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www.sciencemag.org/cgi/content/full/1141194/DC1 Supporting Online Material for SCFFbxl3 Controls the Oscillation of the Circadian Clock by Directing the Degradation of Cryptochrome Proteins Luca Busino, Florian Bassermann, Alessio Maiolica, Choogon Lee, Patrick M. Nolan, Sofia I. H. Godinho, Giulio F. Draetta, Michele Pagano* *To whom correspondence should be addressed. E-mail: [email protected] Published 26 April 2007 on Science Express DOI: 10.1126/science.1141194 This PDF file includes: Materials and Methods Figs. S1 to S10 References Supporting Online Material Materials and Methods Cell culture, synchronization and drug treatment Early-passage Cry1-/-;Cry2 -/- mouse embryonic fibroblasts (MEFs) (S1, S2), MEFs from littermate wild type mice, NIH3T3 cells, HEK293T, and Hela S3 cells were maintained in Dulbecco’s modified Eagle’s medium containing 10% fetal bovine serum (FBS). To synchronize the circadian clock, NIH3T3 cells were grown to confluence and then serum starved for 16 hours in the presence of 0.5% bovine serum (BS). Serum starved cells were then shifted to medium containing 50% adult horse serum and incubated for 90 minutes, after which they were washed twice with PBS and cultured for the additional indicated hours in phenol-red free medium containing 0.5% BS. Synchronization of MEFs was performed as for NIH3T3 cells, except that the serum starvation step was omitted. To test the in vivo interaction between FLAG-tagged F-box proteins and endogenous CRY proteins, the proteasome inhibitor MG132 (Peptide Institute Inc.) (10 µM final concentration) was added for 6 hours prior to harvesting the cells. Purification and analysis of Fbxl3 interactors Hela S3 cells were grown in suspension and retrovirally infected with a pBabe FLAG-HA-Fbxl3 construct (see below). Approximately 5x109 cells were treated with 10 µM MG132 for six hours. Cells were harvested and subsequently lysed in lysis buffer (LB: 50 mM Tris-HCl pH 7.5, 150 mM NaCl, 1 mM EDTA, 50 mM NaF, 0.5% NP40, plus protease and phosphatase inhibitors). Fbxl3 was immunopurified with anti-FLAG agarose resin (Sigma) and washed 5 times with LB (15 minutes each). After washing, proteins were eluted twice by competition with FLAG peptide (Sigma). The eluate was then subject to a second immunopurification with an anti-HA resin (12CA5 monoclonal antibody crosslinked to protein A Sepharose; Covance) prior to a competition with HA peptide (Roche). The final eluate was separated by SDS-PAGE, and proteins were visualized by silver staining. Bands were excised from the gel and digested overnight in situ with Trypsin (Sigma). The acidified peptide solution was desalted and concentrated as described (S3), injected via an Agilent 1100 Nano HPLC onto a C18 column (Reprosil-Pur C18-AQ 3 µm), and packed into a spray emitter (100 µm internal diameter, 8 µm opening, 70 mm length; New Objectives). Peptides were eluted in a gradient from buffer A (5% acetonitrile and 0.5% acetic acid) to buffer B (acetonitrile and 0.5% acetic acid) going from 0% to 20% in 10 min at 300 nl min-1. Spectra were recorded on a QSTAR XL quadrupole time-of-flight (TOF) tandem mass spectrometer (AppliedBiosystems/MDS-Sciex). Biochemical methods Extract preparation, immunoprecipitation, and immunoblotting were previously described (S4, S5). Mouse monoclonal antibodies were from Invitrogen (against βTrcp1, Cul1, Skp1, Skp2, and Emi1), Sigma (anti-FLAG), Covance (anti-HA), BD Transduction Laboratories (anti-c-Jun), Santa Cruz (against Actin, cyclin F, and Skp1). Guinea pig polyclonal antibodies raised against Per1, Per2, Cry1, and Cry2 (S6) and the rabbit polyclonal anti-Cdk1 antibody (S7) were previously described. 2 The anti-Tim antibody was provided by P. Minoo (S8). The anti-Fbxl3 antibody was produced in rabbit by immunization with a peptide corresponding to aa 228-236 (CIDDTPVDDP) of human Fbxl3. This region has high antigenic index and does not show homology to other proteins in the database. A polyclonal antibody against Fbxl10 was generated by immunizing rabbits with a peptide containing amino acids 800-1000 of human Fbxl10. Rabbit polyclonal antibody against Fbxl11 was raised against a peptide containing amino acids 11-24 of human Fbxl11. Plasmids and small hairpin RNAs Per1, Per2, Cry1, Cry2, Clock, and Bmal1 cDNAs were provided by D.M. Virshup and subcloned in pcDNA3.1. Tim cDNA was provided by J. Takahashi and subcloned into pcDNA3.1. For retrovirus production, cDNAs encoding Fbxl3, βTrcp1, Skp2, Fbxl2, Fbxl10, Fbxl11, Fbxl15, Emi1, Fbxo9, Fbx022, Cry1, and Cry2 were subcloned into the retroviral vector pBabe and/or pallino (S9). Fbxl3 cDNA was amplified using a 5’oligo encoding the FLAG-HA epitope tag sequence and subsequently cloned in pBabe (S9). shRNAs targeting human and mouse Fbxl3 were constructed in the Lenti-Lox3.7(pLL3.7) vector. The oligonucleotides used to construct the shRNAs for human Fbxl3 were : (#1) 5’ TGGACATTATTCTCCAAGTATTCAAGAGATACTTGGAGAATAATGTCCTTTTTTC 3’; and (#2) 5’ TGGCCAACAATAGTGATACATTCAAGAGATGTATCACTATTGTTGGCCTTTTTTC 3’. The oligonucleotides used to construct the shRNAs for mouse Fbxl3 were: (#1) 5’ TGAATTGGAATCAGGTATTTTTCAAGAGAAAATACCTGATTCCAATTCTTTTTTC 3’; (#2) 5’ TGGACAGCAGCAAAGAATCATTCAAGAGATGATTCTTTGCTGCTGTCCTTTTTTC 3’; and (#3) 5’ TGCTTGTGAATTGCTCTTTATTCAAGAGATAAAGAGCAATTCACAAGCTTTTTTC 3’. Transient transfections, retrovirus- and lentivirus-mediated DNA transfer HEK293T cells were transfected using the calcium phosphate method as described (S10). For retrovirus production, packaging GP-293 cells (Clontech) were transfected with FuGENE-6 transfection reagent (Roche) according to the manufacturer’s instructions. Forty-eight hours after transfection, the viruscontaining medium was collected and supplemented with 8 µg/ml polybrene (Sigma). Cells were then infected by replacing the cell culture medium with the viral supernatant for six hours. For lentivirus production, HEK293T cells were cotransfected with pLL3.7 and packaging vectors, and the virus-containing medium was collected after 48 - 72 hours and supplemented with 8 µg/ml polybrene. Target cells were incubated with the virus twice a day for three hours for two consecutive days. Recombinant proteins cDNAs encoding the entire coding region of human Fbxl3 and Cry2 were inserted into the baculoviral expression vector pFastBac HTb (Invitrogen). Bacterial vectors for human Ubc3 (His-tagged) (S4), Ubc5 (His-tagged) (S4), and baculoviruses expressing Skp2 (S7), βTrcp1, (S11), Skp1 (His-tagged), Cul1 (HA-tagged), and Roc1 (S7, S12) were previously described. All recombinant proteins were produced in 5B insect cells as described (S13). In vitro ubiquitylation assay The ubiquitylation of Cry2 was performed in a volume of 10 µl containing 50 mM Tris pH 7.6, 5 mM MgCl2, 0.6 mM DTT, 2 mM ATP, 1.5 ng/µl E1 (Boston Biochem), 3 10 ng/µl Ubc3, 10 ng/µl Ubc5, 2.5 µg/µl ubiquitin (Sigma), 1 µM ubiquitin aldehyde, 2 µl of extracts from 5B insect cells expressing human Cry2, Skp1, Cul1, Roc1, and one of the following human F-box proteins: Fbxl3, Skp2, or βTrcp1. In the experiment shown in Fig. 2D, insect cell extracts were substituted with the indicated immunoprecipitates. Where indicated, 2.5 µg/µl methylubiquitin (Boston Biochem) was added instead of ubiquitin. The reactions were incubated at 30°C for the indicated times and analyzed by SDS-PAGE and immunoblotting with an antibody against Cry2. mRNA Analysis RNA was extracted using the RNeasy Kit (Qiagen). cDNA synthesis was performed using Superscript III (Invitrogen). Quantitative PCR analysis was performed according to standard procedures. Primer sequences were: Per1, 5’ ACCTCAGCCAGCATCACCC 3’ and 5’ GAAGAGTCGATGCTGCCAAAG 3’ Per2, 5’ GTCCACCTCCCTGCAGACAAG 3’ and 5’ GCCTTCACCTGCTTCACGC 3’ Cry1, 5’ AAGAGGATGCCCAGAGTGTCG 3’ and 5’ CCTCCCACACGCTTTCGTATC 3’ 18S rRNA, 5’ CGCCGCTAGAGGTGAAATTC 3’ and 5’ CTTTCGCTCTGGTCCGTCTT 3’ Chromatin immunoprecipitations NIH3T3 cells were washed twice with PBS and cross-linked with 1% formaldehyde at room temperature for 10 min. Cells then were rinsed with ice-cold PBS twice and sonicated in lysis buffer (1% SDS, 10 mM EDTA, 50 mM Tris-HCl, pH 8.1, and protease inhibitor cocktail) three times for 10 seconds each, followed by centrifugation for 10 minutes. Supernatants were collected and diluted in buffer (1% Triton X-100, 2 mM EDTA, 150 mM NaCl, 20 mM Tris-HCl, pH 8.1) followed by immunoclearing with 2 µg sheared salmon sperm DNA, 20 µl preimmune serum, and protein G-sepharose for 2 hours at 4°C. Immunoprecipitations were performed by incubating the supernatants overnight at 4°C with an anti-FLAG monoclonal antibody. After immunoprecipitation, protein G-Sepharose, and 2 µg of salmon sperm DNA were added, and the incubation was continued for 1 hour. Precipitates were washed sequentially for 10 minutes each in buffer I (0.1% SDS, 1% Triton X100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 150 mM NaCl), buffer II (0.1% SDS, 1% Triton X-100, 2 mM EDTA, 20 mM Tris-HCl, pH 8.1, 500 mM NaCl), and buffer III (0.25 M LiCl, 1% NP-40, 1% deoxycholate, 1 mM EDTA, 10 mM Tris-HCl, pH 8.1). Precipitates were then washed three times with TE buffer and extracted three times with 1% SDS, 0.1 M NaHCO3. Eluates were pooled and heated at 65°C for at least 6 hours to reverse the formaldehyde cross-linking. DNA fragments were purified with a QIAquick Spin Kit (Qiagen). Quantitative PCR analysis was performed according to standard procedure. Primers sequences were as reported in (S14): Per1, 5'-CAGATGCCAGGAAGAGATCCTTAGCCAACC-3' and 5'-GACTAACCCTAGGATTGCAGCAGGGATCC-3'; Per2, 5'-AGCAGCATCTTCATTGAGGAACCCGGG-3' and 5'-CTCCGCTGTCACATAGTGGAAAACGTGAC-3'; Cry1, 5'-CTGCAACCAGCTCGGGCCGTC-3' and 5'-GATGAATGGGAGGCTGCCGAGGC-3' rDNA, 5'-GGAGTGCGATGGTGTGATCT-3' and 5'-TAAAGATTAGCTGGGCGTGG-3' 4 Supplementary Figures B HA beads 176 97 176 97 66 56 47 66 56 47 36 36 31 31 * FLAG-HA-Fbxl3 HA FT HA eluate HA beads EV FLAG eluate FLAG-HA-Fbxl3 FLAG eluate HA FT HA eluate HA FT HA eluate HA beads FLAG eluate EV Immunoblot (anti-FLAG) HA FT HA eluate HA beads Silver Staining FLAG eluate A * * * * Fbxl3 * Fig. S1. Purification of the Fbxl3 complex. (A) FLAG-HA-tagged Fbxl3 was retrovirally expressed in HeLa S3 cells and subjected to immunopurification using anti-FLAG agarose and anti-HA resin. The FLAG peptide eluate (FLAG eluate), the flow-through from the second column (HA FT), the HA peptide eluate (HA eluate), and the SDS eluate of the material left on the HA resin (HA beads) were separated by SDS-PAGE and visualized by silver staining. Bands were excised from the lane containing the HA eluate and analyzed by mass spectrometry. The mass spectrometry analysis of proteins co-purified with Fbxl3 revealed the presence of two peptides (FLLQCLEDLDANLR and LTDLYKK) corresponding to Cry1 and three peptides (GQPADVFPR, EFFYTAATNNPR, MKQIYQQLSR) corresponding to both Cry1 and Cry2. As a control, a purification was performed from HeLa S3 cells infected with an empty vector (EV). (B) 3% of the material shown in (A) was analyzed by immunoblotting with an anti-FLAG antibody. The asterisks indicate the immunoglobulin chains. 5 HA-Cry2 0 90 180 HA-Cry2 + FLAG-Fbxl3 HA-Cry2 + FLAG-Skp2 HA-Cry2 + FLAG-Fbxl3 + MG132 0 0 0 90 180 90 180 90 180 CHX (min) Cry2 (anti-HA) FBPs (anti-FLAG) Actin Fig. S2. Expression of Fbxl3 accelerates the degradation of Cry2. HEK293T cells were transfected with vectors encoding HA-tagged Cry2 alone or HA-tagged Cry2 in combination with either FLAG-tagged Fbxl3 or FLAG-tagged Skp2. Three hours before lysis, cycloheximide (CHX) was added to the cell culture in the presence or absence of MG132, as indicated. Extracts were then subjected to immunoblotting with antibodies against HA (upper panels) to detect Cry2, against FLAG (middle panels) to detect F-box proteins (FBPs), or against Actin (bottom panels) to show normalization in loading. 6 B Fblx3 shRNA 2 Fbxl3 shRNA 1 LacZ shRNA UI A 0 30 60 120 180 240 0 30 60 120 180 240 CHX(min) Fbxl3 shRNA 1 Fbxl3 shRNA 2 Actin 2 3 Actin LacZ shRNA Fbxl3 1 Cry2 (anti-HA) Fbxl3 shRNA 2 (short exp.) 4 Fig. S3. Downregulation of Fbxl3 stabilizes Cry2 in HEK293T cells. (A) Retrovirally infected HEK293T cells expressing HA-tagged Cry2 were infected with lentiviral constructs that direct the synthesis of shRNAs targeting LacZ (lane 2) or Fbxl3 (lanes 3 and 4, each expressing a different shRNA to human Fbxl3). Lane 1 shows a cell extract from un-infected cells (UI). Twenty-four hours postinfection, cells were trypsinized, replated, and cultured for three additional days. Finally, cells were collected, and protein extracts were probed with antibodies to the indicated proteins. (B) The experiment was performed as in (A), except that cycloheximide (CHX) was added for the indicated times. Notably, since the transcription of exogenous Cry2 was driven by a heterologous promoter, the increase in the level of Cry2 protein observed at time 0 using the shRNA 2 was independent of the transcriptional regulation by Clock:Bmal1. 7 Fbxl3 shRNA 3 LacZ shRNA Fbxl3 shRNA 1 Fbxl3 shRNA 2 A Fbxl3 Actin 1 B 2 3 4 LacZ shRNA +DMSO Fbxl3 shRNA +MG132 +DMSO +MG132 Cry1 (anti-HA) Actin C LacZ shRNA +DMSO Fbxl3 shRNA +MG132 +DMSO +MG132 Cry2 (anti-HA) Actin Fig. S4. Downregulation of Fbxl3 stabilizes CRY proteins in NIH3T3 cells. (A) Retrovirally infected NIH3T3 cells expressing HA-tagged Cry1 were infected with lentiviral constructs that direct the synthesis of shRNAs targeting LacZ (lane 1) or Fbxl3 (lanes 2 - 4, each expressing a different shRNA to murine Fbxl3). Twentyfour hours post-infection, cells were trypsinized, replated, and cultured for three additional days. Finally, cells were collected, and protein extracts were probed with antibodies to the indicated proteins. (B) The experiment was performed as in (A), except that cycloheximide (CHX) was added to the cell culture in the presence or absence of MG132, as indicated. The shRNA construct 3, shown in (A), was used. (C) The experiment was performed as in (B), except retrovirally infected NIH3T3 cells expressing HA-tagged Cry2 were used. 8 Cry2 SCFFbxl3 Ubiquitin Methylubiquitin . . . . in in in. in in. in m m m m 45 45 5m 45 5m 45 + + + + + + + + + + + + + + + Cry2 (Ub)n Cry2 Fig. S5. The high molecular weight forms of Cry2 are polyubiquitylated species of the protein. In vitro ubiquitin ligation assays of recombinant Cry2 protein were conducted in the presence of recombinant SCFFbxl3. Samples were incubated at 30°C for the indicated time, and reactions were then stopped by the addition of Laemmli sample buffer. Where indicated, methylated ubiquitin was added to the reaction mix. Finally, samples were analyzed by SDS-PAGE and immunoblotting analysis using an antibody against Cry2. The bracket on the left side of the panel marks a ladder of bands corresponding to polyubiquitylated Cry2. When methylated ubiquitin (which is chemically modified to block all of its free amino groups) was added to the in vitro reaction, the abundance of the slowest migrating bands decreased. This effect is due to the capping of polyubiquitin chains by methylated ubiquitin that competes with unmodified ubiquitin and terminates the polyubiquitin chains. This experiment formally demonstrates that the high molecular weight forms of Cry2 are polyubiquitylated species of the protein. 9 Fbxl3 shRNA Asynch. 50% HS 6 12 18 24 30 36 42 48 54 Asynch. 50% HS 6 12 18 24 30 36 42 48 54 A LacZ shRNA hours post-serum shock hours post-serum shock Cry1 (long exp.) Cry1 (short exp.) Per1 Per2 Fbxl3 c-Jun Tim1 Actin B hours post-serum shock 250 200 150 100 50 0 36 42 48 54 24 30 Cry1 Per1 Per2 y 50 nch % . H S 6 12 18 fold increase Fbxl3 shRNA As 250 200 150 100 50 0 As y 50 nch % . H S 6 12 18 24 30 36 42 48 54 fold increase LacZ shRNA hours post-serum shock Fig. S6. Fbxl3 promotes the oscillations of Cry1 and the accumulation of PER proteins. (A) NIH3T3 fibroblasts were infected with lentiviral constructs that direct the synthesis of shRNAs targeting LacZ or Fbxl3, as indicated. In this experiment the shRNA construct 3 to mouse Fbxl3 (see fig. S4A) was used. Cells were asynchronously grown to confluence in a medium containing 10% fetal bovine serum (Asynch.). Asynchronous cells were serum starved for 16 hours and then shifted to a medium containing 50% serum for 90 minutes (50% HS). After this period, the serum-rich medium was replaced with 0.5% serum-containing medium and cells were collected at the indicated hours post-serum shock. Cell extracts were analyzed by immunoblotting with antibodies to the indicated proteins. The experiment with this particular shRNA construct to mouse Fbxl3 was repeated two times with similar results. (B) The graphs illustrate the quantification by densitometry of the results shown in (A). The value given for the amount of Cry1, Per1, and Per2 present in asynchronous cells treated with shRNAs targeting LacZ was set as 100%. 10 A y As LacZ shRNA Fbxl3 shRNA nc + h. % 50 SD + + HS Hours post-serum shock 8 4 20 12 + + + + + + + + + + + Cry1 Per2 Fbxl3 Actin B shRNA LacZ shRNA Fbxl3 120 Per2 protein (fold increase) 80 60 40 20 100 50 20 12 8 4 H S . D . ch S. 50 % Hours post-serum shock yn 20 12 8 4 H S D . 50 % S. . ch yn 150 0 0 As 200 As Cry1 protein (fold increase) 100 Hours post-serum shock Fig. S7. Fbxl3 promotes the accumulation of Per2 and controls oscillations of the clock. (A) NIH3T3 fibroblasts were infected with lentiviral constructs that direct the synthesis of shRNAs targeting LacZ or Fbxl3, as indicated. In this experiment the shRNA construct 2 to mouse Fbxl3 (see fig. S4A) was used. Cells were asynchronously grown to confluence in a medium containing 10% fetal bovine serum (Asynch.). Asynchronous cells were serum deprived for 16 hours (SD) and then shifted to a medium containing 50% serum for 90 minutes (50% HS). After this period, the serum-rich medium was replaced with 0.5% serumcontaining medium and cells were collected at the indicated hours post-serum shock. Cell extracts were analyzed by immunoblotting with antibodies to the indicated proteins. The experiment with this particular shRNA construct to mouse Fbxl3 was repeated two times with similar results. (B) The graphs illustrate the quantification by densitometry of the results shown in (A). The value given for the amount of Cry1 and Per2 present in asynchronous cells treated with shRNAs targeting LacZ was set as 100%. 11 LacZ shRNA Fbxl3 shRNA 3 2 1 0 SD 50% HS 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Per1 mRNA (fold increase) 4 hours post-serum shock 3 2 1 0 SD 50% HS 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Per2 mRNA (fold increse) 4 hours post-serum shock 3 2 1 0 SD 50% HS 3 6 9 12 15 18 21 24 27 30 33 36 39 42 45 48 Cry1 mRNA (fold increase) 4 hours post-serum shock Fig. S8. Fbxl3 promotes the accumulation of Clock:Bmal1-regulated mRNAs. The experiment was performed as in Fig. S7, except that whole-cell RNA was prepared to determine the levels of the indicated mRNAs using quantitative RTPCR. The value given for the amount of mRNA present in asynchronous cells treated with shRNAs targeting LacZ was set as 1. Error bars represent + SD (n=3). 12 * Fold Increase 5 * * UI LacZ shRNA Fbxl3 shRNA 4 3 2 1 0 Per2 promoter Per1 promoter Cry1 promoter rDNA promoter Fig. S9. Stabilized Cry1 binds the promoters of Clock:Bmal1-regulated genes. Retrovirally infected NIH3T3 cells expressing FLAG-tagged Cry1 or un-infected NIH3T3 cells (UI) were infected with lentiviral constructs carrying shRNAs against LacZ or Fbxl3 and subsequently used for chromatin immunoprecipitations using an anti-FLAG antibody. DNA precipitated with the anti-FLAG antibody was amplified by real-time PCR using primers flanking the indicated gene promoters. The unrelated rDNA promoter was used as negative control. The value given for the amount of PCR product present in un-infected NIH3T3 cells was set as 1. Error bars represent + SD (n=3). Asterisks indicate a p value <0.01 measured using a t-test. 13 LacZ shRNA Fbxl3 shRNA HA-Cry2 HA-Cry2 HA-Cry2 + Fbxl3 0 1 3 5 0 1 3 5 0 1 3 5 HA-Cry2 +Fbxl3 (C358S) 0 1 3 5 CHX (hours) Cry2 (anti-HA) Cry2 (anti-HA) (short exp.) Fbxl3 Actin Fig. S10. Fbxl3(C358S) does not induce degradation of Cry2. NIH3T3 cells were infected with retroviral vectors encoding HA-tagged Cry2 alone or HA-tagged Cry2 in combination with either FLAG-tagged human Fbxl3 or FLAG-tagged human Fbxl3(C358S), as indicated. Cells were then infected with lentiviral constructs that direct the synthesis of shRNAs targeting LacZ or mouse Fbxl3 (which does not target retrovirally expressed human Fbxl3). Twenty-four hours post-infection, cycloheximide (CHX) was added to the cell culture for the indicated times. Finally, cells were collected, and protein extracts were probed with antibodies to the indicated proteins. 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