Journal: Nucleic Acids Research

Article Title: ATM, KAP1 and the Epstein–Barr virus polymerase processivity factor direct traffic at the intersection of transcription and replication

doi: 10.1093/nar/gkad823

Figure Lengend Snippet: KEY RESOURCES TABLE

Article Snippet: Rabbit anti-EHMT2 antibody , Proteintech , 29303–1-AP.

Techniques: Purification, Gel Extraction, DNA Extraction, RNA Extraction, Plasmid Preparation, SYBR Green Assay, Electroporation, In Vitro, Transfection, Recombinant

Journal: Advanced Science

Article Title: Dynamic Phosphorylation of G9a Regulates its Repressive Activity on Chromatin Accessibility and Mitotic Progression

doi: 10.1002/advs.202303224

Figure Lengend Snippet: Identification of T1045 on G9a is phosphorylated by Plk1 during mitosis. A) Mass spectrum showed that G9a is phosphorylated at T1045. B) Sequence alignment of conserved T1045 sites of G9a in different species. C) In vitro kinase assay showed that T1045A mutation abolished Plk1‐mediated phosphorylation of G9a. D) T1045 phosphorylation of endogenous G9a was examined using an a‐pT1045 antibody. E) Western blots showed that only WT G9a, but not T1045A or T1045E mutant, was phosphorylated at T1045. F,G) Western blots showed that overexpression of WT Plk1, but not K82M mutant or addition of the Plk1 inhibitor BI2536, elevated phosphorylation levels of T1045 in vitro F) or in vivo G). H) Western blots showed that T1045 is phosphorylated at M phase in HeLa cells stably expressing Flag‐hG9a. I) Western blots showed that mitotic HEK293T cells by nocodazole treatment exhibited higher level of T1045 phosphorylation on endogenous G9a.

Article Snippet: The following antibodies and reagents were obtained from commercial sources: a‐Flag antibody (F7425‐2MG) were purchased from Sigma‐Aldrich; a‐G9a/EHMT2 (C6H3, #3306S, D5R4R, #68851), a‐Plk1 (4513S) and a‐Cyclin E1(20808) antibodies were purchased from Cell Signaling Technology; a‐cyclin B1 (1495‐1), a‐histone H3 pS10 (1173‐1) were purchased from Epigenomics; a‐pan‐phosoho‐(Ser/Thr) (ab17464), a‐H3K9me1(ab8896), a‐H3K9me2 (ab1220), and a‐H3K9me3 (ab8898) antibodies were purchased from Abcam; a‐GFP (50430‐2‐AP), a‐Myc (60003‐2‐Ig), and a‐Actin (60008‐1‐Ig) antibodies were purchased from Proteintech; a‐PPP2CB (A3122), a‐PPP3CA (A1063), a‐phospho‐PPP2CA/PPP2CB‐Y307 (AP0927), a‐H3K9me2 (A2359) and mouse a‐GAPDH (glyceraldehyde phosphate dehydrogenase) (AC002) antibodies were purchased from ABclonal; a‐H3 (39163) antibody was purchased from Active Motif; a‐PPP6C (HA720056), a‐PPP1CC (500L38), a‐HA (0906‐1) antibodies was purchased from HuaBio; a‐HRP‐Streptavidin antibody (A0303) were purchased from Beyotime; a‐pT1045‐G9a antibody was generated by Proteintech by immunizing rabbits with a pT1045 peptide (RLQLYRpTAKMGW) conjugated with keyhole limpet hemacyanin (KLH); secondary horseradish peroxidase–conjugated mouse or rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories; a‐FLAG M2 affinity gel (A2220), thymidine (T9250), puromycin (P9620) and nocodazole (M1404) were purchased from Sigma‐Aldrich; the hemagglutinin (HA) affinity gel (SA063001) was purchased from Smart‐Lifesciences; BIX‐01294 (S8006) and BI2536 (S1105) was purchased from Selleck; G418 (E856) were purchased from Biosharp.

Techniques: Sequencing, In Vitro, Kinase Assay, Mutagenesis, Western Blot, Over Expression, In Vivo, Stable Transfection, Expressing

Journal: Advanced Science

Article Title: Dynamic Phosphorylation of G9a Regulates its Repressive Activity on Chromatin Accessibility and Mitotic Progression

doi: 10.1002/advs.202303224

Figure Lengend Snippet: T1045 phosphorylation of G9a attenuated its catalytic ability on H3K9me2. A–C) Schematic diagram A) of sequential phosphorylation‐methylation assays performed by Western blots B) and by liquid scintillation counting C) using recombinant N‐Plk1 and G9a‐SET protein. Cold SAM: non‐radioactive; Isotope SAM: radioactive. CPM represents counts per minute. D–F) Schematic diagram D) of sequential in vitro phosphorylation‐methylation assays performed by Western blots E) using recombinant full‐length Plk1 protein and full‐length Flag‐G9a immunoprecipitated from HEK293T cells. Relative H3K9me2 levels were quantified F). G) Rescue of WT G9a, but not the indicated mutants of G9a, in HeLa S3 G9a‐/‐ cells showed a comparable H3K9me2 level relative to that in WT cells. H) HeLa S3 cells stably expressing Flag‐hG9a released from thymidine block were collected at the indicated time points, and pT1045 levels or H3K9me2 were examined by Western blots. I) Phosphorylation levels of T1045 or various H3K9 methylation states were examined by Western blots in HeLa S3 cells stably expressing Flag‐hG9a treated with or without nocodazole. Error bars denote the mean ± SD from three independent experiments. Unpaired t ‐test, * P < 0.05, ns, not significant.

Article Snippet: The following antibodies and reagents were obtained from commercial sources: a‐Flag antibody (F7425‐2MG) were purchased from Sigma‐Aldrich; a‐G9a/EHMT2 (C6H3, #3306S, D5R4R, #68851), a‐Plk1 (4513S) and a‐Cyclin E1(20808) antibodies were purchased from Cell Signaling Technology; a‐cyclin B1 (1495‐1), a‐histone H3 pS10 (1173‐1) were purchased from Epigenomics; a‐pan‐phosoho‐(Ser/Thr) (ab17464), a‐H3K9me1(ab8896), a‐H3K9me2 (ab1220), and a‐H3K9me3 (ab8898) antibodies were purchased from Abcam; a‐GFP (50430‐2‐AP), a‐Myc (60003‐2‐Ig), and a‐Actin (60008‐1‐Ig) antibodies were purchased from Proteintech; a‐PPP2CB (A3122), a‐PPP3CA (A1063), a‐phospho‐PPP2CA/PPP2CB‐Y307 (AP0927), a‐H3K9me2 (A2359) and mouse a‐GAPDH (glyceraldehyde phosphate dehydrogenase) (AC002) antibodies were purchased from ABclonal; a‐H3 (39163) antibody was purchased from Active Motif; a‐PPP6C (HA720056), a‐PPP1CC (500L38), a‐HA (0906‐1) antibodies was purchased from HuaBio; a‐HRP‐Streptavidin antibody (A0303) were purchased from Beyotime; a‐pT1045‐G9a antibody was generated by Proteintech by immunizing rabbits with a pT1045 peptide (RLQLYRpTAKMGW) conjugated with keyhole limpet hemacyanin (KLH); secondary horseradish peroxidase–conjugated mouse or rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories; a‐FLAG M2 affinity gel (A2220), thymidine (T9250), puromycin (P9620) and nocodazole (M1404) were purchased from Sigma‐Aldrich; the hemagglutinin (HA) affinity gel (SA063001) was purchased from Smart‐Lifesciences; BIX‐01294 (S8006) and BI2536 (S1105) was purchased from Selleck; G418 (E856) were purchased from Biosharp.

Techniques: Methylation, Western Blot, Recombinant, In Vitro, Immunoprecipitation, Stable Transfection, Expressing, Blocking Assay

Journal: Advanced Science

Article Title: Dynamic Phosphorylation of G9a Regulates its Repressive Activity on Chromatin Accessibility and Mitotic Progression

doi: 10.1002/advs.202303224

Figure Lengend Snippet: T1045 phosphomimics of G9a shows slower mitotic progression. A) Representative immunostaining images show the signals of pT1045 (green) and G9a (red) at different cell cycle stages in Flag‐G9a stably‐expressing cells (left panel). The specificity of pT1045 staining (green) is validated by pre‐incubation of pT1045 antibody with a phosphorylated T1045 peptide before immunostaining (right panel). DNA was stained with DAPI (blue). B,C) G9a −/− cells expressing WT or T1045E mutant were synchronized by double‐thymidine (T/T) block and released to the indicated time points. Cell cycle profiles were analyzed using flow cytometry B), and quantification of cell populations at G1 phase or G2/M phase at different released time points were plotted from three biological replicates C). D) T/T released cells expressing WT or T1045E were stained with H3S10p. H3S10p positive cells at the indicated post‐released time points were analyzed by flow cytometry (left panel), and mitotic index was quantified. E) G9a −/− cells expressing WT or T1045E mutant were synchronized by T/T block and released to the indicated time points. Cells were stained with alpha‐tubulin (red) and DAPI (blue) to determine different cell cycle stages. Five different images were captured at each time points from individual cells, and three independent experiments were performed (each dot represents an image). Cells in the indicated mitotic steps were manually counted. Error bars denote the mean ± SEM from three independent experiments. Representative immunostaining images are inserted. F) Western blots showed T1045E mutant delays dephosphorylation progression of H3S10p after post‐nocodazole release to the indicated time points. G) Western blots indicated that T1045E mutant exhibits a short delay of mitotic entry upon T/T release with the indicated time points. Error bars denote the mean ± SD from three biological experiments by unpaired t ‐test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.001, ns, not significant.

Article Snippet: The following antibodies and reagents were obtained from commercial sources: a‐Flag antibody (F7425‐2MG) were purchased from Sigma‐Aldrich; a‐G9a/EHMT2 (C6H3, #3306S, D5R4R, #68851), a‐Plk1 (4513S) and a‐Cyclin E1(20808) antibodies were purchased from Cell Signaling Technology; a‐cyclin B1 (1495‐1), a‐histone H3 pS10 (1173‐1) were purchased from Epigenomics; a‐pan‐phosoho‐(Ser/Thr) (ab17464), a‐H3K9me1(ab8896), a‐H3K9me2 (ab1220), and a‐H3K9me3 (ab8898) antibodies were purchased from Abcam; a‐GFP (50430‐2‐AP), a‐Myc (60003‐2‐Ig), and a‐Actin (60008‐1‐Ig) antibodies were purchased from Proteintech; a‐PPP2CB (A3122), a‐PPP3CA (A1063), a‐phospho‐PPP2CA/PPP2CB‐Y307 (AP0927), a‐H3K9me2 (A2359) and mouse a‐GAPDH (glyceraldehyde phosphate dehydrogenase) (AC002) antibodies were purchased from ABclonal; a‐H3 (39163) antibody was purchased from Active Motif; a‐PPP6C (HA720056), a‐PPP1CC (500L38), a‐HA (0906‐1) antibodies was purchased from HuaBio; a‐HRP‐Streptavidin antibody (A0303) were purchased from Beyotime; a‐pT1045‐G9a antibody was generated by Proteintech by immunizing rabbits with a pT1045 peptide (RLQLYRpTAKMGW) conjugated with keyhole limpet hemacyanin (KLH); secondary horseradish peroxidase–conjugated mouse or rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories; a‐FLAG M2 affinity gel (A2220), thymidine (T9250), puromycin (P9620) and nocodazole (M1404) were purchased from Sigma‐Aldrich; the hemagglutinin (HA) affinity gel (SA063001) was purchased from Smart‐Lifesciences; BIX‐01294 (S8006) and BI2536 (S1105) was purchased from Selleck; G418 (E856) were purchased from Biosharp.

Techniques: Immunostaining, Stable Transfection, Expressing, Staining, Incubation, Mutagenesis, Blocking Assay, Flow Cytometry, Western Blot, De-Phosphorylation Assay

Journal: Advanced Science

Article Title: Dynamic Phosphorylation of G9a Regulates its Repressive Activity on Chromatin Accessibility and Mitotic Progression

doi: 10.1002/advs.202303224

Figure Lengend Snippet: PPP2CB phosphatase is required for dephosphorylation of G9a on T1045 during late mitosis. A) Dot blot assay showed that both the regulatory and catalytic subunits of PPP2CB from cells dephosphorylate pT1045 in vitro. B) Dot blot assay showed that inhibition of PPP2 activity by adding the phosphatase inhibitors blocked dephosphorylation of pT1045 in vitro. C) Western blots showed that overexpression of PPP2CB, but not other phosphatases, dephosphorylates pT1045 of G9a in HEK293T cells. D) Western blots showed that siRNA knockdown of PPP2CB increased pT1045 levels of G9a in HEK293T cells. E) Western blots showed that overexpression of WT, but not catalytic‐inactive of PPP2CB, dephosphorylates pT1045 of G9a. F) Western blots showed that expressing WT PPP2CB, but not catalytic‐dead H118Q mutant in cells in PPP2CB knockdown cells restored elevated pT1045 levels. G) PPP2CB phosphatase activity was examined at different cell cycle stages by immunoblotting with an a‐pY307 PPP2CB antibody. H) PPP2CB and G9a activities were examined in HeLa S3 cells with nocodazole arrest and release for the indicated time points by immunoblotting against anti‐a‐pY307 and anti‐pT1045 and anti‐H3K9me2 antibodies.

Article Snippet: The following antibodies and reagents were obtained from commercial sources: a‐Flag antibody (F7425‐2MG) were purchased from Sigma‐Aldrich; a‐G9a/EHMT2 (C6H3, #3306S, D5R4R, #68851), a‐Plk1 (4513S) and a‐Cyclin E1(20808) antibodies were purchased from Cell Signaling Technology; a‐cyclin B1 (1495‐1), a‐histone H3 pS10 (1173‐1) were purchased from Epigenomics; a‐pan‐phosoho‐(Ser/Thr) (ab17464), a‐H3K9me1(ab8896), a‐H3K9me2 (ab1220), and a‐H3K9me3 (ab8898) antibodies were purchased from Abcam; a‐GFP (50430‐2‐AP), a‐Myc (60003‐2‐Ig), and a‐Actin (60008‐1‐Ig) antibodies were purchased from Proteintech; a‐PPP2CB (A3122), a‐PPP3CA (A1063), a‐phospho‐PPP2CA/PPP2CB‐Y307 (AP0927), a‐H3K9me2 (A2359) and mouse a‐GAPDH (glyceraldehyde phosphate dehydrogenase) (AC002) antibodies were purchased from ABclonal; a‐H3 (39163) antibody was purchased from Active Motif; a‐PPP6C (HA720056), a‐PPP1CC (500L38), a‐HA (0906‐1) antibodies was purchased from HuaBio; a‐HRP‐Streptavidin antibody (A0303) were purchased from Beyotime; a‐pT1045‐G9a antibody was generated by Proteintech by immunizing rabbits with a pT1045 peptide (RLQLYRpTAKMGW) conjugated with keyhole limpet hemacyanin (KLH); secondary horseradish peroxidase–conjugated mouse or rabbit antibodies were purchased from Jackson ImmunoResearch Laboratories; a‐FLAG M2 affinity gel (A2220), thymidine (T9250), puromycin (P9620) and nocodazole (M1404) were purchased from Sigma‐Aldrich; the hemagglutinin (HA) affinity gel (SA063001) was purchased from Smart‐Lifesciences; BIX‐01294 (S8006) and BI2536 (S1105) was purchased from Selleck; G418 (E856) were purchased from Biosharp.

Techniques: De-Phosphorylation Assay, Dot Blot, In Vitro, Inhibition, Activity Assay, Western Blot, Over Expression, Expressing, Mutagenesis