Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202303224

Figure Lengend Snippet: Figure 1. G9a can be phosphorylated by Plk1 both in vitro and in vivo. A,B) Western blots to examine H3K9me2 levels at asynchronous (Asyn), G1/S boundary, or mitosis (M) stage of HeLa S3 cells A), and different H3K9 methylation levels were quantified B). C) Western blots to examine the phosphory- lation state of G9a in HeLa cells stably expressing Flag-hG9a using an a-pSer/Thr antibody. Asterisk represents non-specific bands. D) Examination of the phosphorylation state of G9a by immunoprecipitation of phosphorylated proteins using an a-pSer/Thr antibody. E) In vitro kinase assay examined which kinase can phosphorylate hG9a-SET. The indicated kinases were enriched by immunoprecipitation from HEK293T cells expressing individual construct. Recombinant GST-hG9a-SET was detected by Commassie brilliant blue staining (CBB). Phosphorylation signals were examined by 32P-labelled autora- diography. F,G) Western blots to examine H3K9me2 levels in HEK293T cells overexpressing various Plk1 constructs F), and relative H3K9me2 levels were quantified G). H,I) Western blots to examine various H3K9 methylation levels in HeLa S3 cells with knockdown of PLK1 H), and relative H3K9me2 levels were quantified I). J) In vitro kinase assay to show that the N-terminus of Plk1 containing the kinase domain (N-Plk1) can phosphorylate G9a-SET protein.

Article Snippet: Plasmid Construction: Human G9a (hG9a, Addgene #33025) was subcloned into a pCS2-3× Flag, pCS2-3× HA, pCW-3× Flag, or pGEX-6P-1 vector.

Techniques: In Vitro, In Vivo, Western Blot, Methylation, Stable Transfection, Expressing, Phospho-proteomics, Immunoprecipitation, Kinase Assay, Construct, Recombinant, Staining, Knockdown

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202303224

Figure Lengend Snippet: Figure 2. 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: Plasmid Construction: Human G9a (hG9a, Addgene #33025) was subcloned into a pCS2-3× Flag, pCS2-3× HA, pCW-3× Flag, or pGEX-6P-1 vector.

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

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202303224

Figure Lengend Snippet: Figure 3. 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. Rel- ative 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: Plasmid Construction: Human G9a (hG9a, Addgene #33025) was subcloned into a pCS2-3× Flag, pCS2-3× HA, pCW-3× Flag, or pGEX-6P-1 vector.

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

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202303224

Figure Lengend Snippet: Figure 4. T1045 phosphorylation of G9a attenuated its repressive activity on gene repression. A) Schematic diagram of a reporter system to measure the effect of G9a on gene expression of luciferase (LUC gene) by targeting the UHRF1 promoter. GAL4-driven G9a construct specifically recognizes TATA boxes in the 5’-UAS regions. B,C) Relative luciferase activities in cells expressing various G9a constructs were measured from three independent replicates with three technical repeats B), and protein levels of the indicated G9a constructs were examined by Western blots C). D,E) Relative luciferase activities in HEK293Tcells expressing the indicated Plk1 constructs were measured from three independent experiments each with three technical repeats D), and protein levels of the indicated cells were examined by Western blots E). F) Venn diagram showed the number of unique or shared genes downregulated in WT cells compared to cells expressing empty vector (Vec) or T1045E mutant, respectively [Log2(Fold change, FC) < −1, P < 0.05]. G) Scatter plots showed the downregulated genes in WT cells relative to HEK293T cells expressing Vec or T1045E mutant (n = 174). Red dots represent the overlapped genes between two groups. H) Heatmap depicted expression patterns of the ten overlapped genes in the indicated cells. Two independent replicates for each cell were presented. I) Genome browser tracks showed RNA-seq signals at FAM83H and GBA gene loci in the indicated cells. J,K) Relative luciferase activities in HEK293T cells expressing the indicated G9a constructs were measured from multiply independent experiments J), and protein levels of the indicated cells were examined by Western blots K). Error bars denote the mean ± SD from three independent experiments. Unpaired t-test, * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001, ns, not significant.

Article Snippet: Plasmid Construction: Human G9a (hG9a, Addgene #33025) was subcloned into a pCS2-3× Flag, pCS2-3× HA, pCW-3× Flag, or pGEX-6P-1 vector.

Techniques: Phospho-proteomics, Activity Assay, Gene Expression, Luciferase, Construct, Expressing, Western Blot, Plasmid Preparation, Mutagenesis, RNA Sequencing

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202303224

Figure Lengend Snippet: Figure 5. T1045 phosphorylation of G9a reduced H3K9me2 occupancy and increased chromatin accessibility at gene promoter regions. A) Averaged signal intensities (top) and heatmaps (bottom) showed the signals of H3K9me2 ± 3 kb from the center of peaks (n = 87,682) in the indicated cells. Each profile was plotted from two biological replicates. B) Profiles showed that averaged intensities of H3K9me2 ChIP-seq in the gene coding regions ± 3 kb away from the normalized transcription start sites (TSS) and transcription end sites (TES) in the indicated cells. C) Averaged signal intensities (top) and heatmaps (bottom) showed the ATAC-seq signals ± 3 kb away from TSS and TES in the indicated cells. Each profile was plotted from two biological replicates. D) Profiles showed that averaged intensities of ATAC-seq in the gene coding regions ± 3 kb away from TSS and TES of the indicated cells. E) Volcano plots showed differential peaks from ATAC-seq in G9a−/−cells stably expressing WT G9a relative to T1045E mutant. The downregulated ATAC-seq peaks (n = 5,813) in WT sample were highlighted with green frame. F) Heatmaps showed ATAC-seq differential signals from the center of ATAC-seq peaks in T1045E mutant relative to that in WT cells. Each profile was plotted from two biological replicates. G) Genome browser tracks showed representative patterns of H3K9me2 ChIP-seq and ATAC-seq in 5 different genome loci of the indicated cells. H) Box plots compared the signals of ATAC-seq and H3K9me2 peaks in WT and T1045E cells. P-values were calculated by two-sided t-test.

Article Snippet: Plasmid Construction: Human G9a (hG9a, Addgene #33025) was subcloned into a pCS2-3× Flag, pCS2-3× HA, pCW-3× Flag, or pGEX-6P-1 vector.

Techniques: Phospho-proteomics, ChIP-sequencing, Stable Transfection, Expressing, Mutagenesis

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202303224

Figure Lengend Snippet: Figure 6. 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

Article Snippet: Plasmid Construction: Human G9a (hG9a, Addgene #33025) was subcloned into a pCS2-3× Flag, pCS2-3× HA, pCW-3× Flag, or pGEX-6P-1 vector.

Techniques: Immunostaining, Stable Transfection, Expressing, Staining, Incubation, Mutagenesis, Blocking Assay, Cytometry

Journal: Advanced science (Weinheim, Baden-Wurttemberg, Germany)

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

doi: 10.1002/advs.202303224

Figure Lengend Snippet: Figure 7. 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: Plasmid Construction: Human G9a (hG9a, Addgene #33025) was subcloned into a pCS2-3× Flag, pCS2-3× HA, pCW-3× Flag, or pGEX-6P-1 vector.

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