Recruitment of HBO1 Histone Acetylase and Blocks

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Anthony Dorsey
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1 Molecular Cell, Volume 44 Supplemental Information JNK1 Phosphorylation of Cdt1 Inhibits Recruitment of HO1 Histone cetylase and locks Replication Licensing in Response to Stress enoit Miotto and Kevin Struhl

2 Figure S1. Related to Figure 1 Representation of HO1 ChIP-qPCR data in percentage of total DN Title of each panel indicate the figure associated with these data. See figure legend for information regarding the samples. In each panel HO1 ChIP and IgG data are presented for the MCM4 replication origin and the GPDH region. HO1 ChIP/MCM4 origin IgG/MCM4 origin HO1 ChIP/GPDH IgG/GPDH % total 1,9,7,9,7,9,7 % total,9,7 % total DN Figure 1 urea aniso DTT Sorb. ctd HU Figure 2C % total Figure 3 sorbitol Sorbitol+MG132 sorbitol Recovery Recovery.5h 1h Figure 3D DTT niso Urea H ctd Cisplsynch. % total 1,2 % total 1 Figure 1G urea niso. DT Sorbitol 1,4 1,2 1 Figure 3 - SP JNKIIp38 TMi Sorbitol+ inhibitor % total DN 1 % total Figure 3C - ctr JNK1 JNK2 Sorbitol + sirn Figure 5D empty WT T29 T29 T29 Cy Empty WT T29 Ori -1kb Ori +1kb Sorbito

3 Figure S2. Related to Figure 1 HO1 ChIP cjun cfos cjun sp 5 Occupancy Occupancy MCM4 TOP1 Increasing cjun, cfos or a constitutive form of cjun in HeLa cells does not perturb HO1 binding at origins HeLa cells in their exponential phase of growth were transfected with plasmids encoding cjun, cfos or a constitutive JNK-phosphorylated form of cjun. 48 hours after transfection, cells were harvested and cross-linked with formaldehyde. HO1 binding at MCM4 and TOP1 origins was investigated by ChIP (n=3). G/G1 S G2/M 4N urea anisomycin 4N 4N sorbitol 4N DTT 4N ctinomycin D 4N %G/G urea 49 anisomycin 48.7 sorbitol 48.2 DTT 51.3 ctinomycin D 51 %S %G2/M Cell cycle distribution of HeLa cells exposed to different stress agents HeLa cells in their exponential phase of growth were exposed to stress agents (in presence of serum). Cells were stained with 2 g/ml propidium iodide (Sigma) in presence of 5 g/ml of RNase H for 3 minutes at room temperature in the dark and extensively washed in PS. 1x1 6 cells were subsequently analyzed for each condition using a FCSCalibur flow cytometer and data were processed using the CellQuest software (ecton Dickinson).

4 Figure S3. Related to Figure 1 O cc u pa nc y HeLa cells sorbitol 3mM 1 hour O ORC2 ChIP cc u 1 pa 5 nc MCM4 TOP1 y MCM4 TOP1 3 1 MCM3 ChIP O cc u pa nc y IgG MCM4 TOP1 Whole cell extracts HeLa cells Sorbitol HO1 Chromatin fractions + ORC2 MCM Complex MCM2 MCM3 MCM5 Cdc6 Cdt1 Geminin DN licensing defect in HeLa cells in hyper-osmotic condition () ChIP analysis of ORC2 and MCM3 binding at MCM4 and TOP1 replication origins in HeLa cells in their exponential phase of growth treated with 3mM sorbitol for 1 hour (n=3). () Western blot analysis of DN licensing factors expression and chromatin loading in HeLa cells treated with sorbitol or left untreated.

5 Figure S4. Related to Figure 2 Sorbitol Input (1%) IgG IP: H-Cdt1 Input (1%) IgG IP: H-Cdt1 PCN Skp2 Cdt1 interaction with PCN and Skp2 is not promoted by hyper-osmotic shock Western blot analysis of H-Cdt1 immunoprecipitates in and sorbitol treated cells. Nuclear extracts from HeLa cells expressing H-Cdt1 and treated with sorbitol or not were incubated with anti-h resin. fter extensive wash, Skp2 and PCN presence in the immunoprecipitates was tested by Western blot using specific antibodies.

6 Figure S5. Related to Figure 3 TM IP Extract caffeine SP2358 P-TM P-CRE TM GPDH C JNK1 P-S/T GSTcJun(Nter) JNK1 IP SP6125 D JNK1 IP JNK Inh. I P-JNK JNK1 Efficient inhibition of TM, p38 and JNK activity in cells treated with specific inhibitors prior to sorbitol treatment HeLa cells in their exponential phase of growth were treated with TM, JNK and p38 inhibitors, 3 minutes, prior to exposure to 3mM sorbitol for 1 hour. () TM activity was monitored in cells by analysis of its phosphorylation level after immuno-precipitation. () p38 activity was assessed by quantifying the phosphorylation of its substrate CRE (Iordanov et al., 1997). (C) JNK activity following SP6125 treatment was assessed by quantifying the phosphorylation of its substrate GST-cJun(Nter) in an IP-Kinase assay. (D) JNK activity following JNK Inhibitor I treatment was monitored in cells by analysis of its phosphorylation level after immuno-precipitation. The panels are representative of cell samples used in Figure 3 for chromatin immunoprecipitation analysis.

7 Figure S6. Related to Figure 3 R el ati ve bi n di n g HO1 ChIP HeLa cells sorbitol Cdk2 inhibitor then sorbitol R el ati ve bi n di n g MCM4 cmyc 35 HCT116 cells sorbitol p38 inhibitor then sorbitol JNK inhibitor I then sorbitol JNK inhibitor II then sorbitol 1 5 MCM4 cmyc Effect of Cdk2 kinase and p53 transcription factor on HO1 binding at MCM4 origin () HeLa cells in their exponential phase of growth were treated with a Cdk2 kinase specific inhibitor (sc-22149; Santa Cruz), prior to exposure to sorbitol. HO1 binding was analyzed by ChIP-qPCR at the MCM4 origin (n=3). () HCT116 cells, p53 positive, in their exponential phase of growth were treated with a JNK or p38 kinase specific inhibitor prior to exposure to sorbitol. HO1 binding was analyzed by ChIP-qPCR at the MCM4 origin (n=3).

8 Figure S7. Related to Figure 5 1 TP SP SV TP SP SP SP MEQRRVTDFF RRRPGPPRI PPKLCRTP SPRPLRP STSGSRKR RPPPGRD QRPPRRRL RLSVDEVSSP STPEPDIP CPSPGQKIKK STPGQPPH LTSQDQDTI SELSCLQR RELGRVRL KSQDGES CTPEEGRPE EPCGEKPY QRFHLQPG LPGLVLPYKY QVLEMFRSM DTIVGMLHNR SETPTFKVQ RGVQDMMRRR FEECNVGQIK TVYPSYRFR QERSVPTFKD GTRRSDYQLT IEPLLEQED GPQLTSR LLQRRQIFSQ KLVEHVKEHH KFLSLSP MVVPEDQLTR WHPRFNVDEV PDIEPLPQ PPTEKLTT QEVLRRNL ISPRMEKLS QLLRSPS SPGSPRPLP TPPTPP SPSLKGVSQ DLLERIRKE QKQLQMTR CPEQEQRLQR LERLPELRV LRSVFVSERK PLSMEVC RMVGSCCTIM SPGEMEKHLL LLSELLPDWL SLHRIRTDTY VKLDKDL HITRLHQT REEGL C MEQRRVTDFF RRRPGPPRI PPKLCRTP SPRPLRP STSGSRKR RPPPGRD QRPPRRRL RLSVDEVSSP STPEPDIP CPSPGQKIKK STPGQPPH LTSQDQDTI SELSCLQR RELGRVRL KSQDGES CTPEEGRPE EPCGEKPY QRFHLQPG LPGLVLPYKY QVLEMFRSM DTIVGMLHNR SETPTFKVQ RGVQDMMRRR FEECNVGQIK TVYPSYRFR QERSVPTFKD GTRRSDYQLT IEPLLEQED GPQLTSR LLQRRQIFSQ KLVEHVKEHH KFLSLSP MVVPEDQLTR WHPRFNVDEV PDIEPLPQ PPTEKLTT QEVLRRNL ISPRMEKLS QLLRSPS SPGSPRPLP TPPTPP SPSLKGVSQ DLLERIRKE QKQLQMTR CPEQEQRLQR LERLPELRV LRSVFVSERK PLSMEVC RMVGSCCTIM SPGEMEKHLL LLSELLPDWL SLHRIRTDTY VKLDKDL HITRLHQT REEGL Threonine 29 is a JNK- and stress-regulated phosphorylation site () Summary of previously described phosphorylation sites onto Cdt1 (Takeda et al., 28; Chen et al., 29; eausoleil et al., 24; Dephourne et al., 28). The following residues of each phosphorylation site is also indicated. Single bars represent additional SP and TP motif, matching the JNK consensus target site. () Phosphopeptide analysis of His-Cdt1 incubated with JNK1 in vitro. His-Cdt1 incubated with bulk IP (IgG) was used as a for non-phospho-sites. Residues recovered during the mass-spectrometry analysis in both samples are indicated in bold and the phospho-sites underlined and in red. (C) Phosphopeptide analysis of Flag-Cdt1 immunoprecipitated from HeLa cells incubated with sorbitol, sorbitol and JNK inhibitor I or left untreated. Residues recovered during the mass-spectrometry analysis in the three samples are indicated in bold. Phospho-sites are underlined and JNK-dependent phospho-site appear in red.

SUPPLEMENTARY INFORMATION

SUPPLEMENTARY INFORMATION Supplementary Discussion The cell cycle machinery and the DNA damage response network are highly interconnected and co-regulated in assuring faithful duplication and partition of genetic materials into