Fig 2: G9a negatively regulates UHRF1 transcription. (A) Formalin-fixed tissue array slides were used in immunohistochemistry experiments. Tissue array slides from lymph nodes, esophagus, colon, rectum and uterine cervix were used for immunohistochemical 3,3-diaminobenzidine (DAB) staining for G9a or UHRF1. Numbers in the graphs represent fold-changes relative to normal tissues. (B and C) G9a and UHRF1 mRNA levels were analyzed using real-time PCR in stable G9a-knockdown H1299 cells and G9a-/- MEF cells. Cells were lysed and immunoblotted with anti-G9a and anti-UHRF1 antibodies. ß-actin was used as a loading control. Results are shown as means ± SDs; n = 3. **P < 0.01, ***P < 0.001. (D) 293T cells were transfected with the pGL3-UHRF1 promoter reporter and the indicated DNA constructs and shRNAs, and their cell extracts were assayed for luciferase activity (left panel). G9a-mediated UHRF1 restored transcriptional repression by BIX01294. The pGL3-UHRF1 promoter and Flag-G9a were cotransfected into the 293T cells. Twenty-four hours after transfection, they were treated with BIX01294 (10 µM) for 24 h, and the luciferase activity was subsequently measured (right panel). Luciferase activity was normalized to that of ß-galactosidase. Each P-value represents the mean of five replicates from a single assay. All results represent at least three independent experiments (±SD). *P < 0.05, **P < 0.01. Immunoblot analyses of the expression levels of endogenous and exogenous G9a in shG9a construct 1- or 2-transfected 293T cells are shown using anti-G9a, anti-Flag, and anti-ß-actin antibodies. (E) Immunoblot analyses show the relative expression levels of UHRF1 in G9a knockdown and G9a WT and ?SET rescued H1299 cells. The expression levels are normalized to ß-actin. (F) Schematic diagram of primer pairs in ChIP analysis (upper panel). Arrows indicate the primers used for real-time PCR amplification. ChIP analyses of the UHRF1 promoter in stable G9a knockdown H1299 cells were conducted using anti-G9a and anti-H3K9-me2, which were examined via real-time PCR. Results are shown as means ± SDs; n = 3. (G) The expression levels of G9a and UHRF1 in K562 cells with transient G9a knockdown were detected via real-time PCR and immunoblot analysis. The error bars represent 2-??CT ± the SD of three independent experiments. *P < 0.05, ***P < 0.001.

Fig 3: Regulation of DNA methylation during meiotic prophase I in males and females.(A) Violin plots showing expression of selected enzymes involved in the regulation of DNA methylation. Red dots mark mean expression per cluster. (B) RNA FISH of DNMT3A (left) or TET2 (right) together with DDX4 in 18WPF ovaries. Orange arrows indicate dictyate/diplotene oocytes. (C-E) Immunofluorescence for DNMT3A (C), DNMT3B (D) and UHRF1 (E) with DDX4 in 18WPF ovaries, 19.5WPF testes and adult testes. Orange arrows in 18WPF ovaries indicate dictyate/diplotene oocytes and in testes indicate representative DDX4+ germ cells. Scale bars are 20µm. (F) Immunofluorescence for UHRF1 and PCNA in 18WPF ovaries. Orange arrows indicate representative UHRF1+PCNA+ proliferating gonadal somatic cells. Scale bars are 20µm. (G) Immunofluorescence for 5-hydroxymethylcytosine (5hmC) and 5-methylcytosine (5mC) in 19WPF ovaries. Orange arrows indicate dictyate/diplotene oocytes. Scale bars are 20µm.

Fig 4: UHRF1 is downregulated by G9a during leukemia cell differentiation. (A) HL-60 cells were treated with TPA or DMSO. After 48 h, real-time PCR was performed to compare the expression levels of UHRF1. All results represent at least three independent experiments (±SDs). *** P < 0.001. Cells were lysed and immunoblotted with anti-G9a, anti-LMO2, and anti-UHRF1 antibodies. ß-actin was used as a loading control. (B) ChIP analyses of the UHRF1 promoter in TPA-treated HL-60 cells were conducted using anti-G9a, anti-H3K9-me2, anti-H3K27-me3, anti-Pol II, anti-Acetyl-H3, anti-HDAC1, anti-HDAC2, anti-YY1, and anti-IgG and were examined via real-time PCR analysis. All results represent at least three independent experiments (± SD). * P <0.05, *** P <0.001.

Fig 5: G9a-mediated transcriptional repression of UHRF1 is YY1 dependent. (A) 293T cells were cotransfected with the pGL3-UHRF1 promoter, Flag-G9a and Flag-YY1. Following transfection, cells were grown for 48 h, and cell extracts were assayed for luciferase activity. Expression of the transfected constructs is shown in the immunoblot analysis. (B) pGL3-UHRF1 promoter and the indicated constructs were cotransfected into 293T cells. Twenty-four hours after transfection, 330 nM TSA or 5 mM NIA was added for 24 h, and luciferase activities were subsequently measured. G9a, HDAC1 and HDAC2 expression was confirmed by immunoblot analysis. (C) 293T cells were cotransfected with the pGL3-UHRF1 promoter, Flag-G9a, siCTL RNA and siYY1 RNAs (100 nM). Luciferase activity was measured 48 h after transfection. G9a overexpression and YY1 knockdown by two different siYY1 RNAs are shown in the immunoblot analysis. (A–C) Luciferase activity was normalized to that of ß-galactosidase, and the results are presented as means ± SD (n = 3). *P < 0.05, **P < 0.01, ***P < 0.001. (D) 293T cells were transfected with siCTL RNA or siYY1 RNAs (100 nM). YY1, G9a, and UHRF1 expression levels were confirmed using real time-PCR and immunoblot analysis. YY1 knockdown by siYY1 RNA is shown in the immunoblot analysis. All results are representative of at least three independent experiments (±SDs). **P < 0.01, ***P < 0.001. (E) 293T cells were transfected with siCTL RNA or siYY1 RNAs. ChIP analysis was performed using anti-G9a, anti-YY1, and anti-H3K9-me2 antibodies, and the results were confirmed by real-time PCR. Recruitment of G9a, YY1 and H3K9-me2 to the UHRF1 promoter and distal region was normalized by input. All results represent at least three independent experiments (±SDs). *P < 0.05, **P < 0.01, ***P < 0.001.