hoechst | Sigma-Aldrich | Cat#B2261 | 10 µg/µl |
| Software, algorithm | ImageJ | ImageJ | https://imagej.net/Fiji/Downloads | Quantification of western blots |
| Software, algorithm | Graphpad Prism 6.0 | Graphpad | https://www.graphpad.com/ scientific-software/prism// | Statistical analysis and graphs representation |
| Software, algorithm | FlowJo | BD Life Sciences | RRID:SCR_008520 |
Table 1
List of strains used in the study.
| Strain | Genotype | Source |
|---|---|---|
| ROP238 | h + ade6 M216 arg3-D4 his3-D1 leu1-32 ura4-D18 hst4-TAP:KanR | Lab stock |
| ROP191 | h_ ade6-210 arg3-D4 his3-D1 leu1-32 ura4-D18 | Lab stock |
| DHP38 | h + cdc25-22 ade6-216 arg3-D4 his3-D1 leu1-32 ura4-D18 hst4-TAP:KanR | This Study |
| DHP37 | h- ade6-216 arg3-D4 his3-D1 leu1-32 ura4-D18 hst4-TAP:KanR mts2-1 | This Study |
| ROP57 | h + ade6 M216 arg3-D4 his3-D1 leu1-32 ura4-D18 hst4∆-his3+ | Lab stock |
| DHP57 | h + skp1-94 his3-D1 arg3-D4 leu1-32 ura4-D18 ade? hst4-TAP:KanR | This Study |
| DHP56b | h- skp1-A4 his3-D1 arg3-D4 leu1-32 ura4-D18 ade? hst4-TAP:KanR | This Study |
| SKP471 | h− leu1-32 ura4-D18 pof3Δ::kan | Takashi Toda |
| TP403-2B | h90 leu1-32 ura4-D18 pop1::ura+ | Takashi Toda |
| DHP35 | h- arg3-D4 his3-D1 leu1-32 ura4-D18 pof3::kanR hst4-TAP:KanR | This Study |
| DHP78 | h + hsk1-89:ura4+ his3-D1arg3-D4 leu1-32 ura4-D18 ade? hst4-TAP:KanR | This Study |
| ROP247 | h + h3.2 K56R h3.1∆/h4.1∆::his3+ h3.3∆ /h4.3∆::arg3+ leu1-32 ura4-D18 his3-D1 arg3-D4 ade6-210 | Lab collection |
| DHP77 | h + ade6-216 arg3-D4 his3-D1 leu1-32 ura4-D18 hst4-TAP:KanR gsk3::ura4+ | This Study |
| ENY3684 | h + pof3-myc:hphMX6 pol2-3FLAG:kanMX6 leu1-32 ura4-D18 | Eishi Noguchi |
| yFS988 | h- ura4-D18 ade-? cdc10-M17 leu1-32::pYJ294(leu1 cds1-6his2HA) | Nick Rhind |
| FY4225 | h- cdc25-22 leu1::hENT-leu1+ his7-366::hsv-tk-his7+ ura4? ade6-M210 | Susan.L.Forsburg |
| FY1077 | h- hsk1HA::ura4+ ura4-D18 leu1-32 ade6-M216 | Susan Forsburg |
| FY1763 | h + leu1:dfp1 +6his3HA leu1+ dfp1-D1 ura4-D18 ade6-M216 | Susan Forsburg |
| DHP146 | h- ade6-216 arg3-D4 his3-D1 leu1-32 ura4-D18 4SA-hst4-TAP:KanR | This Study |
| DHP148 | h- cdc25-22 leu1::hENT-leu1+ his7-366::hsv-tk-his7+ ura4? ade6-M210 4SA-hst4-TAP::KanR | This Study |
| DHP168 | h- h3.1/h4.1Δ::his3+ h3.3/h4.3Δ::arg3+ leu1-32 ura4-D18 arg3-D ade6-M210 swi1-3FLAG::KanR | This Study |
| DHP169 | h + H3.2 K56R h3.1/h4.1Δ::his3+ h3.3/h4.3Δ::arg3+ leu1-32 ura4-D18 arg3-D ade6-M210 swi1-3FLAG::KanR | This Study |
| MS193 | h- leu1-32 ura4-D18 cdc21 | Hisao Masai |
| DHP172 | h + leu1-32 ura4-D18 ade6-M210 cdc21 (mcm4) hst4-TAP::Kan | This Study |
| DHP173 | h + ura4-D18 ade-? leu1-32::pYJ294(leu1 cds1-6his2HA) rad3::ura4+ | This Study |
| DHP174 | h- ura4-D18 ade-? leu1-32::pYJ294(leu1 cds1-6his2HA) 4SA-hst4-TAP::Kan | This Study |
| EN3182 | h− swi1::Kanr leu1-32 ura4-D18 | Eishi Noguchi |
| EN3366 | h− swi3::Kanr leu1-32 ura4-D18 | Eishi Noguchi |
| EN3381 | h− swi1-3FLAG:Kanr leu1-32 ura4-D18 | Eishi Noguchi |
| EN3382 | h− swi3-3FLAG:Kanr leu1-32 ura4-D18 | Eishi Noguchi |
| DHP143 | h + leu1-32 ura4-D18 arg3-D4 ade6-M216 swi1-3FLAG::KanR | This Study |
| DHP144 | h- leu1-32 ura4-D18 arg3-D4 ade6-M216 swi1-3FLAG::KanR 4SA-hst4-TAP::KanR | This Study |
| DHP177 | h- leu1-32 ura4-D18 arg3-D4 ade6-M216 mrc1-13myc::Kan 4SA-hst4-TAP::Kan | This Study |
| DHP115a | h + hsk1HA::ura4+ ura4-D18 leu1-32 ade6-M216 dfp1-13 myc:G418r | This Study |
| DHP152 | h- leu1-32 ura4-D18 mrc1-13myc::Kan | Hisao Masai |
Additional files
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Transparent reporting form
- https://cdn.elifesciences.org/articles/70787/elife-70787-transrepform1-v2.pdf
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Source data 1
Source data for all figures.
- https://cdn.elifesciences.org/articles/70787/elife-70787-supp1-v2.pdf
