Active Recombinant Human PRC2 Complex, Flag-tagged

• Cat.No.: PRC2-28H
• Product Overview: Recombinant Human PRC2 Complex that includes full length EZH2, SUZ12, EED and RbAp46/48 (accession numbers NP_001190176.1, NP_056170, NP_003788.2, NP_002884.1, and NP_005601.1, respectively) was expressed in Sf9 and contains an N-terminal FLAG tag at the N-terminus of EZH2.

Specification

• Species: Human
• Source: Insect Cells
• Tag: Flag
• Description: PRC2 (Polycomb Repressive Complex 2) is one of the two classes Polycomb-group, or PcG proteins (the other being PRC1) that are important epigenetic determinants of stem cell identity. They play an important role in long-term epigenetic silencing of genes during cell fate determination and differentiation. PRC2 functions as a repressor of chromatin. PRC2 is required to target recruitment to specific DNA sequences (termed Polycomb Response Elements or PREs) of genomic regions to be silenced. Once associated with chromatin, the PRC2 subunit EZH2 has histone methyltransferase activity that catalyzes the trimethylation of histone H3 at lysine 27. H3K27me3 is well established as a hallmark of regions of repressed chromatin. Trimethylation of lysine 27 leads to the recruitment of PRC1 through the binding of H3K27me3 by chromodomain-containing proteins in PRC1. PRC1 is responsible for long-term gene silencing after cellular differentiation.
• Form: 25 mM HEPES pH 7.5, 120 mM NaCl, 5% glycerol and 0.2 mg/ml 3X FLAG peptide.
• Bio-activity: H3K27me(1-3) methyltransferase.
• Molecular Mass: The molecular weights of expressed EZH2, SUZ12, EED and RbAp46/48 are 87 kDa, 83 kDa, 50.2 kDa, 47.8 kDa and 47.7 kDa, respectively.
• Applications: PRC2 Complex is suitable for use in the study of enzyme kinetics, inhibitor screening and selectivity profiling.
• Storage: Recombinant proteins in solution are temperature sensitive and must be stored at -80°C to prevent degradation. Avoid repeated freeze/thaw cycles and keep on ice when not in storage.
• Concentration: 0.65 mg/ml
• Full Length: Full L.
• Publications:
  SUMOylation of E2F1 Regulates Expression of EZH2 (2020)

Citation

SUMOylation of E2F1 regulates expression of EZH2

Journal: Cancer research    PubMed ID: 32816857    Data: 2022/1/7

Authors: Li Du, Marwan G. Fakih, Yuan Chen

Article Snippet:In CDH1 promoter luciferase assay, both WT and 4KR EZH2 showed similar dose-dependent suppression of the CDH1 promoter ( ).showed similar dose-dependent suppression of the CDH1 promoter ( ). ... Taken together, these data suggest that SUMOylation regulates EZH2 function mainly through its expression and not through direct SUMO modification of EZH2. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 6. caption a7 caption a8 EZH2 SUMOylation doesn’t affect its histone methyltranferase activity, protein stability or its suppression of CDH1 promoter. (A) A representative blot of in vitro SUMOylation of purified PRC2 (Creative-Biomart) by incubating with recombinant SUMO E1 (SAE1/SAE2), SUMO E2 enzyme (UBC9), and SUMO1 or SUMO2, and without or with RanBP2 (an E3) at 30oC for 3 h or 16 h. EZH2 was detected by western blot. (B) A representative blot of in vitro histone methytransferase assay with PRC2 after PRC2 in vitro SUMOylation.. SUMOylated PRC2 from in vitro SUMOylation was incubated with or without SENP1 for 30 min, and then histone H3 and S-adenosyl methionine were added for 60 min. Then, western blots were performed to detect H3K27me3 level. (C) Top, schematic diagram indicating the three detected and one predicted SUMOylation sites of EZH2—K20, K307, K419 and K421.SUMOylated PRC2 from in vitro SUMOylation was incubated with or..

(A) A representative blot of in vitro SUMOylation of purified PRC2 (Creative-Biomart) by incubating with recombinant SUMO E1 (SAE1/SAE2), SUMO E2 enzyme (UBC9), and SUMO1 or SUMO2, and without or with RanBP2 (an E3) at 30oC for 3 h or 16 h. EZH2 was detected by western blot. (B) A representative blot of in vitro histone methytransferase assay with PRC2 after PRC2 in vitro SUMOylation. SUMOylated PRC2 from in vitro SUMOylation was incubated with or without SENP1 for 30 min, and then histone H3 and S-adenosyl methionine were added for 60 min. Then, western blots were performed to detect H3K27me3 level. (C) Top, schematic diagram indicating the three detected and one predicted SUMOylation sites of EZH2—K20, K307, K419 and K421. 293T cells were transfected with plasmids expressing HA-tagged WT or mutant EZH2 with 4 SUMOylation sites mutated to arginine (4KR) along with an UBC9-expressing vector. Then, the expression of both WT and mutant EZH2 were detected by western blots with an anti-HA antibody. Cell lysates were immunoprecipitated with an anti-HA antibody under denaturing condition followed by immunoblotting with an anti-HA antibody and SUMO1 antibody. (D) SUMOylation did not affect EZH2 protein stability. Representative western blot of EZH2 in HCT116 cells transfected with HA-tagged EZH2 WT or 4KR mutant expression plasmid for 2 days, followed by treatment with 100 μg ml?1 CHX for indicated time. GAPDH was used as a loading control. (E) SUMOylation doesn’t affect EZH2 function in suppressing CDH1 promoter. HCT116 cells were transfected with empty vector (Ctrl), EZH2 WT or 4KR mutant together with CDH1 promoter reporter plasmid and renilla plasmid. EZH2 WT or 4KR plasmids were transfect with different doses (100 ng/well “+” and 200 ng/well “++”). CDH1 promoter activity was measured 48 h post transfection and normalized to renilla. Estimated variation is indicated as SD, p values were derived using a two-tailed Student’s t-test or ANOVA. ns, not significant, * p < 0.05, ** p < 0.01, *** p < 0.001.