Chromatin Fractionation

-​ Main approach and methods -​ Main experiments - techniques -​ Generation of Phosphorylated ORC2 Antibodies -​ Phospho-specific antibodies were created using synthetic peptides corresponding to specific amino acid sequences (112–120 and 221–230) of ORC2. -​ These peptides were used for generating anti-phospho-Thr-116 (-pT116) and anti-phospho-Thr-226 (-pT226) antibodies. -​ The antibodies were produced by Peptron Corp. -​ Immunizing sera were purified through a two-step affinity purification process using resins linked to both phosphopeptide and non-phosphopeptide. -​ Chromatin Fractionation -​ Synchronized HeLa cells were washed with phosphate-buffered saline and then resuspended in buffer A containing various components including protease inhibitor mixture and calyculin A. -​ This suspension was homogenized using a Dounce homogenizer after being kept on ice for 15 minutes. -​ Nuclei were obtained by centrifugation, followed by lysis with buffer A containing 0.5% Nonidet P-40, and then centrifuged again. -​ The soluble fraction was collected as the supernatant, while the precipitate was eluted successively with buffer B containing different concentrations of NaCl. -​ Alternatively, for Fig. 5B, cells were resuspended in buffer C and lysed on ice for 10 minutes, followed by centrifugation. -​ The resulting supernatant was further processed to obtain a soluble fraction, and the precipitate was washed to obtain the chromatin-enriched fraction. -​ Tet-On Inducible Cell Lines -​ FLAG-tagged ORC2 wild type (WT), T116A/T226A (AA), or T116D/T226D (DD) mutant genes were inserted into a pTRE2hyg vector and transfected into HeLa Tet-On cells. -​ These cells already contained a stably expressed reverse tetracycline-responsive transcriptional activator. -​ Hygromycin-resistant cells were selected under 200 g/ml hygromycin for 2–4 weeks for subsequent experiments. -​ FLAG-ORC2 proteins were induced by the addition of 2 g/ml doxycycline. -​ ChIP Assay -​ Soluble chromatin fraction obtained after sonication was used for the ChIP assay. -​ One-tenth of this fraction was kept as input control for PCR. -​ -​ The remaining fraction was precipitated with anti-FLAG antibody and protein A-Sepharose beads, followed by sequential washes in different buffers. The immunocomplexes were eluted, reverse cross-linked, and used for PCR with specific primers. -​ Results - Edwin Human ORC2 is phosphorylated at Thr 116 and Thr 226 -​ Endogenous ORC2 was isolated using immunoprecipitation -​ Phosphoantibodies used to verify the phosphorylation at Thr116 and 226 -​ Antibodies designed to recognize phospho-Thr-116 and phospho-Thr-226 -​ Neither phosphoantibody recognized T to A substitutions -​ Phosphoantibodies recognized before phosphatase tratment or when there was RNA interference -​ Double thymidine blocks - arrests the cell at the G1/S boundary -​ Nocodazole - arrest of the cell at prometaphase -​ Nodacozole treated cells have more phosphorylated ORC2 than double thymidine block treated cells -​ CONCLUSION: different phosphorylation levels during different stages of the cell cycle Phosphorylation of ORC2 in S phase -​ When we release the thymidine block - there is synchronized release into the S phase -​ Using FACS analysis and cell cycle markers, found that S phase happens at 6-8h after release, division at 10-12 hours and next G1 phase at 14 hours -​ Find that the level of ORC2 is constant, but the level of phospho-ORC2 changes -​ Increase at the 2h mark, peaks at 6h and maintains steady until 12 hours where it decreases after 14 hours -​ Cycle is similar to the cycle of cyclin A -​ Release from nodocazole -​ Unphosphorylated, and phosphrylated in M and S phases -​ CONCLUSION: Phosphorylation at Thr-116 and Thr-226 happens in S phase - persists until the M phase When does the ORC2 get phosphorylated? -​ The-116 and Thr-226 are within the TPXK consensus sequence for CDK -​ Roscovitine is a cyclin dependent kinase (CDK) inhibitor -​ Condition of overexpression of cyclin A -​ `cyclin A is expressed ans associated during the S phase -​ When we overexpress cyclin A there is increased phosphorylation of Thr-116 and Thr-226 -​ Condition of overexpression of cyclin E -​ Cyclin E activates CDK2 in the G1 phase -​ No increase in phosphorylation -​ IP with Orc2 and anti-Orc2 AB -​ Co-precipitated cyclinA and CDK2 in S and G2 phase Phosphorylation of ORC2 does not affect formation of ORC complex -​ When we do IP on ORC2 with anti-ORC2 AB, coprecipittated the ORC 1, 3, 4, 5 subunits as well -​ Also works with anti-phospho thr 116 and anti-phospho thr 116 antibodies -​ Level of coprecipitaiton of other ORC subunits -​ Similar to the level of ORC2 -​ Suggests that phosphorylation of ORC2 does not affect complex formation Phosphorylation of ORC2 dissociates ORC from Chromatin -​ Chromatin fractionation performed from synchronized G1 cells or in stages of the S phase -​ Phosphorylated ORC2 easily dephosph. During cell lysis and fractionation -​ Calyculin A is an inhibitor of protein phosphatases -​ Will prevent dephosphorylatiom -​ Since there is easy dephosphorylation during lysis and fractionation, add calyculin A during these phases -​ 0.1M and 0.25M NaCl eluates of chromatin contained similar levels of non-phosphorylated Orc2-5 subunits -​ Shows that phosphorylated ORC2 proteins distributed to the 0.1M eluate over the 0.25M eluate -​ Shows that phosphorylated ORC2 dissociates easier than normal -​ ORC2 proteins released from chromatin at a lower NaCl level than non-phosphorylated proteins -​ CONCLUSION that phosphorylated ORC2 is released at a lower NaCl level than nonphosphorylated ORC2 -​ S phase progression -​ Cell fractions have more phospho Thr 116 and 226 (generally more phosphorylation) -​ Soluble fractions and 0.1M eluates contained more amounts of ORC2-5 than 0.25M eluates -​ co-IP of ORC2 of G2/M cells using anti-ORC4 antibody shows that the soluble fraction contains more phospho116 and 226 than the 0.25M fraction -​ Avoid dephosphorylation - add calyculin A -​ Addition of calyculin A to G1 phase cells did not increase phosphorylation or the fractionation pattern - since it is not phosphorylated in G1 it is more affinity to the chromatin -​ Calyculin A treatment reduced ORC1 amounts in S and G1 phase but not affecting ORC2-5 + phosphorylation had no effect on ORC formation -​ Therefore, phosphorylation during S phase reduces affinity of Orc2-5 to chromatin and dephosphorylation increases affinity -​ Phosphodefective and phosphomimetic forms of ORC2 -​ Use detergent to remove non-chromatin-associated proteins -​ Pre-extraction; removed phosphomimetic ORC2 -​ Phosphomimetic ORC2 decreased in the chromatin-enriched fraction -​ Increased in the soluble fraction -​ Basically the point is that G1 cells have similar ORC levels under both concentrations of salt but with phosphorylated ORC distributed to the 0,1M eluate more than the 0.25M eluate released from chromatin at lower salt concentration than non-phosphorylated -​ SUCCESSIVE ELUTION OF PPT Phosphorylation of ORC2 Dissociates ORC from ori -​ Tagged with FLAG-ORC2 -​ Dissociate from chromatin in the early S phase and remained dissociated in the G2 and M phase -​ Associated with chromatin in the G1 phase of the cell cycle -​ Correlated with cell-cycle dependent phosphorylation pattern of endogenous ORC2 -​ Phosphomimetic ORC2 did not dissociate, and phosphodefective protein reduced -​ Support hypothesis that phosphorylation disscoiates ORC from chromatin in a cell cycle dependent manner ​