Journal: eLife

Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

doi: 10.7554/eLife.12785

Figure Lengend Snippet: CCNE1 (Cyclin E) gene transcription is up-regulated upon ORC1 depletion. ( A – D ) Quantitative PCR analysis of ORC1, CCNE1 (Cyclin E), CCNA2 (Cyclin A) and GAPDH transcript levels in U2OS cells transfected with either control siRNA targeting GFP (blue bar) or ORC1 siRNA (grey bar). The siRNA treated cells were released after nocodazole arrest and mRNA levels estimated at different time points as indicated in hours. The values shown are average fold change (mean±SEM) from three independent experiment normalized to β-actin transcripts. Statistical analysis was performed using the Student’s t test. *p<0.01; **p<0.001; ***p<0.0001; NS, not significant. ORC, Origin Recognition Complex; RB, Retinoblastoma; GST, Glutathione S transferase. DOI: http://dx.doi.org/10.7554/eLife.12785.004

Article Snippet: To generate Glutathione S transferase (GST) fusion proteins, wild type RB or its mutants (R661W and N757F), HDAC1, SUV39H1 or its fragments, HP1α or CDC6 were cloned into the bacterial expression vector pGEX6P1 (GE Healthcare Life Sciences, NJ).

Techniques: Real-time Polymerase Chain Reaction, Transfection

Journal: eLife

Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

doi: 10.7554/eLife.12785

Figure Lengend Snippet: Cyclin E-CDK2 phosphorylation controls ORC1 and RB interaction. ( A ) Interaction between purified RB and ORC1 in a MBP pull down assay. MBP-fused wild-type RB was bound to amylose resin and further incubated with in vitro translated, S 35 -labeled wild type ORC1 or its mutants in the presence or absence of Cyclin E-CDK2 and 1 mM ATP. Beads were isolated and bound proteins were separated by gel electrophoresis. MBP was used as a control in the assay. ( B ) Alignment of ORC1 sequences is shown with conserved LxCxE motif. The conserved residues of LxCxE motif are indicated with different colors. The alignment shows conserved residues in ORC1 from different species in vertebrate and invertebrate classes (Invertebrates: Brugia malayi , Caenorhabditis briggsae, Caenorhabditis elegans, Strongylocentrotus purpuratus, Culex quinquefasciatus, Apis mellifera, Drosophila melanogaster, Aedes aegypti , and Pediculus humanus ; Vertebrates: Danio rerio, Xenopus laevis, Xenopus tropicalis, Gallus gallus, Taeniopygia guttata, Mus musculus and Homo sapiens) . ( C ) Interaction between ORC1 and RB in a MBP pull-down assay. GST-fused wild-type RB or its mutant proteins were incubated with wild type MBP-ORC1 in the presence or absence of Cyclin E-CDK2 and/or 1 mM ATP. Amylose-bead-bound proteins were isolated and bound proteins were separated by gel electrophoresis followed by immunoblotting with anti-GST antibody. Recombinant MBP was used as a control in the assay. ORC, Origin Recognition Complex; RB, Retinoblastoma; GST, Glutathione S transferase; MBP, Maltose binding protein. DOI: http://dx.doi.org/10.7554/eLife.12785.005

Article Snippet: To generate Glutathione S transferase (GST) fusion proteins, wild type RB or its mutants (R661W and N757F), HDAC1, SUV39H1 or its fragments, HP1α or CDC6 were cloned into the bacterial expression vector pGEX6P1 (GE Healthcare Life Sciences, NJ).

Techniques: Purification, Pull Down Assay, Incubation, In Vitro, Labeling, Isolation, Nucleic Acid Electrophoresis, Mutagenesis, Western Blot, Recombinant, Binding Assay

Journal: eLife

Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

doi: 10.7554/eLife.12785

Figure Lengend Snippet: ( A ) Purified MBP-ORC1 and various GST-fused proteins were mixed and proteins bound in a GST-pull down were detected by immunoblotting with anti-MBP antibodies. The purified proteins are shown in . ( B ) U2OS and MCF7 cell lysates were immunoprecipitated with SUV39H1 antibody and immunoblotted with the indicated antibodies. Rabbit IgG served as control antibody. Asterisk indicates the cross-reacting antibody band; arrow indicates the SUV39H1 protein. ( C ) Immunoprecipitation from RB-negative SaOS-2 cell lysates with ORC1 antibody or IgG and immunoblotted with antibodies against ORC1, SUV39H1 or ORC3. ( D ) HEK293 cells were transiently co-transfected with GFP, GFP-ORC1 or GFP-RB plus T7-SUV39H1 plasmids (2.5 μg each). GFP antibody immunoprecipitates were immunoblotted with the indicated antibodies. The interaction between ORC1 and SUV39H1 and between RB and SUV39H1 is shown with purified proteins and quantitated in . Higher levels of RB are required to demonstrate an interaction with SUV39H1 in vivo and ORC1 interacts with the SET domain of SUV39H1, . ( E ) MBP-ORC1 was incubated with bead-bound histone peptides with or without the indicated modifications and bound MBP-ORC1 was observed by immunoblotting with anti-MBP antibody (lower box) or silver staining (upper box). ( F – G ) Wild-type CCNE1 -luciferase reporter assay in U2OS cells. U2OS cells were transiently co-transfected with 500 ng of 10–4 CCNE1 promoter, 50 ng E2F1, 50 ng DP1 and 20 ng pCMV-LacZ plasmids along with the indicated amounts ORC1 and/or SUV39H1 plasmids. ( F ) Increasing amounts of ORC1-Flag or T7-SUV39H1 repress Cyclin E gene promoter. Experiments were carried out in triplicate. Expression of proteins was confirmed by Immunoblot; α-Tubulin as loading control. Statistical analysis was performed using the Student’s t test. *p<0.05; **p<0.005; ***p<0.001. ( G ) ORC1-Flag cooperates with wild type but not mutant SUV39H1 to repress CCNE1 gene expression. The experiments were carried out in triplicate. Expression of proteins was confirmed by Western blots. α-Tubulin served as a control for equal loading of each sample. Statistical analysis was performed using the Student’s t test. *p<0.01; **p<0.005; ***p<0.0001. Repression of transcription by ORC1 and SUV39H1 was also demonstrated using an artificial promoter and tethering the proteins via the GAL4 DNA binding domain in . ORC, Origin Recognition Complex; MBP, Maltose binding protein; GST, Glutathione S transferase. DOI: http://dx.doi.org/10.7554/eLife.12785.006

Article Snippet: To generate Glutathione S transferase (GST) fusion proteins, wild type RB or its mutants (R661W and N757F), HDAC1, SUV39H1 or its fragments, HP1α or CDC6 were cloned into the bacterial expression vector pGEX6P1 (GE Healthcare Life Sciences, NJ).

Techniques: Purification, Western Blot, Immunoprecipitation, Transfection, In Vivo, Incubation, Silver Staining, Luciferase, Reporter Assay, Expressing, Mutagenesis, Binding Assay

Journal: eLife

Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

doi: 10.7554/eLife.12785

Figure Lengend Snippet: Bacteria expressed and purified recombinant proteins. Silver stain of purified GST, GST-RB, GST-HDAC1, GST-SUV39H1, GST-HP1α, GST-CDC6 and MBP-ORC1 proteins. MW stands for protein molecular weight marker in kilodalton. ORC, Origin recogntion complex; MBP, Maltose binding protein; RB, Retinoblastoma; GST, Glutathione S transferase. DOI: http://dx.doi.org/10.7554/eLife.12785.007

Article Snippet: To generate Glutathione S transferase (GST) fusion proteins, wild type RB or its mutants (R661W and N757F), HDAC1, SUV39H1 or its fragments, HP1α or CDC6 were cloned into the bacterial expression vector pGEX6P1 (GE Healthcare Life Sciences, NJ).

Techniques: Purification, Recombinant, Silver Staining, Molecular Weight, Marker, Binding Assay

Journal: eLife

Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

doi: 10.7554/eLife.12785

Figure Lengend Snippet: SUV39H1 interaction with ORC1 and RB. ( A ) Interaction between purified MBP-ORC1 or MBP-RB with GST-SUV39H1 in a MBP pull down assay. MBP-fused proteins were bound to amylose resin and incubated with GST-SUV39H1. Bound proteins were separated by gel electrophoresis followed by immunoblotting with either anti-GST or anti-SUV39H1 antibodies. Recombinant MBP was used as a control. ( B , C ) GST-SUV39H1 protein was bound to the resin and incubated with either MBP-ORC1 or MBP-RB proteins. Bead-bound proteins were immunoblotted with anti-MBP antibody. Recombinant GST was used as a control. ( D ) Concentration-dependent interaction with increasing levels of GST-SUV39H1 and either 100 nM of MBP or MBP-ORC1 or MBP-RB followed by pull down with amylose beads and subsequently immunoblotted with anti-GST antibody. Bands were quantified and represented in a graph after background subtraction with MBP control protein. ORC, Origin recogntion complex; MBP, Maltose binding protein; GST, Glutathione S transferase. DOI: http://dx.doi.org/10.7554/eLife.12785.008

Article Snippet: To generate Glutathione S transferase (GST) fusion proteins, wild type RB or its mutants (R661W and N757F), HDAC1, SUV39H1 or its fragments, HP1α or CDC6 were cloned into the bacterial expression vector pGEX6P1 (GE Healthcare Life Sciences, NJ).

Techniques: Purification, Pull Down Assay, Incubation, Nucleic Acid Electrophoresis, Western Blot, Recombinant, Concentration Assay, Binding Assay

Journal: eLife

Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

doi: 10.7554/eLife.12785

Figure Lengend Snippet: ORC1 interaction with SET domain of SUV39H1 does not involve other ORC subunits. ( A ) HEK293 cells were transiently co-transfected with GFP, GFP-ORC1 or GFP-RB and T7-SUV39H1-expressing plasmids at the indicated amounts in micrograms. The whole cell lysate prepared from HEK293 cells expressing the indicated constructs were immunoprecipitated with GFP antibody followed by immunoblotting with specific antibodies. ( B ) GFP-tagged wild-type ORC1 or its mutants (A-A: [ORC 235 ARA 237 'Cy' motif mutant] or ORC1S258A,S273A,T375A [CDK: with mutants in CDK target sites]) were co-transfected in HEK293 cells with either Flag-SUV39H1 or its empty vector. Immunoprecipitation with anti-Flag antibody from cell lysates of HEK293 cells overexpressing the indicated constructs followed by immunoblotting with the indicated antibodies. ( C ) Schematic showing domains of human SUV39H1 protein. In the GST-pull down assay, GST-SUV39H1 or its truncation mutant proteins were incubated with MBP-ORC1 protein as indicated and immunoblotted with anti-MBP antibody. GST protein served as negative control. ORC, Origin recogntion complex; MBP, Maltose binding protein; GST, Glutathione S transferase. DOI: http://dx.doi.org/10.7554/eLife.12785.009

Article Snippet: To generate Glutathione S transferase (GST) fusion proteins, wild type RB or its mutants (R661W and N757F), HDAC1, SUV39H1 or its fragments, HP1α or CDC6 were cloned into the bacterial expression vector pGEX6P1 (GE Healthcare Life Sciences, NJ).

Techniques: Transfection, Expressing, Construct, Immunoprecipitation, Western Blot, Mutagenesis, Plasmid Preparation, Pull Down Assay, Incubation, Negative Control, Binding Assay

Journal: eLife

Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

doi: 10.7554/eLife.12785

Figure Lengend Snippet: Purified Proteins. Coomassie Brilliant Blue stained gel of purified MBP, MBP-GFP-ORC1, MBP-RB and GST-CDC6 proteins. MW stands for protein molecular weight marker in kilodalton. MBP, Maltose binding protein; RB, Retinoblastoma; GST, Glutathione S transferase. DOI: http://dx.doi.org/10.7554/eLife.12785.014

Article Snippet: To generate Glutathione S transferase (GST) fusion proteins, wild type RB or its mutants (R661W and N757F), HDAC1, SUV39H1 or its fragments, HP1α or CDC6 were cloned into the bacterial expression vector pGEX6P1 (GE Healthcare Life Sciences, NJ).

Techniques: Purification, Staining, Molecular Weight, Marker, Binding Assay

Journal: eLife

Article Title: Opposing roles for DNA replication initiator proteins ORC1 and CDC6 in control of Cyclin E gene transcription

doi: 10.7554/eLife.12785

Figure Lengend Snippet: ( A – C ) Equimolar amounts of MBP-GFP-ORC1 and MBP-RB proteins were incubated with increasing amounts of GST-CDC6 and/or Cyclin E-CDK2. MBP-GFP-ORC1 protein was immunoprecipitated with GFP antibody, then immunoblotted with the indicated antibodies. The purified proteins used in these experiments are shown in ( A ) MBP-GFP-ORC1 binds GST-CDC6. MBP protein served as control. ( B ) The binding of MBP-GFP-ORC1 protein to either GST-CCD6 in the presence of Cyclin E-CDK2 (left section) or MBP-RB (right section). ( C ) The binding of MBP-GFP-ORC1 to MBP-RB in the presence of increasing molar amounts of Cyclin E-CDK2 (right section) or both GST-CDC6 and Cyclin E-CDK2 (left section). ( D ) GST-pull down assay using GST-CDC6 wild type or CDC6 94 ARA 96 mutant (CDC6A-A) with purified Cyclin E-CDK2 protein followed by Immunoblotting with Cyclin E antibody. GST protein served as control. ( E ) Nocodazole arrested U2OS cells were transfected with 500 ng of GFP, GFP-CDC6 wild type or CDC6 94 ARA 96 mutant (CDC6A-A) plasmids, then released into the next cell cycle. At indicated times, whole cell extracts were immunoblotted with specific antibodies against GFP and Cyclin E. α-Tubulin served as loading control. ( F ), CCNE1 promoter-luciferase reporter assay in U2OS cells. Cells transiently co-transfected with 500 ng of 10–4 CCNE1 promoter, 50 ng E2F1, 50 ng DP1 and 20 ng pCMV-LacZ plasmids together with increasing amounts GFP-CDC6 WT or CDC6 94 ARA 96 plasmids for 24 hr. Relative luciferase activity was normalized to co-transfected LacZ control. Experiments in triplicate. Protein expression determined by immunoblot; α-Tubulin as loading control. Statistical analysis was performed using the Student’s t test. *p<0.05; **p<0.001; ***p<0.0005. GFP, Green fluorescent protein; MBP, Maltose binding protein; GST, Glutathione S transferase. DOI: http://dx.doi.org/10.7554/eLife.12785.013

Article Snippet: To generate Glutathione S transferase (GST) fusion proteins, wild type RB or its mutants (R661W and N757F), HDAC1, SUV39H1 or its fragments, HP1α or CDC6 were cloned into the bacterial expression vector pGEX6P1 (GE Healthcare Life Sciences, NJ).

Techniques: Incubation, Immunoprecipitation, Purification, Binding Assay, Pull Down Assay, Mutagenesis, Western Blot, Transfection, Luciferase, Reporter Assay, Activity Assay, Expressing