Journal: bioRxiv

Article Title: Site-specific DNA demethylation during spermatogenesis presets the sites of nucleosome retention in mouse sperm

doi: 10.1101/2025.01.10.632457

Figure Lengend Snippet: (A) Mononucleosome enrichment in spermatozoa at genomic sites of MethylCap-seq peaks of KIT + spermatogonia. Class I (green): genomic sites that are demethylated at the mitosis-to-meiosis transition, and Class II (red): all other sites. (B) H3.3 enrichment in the two classes of genomic sites in sperm. (C) Enrichment of H3.3 and nucleosomes (D) Enrichment of SCML2 in cultured germline stem (GS) cells, and H3.3 enrichment in sperm (left) and mononucleosome enrichment in sperm (right) (E) Enrichment of H3K27me3 and H3K4me3 during spermatogenesis and in spermatozoa. (F) RNA-seq analysis in embryos for three groups of genes. Top 1,000 nucleosome-enriched genes among class I and II peak-containing genes at promoters (7,577 and 7,261 genes) and bottom 3,000 nucleosome-enriched genes (Nucleosome-depleted genes).

Article Snippet: To this end, we focused on germline-specific Polycomb protein SCML2, which has a high affinity to hypomethylated DNA and is a critical regulator of germline transcriptomes and bivalent chromatin ( ; Maezawa, Hasegawa, Alavattam, et al., 2018; ; Maezawa, Yukawa, et al., 2018).

Techniques: Cell Culture, RNA Sequencing

Journal: bioRxiv

Article Title: Site-specific DNA demethylation during spermatogenesis presets the sites of nucleosome retention in mouse sperm

doi: 10.1101/2025.01.10.632457

Figure Lengend Snippet: Genomic regions that are demethylated during the transition from mitotic spermatogonia to meiotic spermatocytes acquire H3K4me3 in meiotic spermatocytes, leading to SCML2-mediated deposition of H3K27me3, thereby establishing persisting bivalent marks at these hypomethylated nucleosome retention sites in spermatozoa.

Article Snippet: To this end, we focused on germline-specific Polycomb protein SCML2, which has a high affinity to hypomethylated DNA and is a critical regulator of germline transcriptomes and bivalent chromatin ( ; Maezawa, Hasegawa, Alavattam, et al., 2018; ; Maezawa, Yukawa, et al., 2018).

Techniques:

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: Strategies to identify SCML2 as a YAP1-associated protein (A) Diagram showing the proteomics analysis of YAP1-associated proteins from the nuclear extracts (NE) from LNCaP and C4-2 cells after treatment with dihydrotestosterone (DHT, 10 nM) or enzalutamide (ENZ, 20 μM) for 16h in DCC (dextran-coded charcoal-stripped) serum-fed conditions. (B) Venn diagram shows the YAP1 interacting proteins in LNCaP and C4-2 cells. (C) The table shows YAP1 interacting proteins that are transcriptional regulators, localize to the nucleus, and function as repressors. (D) YAP1 protein network in connection with SCML2 and AR. Protein networks were constructed using the GeneMania web portal.

Article Snippet: SCML2 (G-1) mouse mAb , Santa Cruz Biotechnology (SCBT) , SCBT, Cat# sc-271228; RRID: AB_10613441.

Techniques: Construct

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: Androgen regulates the SCML2 subcellular localization (A) Immunoreactivity of SCML2 and YAP1 proteins total cell lysates isolated from the AR-positive and the AR-negative human cell lines grown under steady-state conditions. SCML2 blot was generated using SCML2 (G1) mouse monoclonal antibody. The beta-actin blot was included as a loading control. (B) Quantification of the SCML2 and YAP1 blots normalized to the beta-actin. (C and D) Western blots of SCML2 and AR in LNCaP and C4-2 cells. SCML2 blot was generated with the SCML2 (C-7) antibody. GAPDH and Lamin A/B blots were used for cytoplasmic (C) and nuclear (N) protein markers, respectively. (E and F) Immunofluorescence (IF) imaging of SCML2 in LNCaP and C4-2 cells. LNCaP and C4-2 cells were treated with ethanol (EtOH) vehicle, DHT (dihydrotestosterone, 10 nM), and ENZ (enzalutamide, 20 μM) 16h in DCC serum-fed conditions. IF data were generated using SCML2 (G1) antibody. Data are represented as mean ± SEM of three independent experiments. The size bar: 10 μm.

Article Snippet: SCML2 (G-1) mouse mAb , Santa Cruz Biotechnology (SCBT) , SCBT, Cat# sc-271228; RRID: AB_10613441.

Techniques: Isolation, Generated, Control, Western Blot, Immunofluorescence, Imaging

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: Protein complex formation between SCML2 and YAP1 under varying conditions (A) Native SCML2 is co-immunoprecipitated with YAP1 protein in C4-2 cells grown in serum-fed conditions. (B) Co-IP of YAP1 with HA-Mock Vector (Vec), HA-SCML2-WT, or HA-SCML2-ΔRBR protein ectopically expressed in HEK-293 cells. ΔRBR denotes the deletion of RNA binding region. H: High exposure, L: Low exposure. (C) Proximity ligation assay (PLA) showing protein-protein interaction (the foci in red) between SCML2 and YAP1 in LNCaP cells, as visualized by confocal microscopy. The interaction of SMCL2 with YAP1 was assessed using SCML2 (G1) antibody. The size bar: 10 μm. (D) The graph shows the quantification of the foci. Cells were treated with EtOH, DHT (10 nM), and ENZ (20 μM) in DCC serum-fed conditions for 16h. (E and F) GST pulldown assay with the recombinant GST-YAP1 peptide fragment. Nuclear extract isolated from LNCaP cells under steady-state growth conditions was used as a source of SCML2 protein in GST pulldown assay. PM: protein marker. Data are represented as mean ± SEM of at least three independent experiments.

Article Snippet: SCML2 (G-1) mouse mAb , Santa Cruz Biotechnology (SCBT) , SCBT, Cat# sc-271228; RRID: AB_10613441.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, RNA Binding Assay, Proximity Ligation Assay, Confocal Microscopy, GST Pulldown Assay, Recombinant, Isolation, Marker

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: YAP1 and SCML2 activity inversely correlate (A) Immunoblots of YAP1 and SCML2 protein with or without SCML2 knockdown conditions. SCML2 blot was generated with the SCML2 (G1) antibody. (B) YAP1-mediated promoter-luciferase (Luc) reporter gene activity with or without SCML2 or YAP1 knockdown conditions, ∗ , ∗∗p > 0.01. (C) Quantitative PCR analysis of STK4-encoded MST1 transcripts in LNCaP cells with or without SCML2 knockdown by siRNA, ∗p > 0.001. (D) SCML2 and STK4 transcripts positively correlate in human prostate tumor tissues, Pearson correlation: 0.29, p = 2.46e-11. (E) Immunoblots of YAP1 and SCML2 in LNCaP cells after treatment with vehicle (−) or DHT with (+) and transiently transfected with the scramble (scram) or YAP1 siRNA under 5% DCC serum-fed growth condition. Beta-actin blots were used as a loading control. (F) The graph shows the quantification of the SCML2 blot in panel E, ∗p > 0.01. Data are represented as mean ± SEM of three independent experiments.

Article Snippet: SCML2 (G-1) mouse mAb , Santa Cruz Biotechnology (SCBT) , SCBT, Cat# sc-271228; RRID: AB_10613441.

Techniques: Activity Assay, Western Blot, Knockdown, Generated, Luciferase, Real-time Polymerase Chain Reaction, Transfection, Control

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: SCML2 regulates cell growth in a context-dependent manner (A–D) Analysis of SCML2 in LNCaP and C4-2 cells under Scrambled (control) siRNA (siR-Scram) or SCML2 siRNA knockdown, followed by treatment with EtOH (vehicle), DHT (D: 10 nM), or ENZ (20 μM) in DCC serum-fed growth conditions. The SCML2 blot was generated using the SCML2 (G1) antibody. AR blots were included in these experiments to monitor the responsiveness of androgen hormone signaling. Beta-actin blots were included as a loading control. Cell growth was assessed by CCK-8 assay at 48h post-treatment in DCC serum-fed conditions. Data are three independent experiments in triplicates each. (E) Analysis of the gene regulatory histone marks in LNCaP and C4-2 cells with or without SCML2 and YAP1 knockdown conditions. Data are represented as mean ± SEM of three independent experiments.

Article Snippet: SCML2 (G-1) mouse mAb , Santa Cruz Biotechnology (SCBT) , SCBT, Cat# sc-271228; RRID: AB_10613441.

Techniques: Control, Knockdown, Generated, CCK-8 Assay

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: A significant number of the SCML2 target genes overlap with the YAP1 targets (A) Venn diagram shows the unique and overlapping targets of SCML2 and YAP1. (B) The 226 overlapping targets of SCML2 and YAP1 are associated with diverse biological processes. (C) The protein network of the 20 gene set. (D) Percent alteration of the 20 genes in prostate adenocarcinoma (PRAD) dataset in The Cancer Genome Atlas (TCGA).

Article Snippet: SCML2 (G-1) mouse mAb , Santa Cruz Biotechnology (SCBT) , SCBT, Cat# sc-271228; RRID: AB_10613441.

Techniques:

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet:

Article Snippet: SCML2 (G-1) mouse mAb , Santa Cruz Biotechnology (SCBT) , SCBT, Cat# sc-271228; RRID: AB_10613441.

Techniques: Virus, Bacteria, Recombinant, Western Blot, Protease Inhibitor, Proximity Ligation Assay, Luciferase, Reporter Assay, Bicinchoninic Acid Protein Assay, CCK-8 Assay, Small Interfering RNA, Plasmid Preparation, Software

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: Strategies to identify SCML2 as a YAP1-associated protein (A) Diagram showing the proteomics analysis of YAP1-associated proteins from the nuclear extracts (NE) from LNCaP and C4-2 cells after treatment with dihydrotestosterone (DHT, 10 nM) or enzalutamide (ENZ, 20 μM) for 16h in DCC (dextran-coded charcoal-stripped) serum-fed conditions. (B) Venn diagram shows the YAP1 interacting proteins in LNCaP and C4-2 cells. (C) The table shows YAP1 interacting proteins that are transcriptional regulators, localize to the nucleus, and function as repressors. (D) YAP1 protein network in connection with SCML2 and AR. Protein networks were constructed using the GeneMania web portal.

Article Snippet: SCML2 (C-7) mouse mAb , Santa Cruz Biotechnology , SCBT, Cat# sc-398400; RRID: AB_2938698.

Techniques: Construct

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: Androgen regulates the SCML2 subcellular localization (A) Immunoreactivity of SCML2 and YAP1 proteins total cell lysates isolated from the AR-positive and the AR-negative human cell lines grown under steady-state conditions. SCML2 blot was generated using SCML2 (G1) mouse monoclonal antibody. The beta-actin blot was included as a loading control. (B) Quantification of the SCML2 and YAP1 blots normalized to the beta-actin. (C and D) Western blots of SCML2 and AR in LNCaP and C4-2 cells. SCML2 blot was generated with the SCML2 (C-7) antibody. GAPDH and Lamin A/B blots were used for cytoplasmic (C) and nuclear (N) protein markers, respectively. (E and F) Immunofluorescence (IF) imaging of SCML2 in LNCaP and C4-2 cells. LNCaP and C4-2 cells were treated with ethanol (EtOH) vehicle, DHT (dihydrotestosterone, 10 nM), and ENZ (enzalutamide, 20 μM) 16h in DCC serum-fed conditions. IF data were generated using SCML2 (G1) antibody. Data are represented as mean ± SEM of three independent experiments. The size bar: 10 μm.

Article Snippet: SCML2 (C-7) mouse mAb , Santa Cruz Biotechnology , SCBT, Cat# sc-398400; RRID: AB_2938698.

Techniques: Isolation, Generated, Control, Western Blot, Immunofluorescence, Imaging

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: Protein complex formation between SCML2 and YAP1 under varying conditions (A) Native SCML2 is co-immunoprecipitated with YAP1 protein in C4-2 cells grown in serum-fed conditions. (B) Co-IP of YAP1 with HA-Mock Vector (Vec), HA-SCML2-WT, or HA-SCML2-ΔRBR protein ectopically expressed in HEK-293 cells. ΔRBR denotes the deletion of RNA binding region. H: High exposure, L: Low exposure. (C) Proximity ligation assay (PLA) showing protein-protein interaction (the foci in red) between SCML2 and YAP1 in LNCaP cells, as visualized by confocal microscopy. The interaction of SMCL2 with YAP1 was assessed using SCML2 (G1) antibody. The size bar: 10 μm. (D) The graph shows the quantification of the foci. Cells were treated with EtOH, DHT (10 nM), and ENZ (20 μM) in DCC serum-fed conditions for 16h. (E and F) GST pulldown assay with the recombinant GST-YAP1 peptide fragment. Nuclear extract isolated from LNCaP cells under steady-state growth conditions was used as a source of SCML2 protein in GST pulldown assay. PM: protein marker. Data are represented as mean ± SEM of at least three independent experiments.

Article Snippet: SCML2 (C-7) mouse mAb , Santa Cruz Biotechnology , SCBT, Cat# sc-398400; RRID: AB_2938698.

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Plasmid Preparation, RNA Binding Assay, Proximity Ligation Assay, Confocal Microscopy, GST Pulldown Assay, Recombinant, Isolation, Marker

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: YAP1 and SCML2 activity inversely correlate (A) Immunoblots of YAP1 and SCML2 protein with or without SCML2 knockdown conditions. SCML2 blot was generated with the SCML2 (G1) antibody. (B) YAP1-mediated promoter-luciferase (Luc) reporter gene activity with or without SCML2 or YAP1 knockdown conditions, ∗ , ∗∗p > 0.01. (C) Quantitative PCR analysis of STK4-encoded MST1 transcripts in LNCaP cells with or without SCML2 knockdown by siRNA, ∗p > 0.001. (D) SCML2 and STK4 transcripts positively correlate in human prostate tumor tissues, Pearson correlation: 0.29, p = 2.46e-11. (E) Immunoblots of YAP1 and SCML2 in LNCaP cells after treatment with vehicle (−) or DHT with (+) and transiently transfected with the scramble (scram) or YAP1 siRNA under 5% DCC serum-fed growth condition. Beta-actin blots were used as a loading control. (F) The graph shows the quantification of the SCML2 blot in panel E, ∗p > 0.01. Data are represented as mean ± SEM of three independent experiments.

Article Snippet: SCML2 (C-7) mouse mAb , Santa Cruz Biotechnology , SCBT, Cat# sc-398400; RRID: AB_2938698.

Techniques: Activity Assay, Western Blot, Knockdown, Generated, Luciferase, Real-time Polymerase Chain Reaction, Transfection, Control

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: SCML2 regulates cell growth in a context-dependent manner (A–D) Analysis of SCML2 in LNCaP and C4-2 cells under Scrambled (control) siRNA (siR-Scram) or SCML2 siRNA knockdown, followed by treatment with EtOH (vehicle), DHT (D: 10 nM), or ENZ (20 μM) in DCC serum-fed growth conditions. The SCML2 blot was generated using the SCML2 (G1) antibody. AR blots were included in these experiments to monitor the responsiveness of androgen hormone signaling. Beta-actin blots were included as a loading control. Cell growth was assessed by CCK-8 assay at 48h post-treatment in DCC serum-fed conditions. Data are three independent experiments in triplicates each. (E) Analysis of the gene regulatory histone marks in LNCaP and C4-2 cells with or without SCML2 and YAP1 knockdown conditions. Data are represented as mean ± SEM of three independent experiments.

Article Snippet: SCML2 (C-7) mouse mAb , Santa Cruz Biotechnology , SCBT, Cat# sc-398400; RRID: AB_2938698.

Techniques: Control, Knockdown, Generated, CCK-8 Assay

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet: A significant number of the SCML2 target genes overlap with the YAP1 targets (A) Venn diagram shows the unique and overlapping targets of SCML2 and YAP1. (B) The 226 overlapping targets of SCML2 and YAP1 are associated with diverse biological processes. (C) The protein network of the 20 gene set. (D) Percent alteration of the 20 genes in prostate adenocarcinoma (PRAD) dataset in The Cancer Genome Atlas (TCGA).

Article Snippet: SCML2 (C-7) mouse mAb , Santa Cruz Biotechnology , SCBT, Cat# sc-398400; RRID: AB_2938698.

Techniques:

Journal: iScience

Article Title: Discordant interactions between YAP1 and polycomb group protein SCML2 determine cell fate

doi: 10.1016/j.isci.2023.107964

Figure Lengend Snippet:

Article Snippet: SCML2 (C-7) mouse mAb , Santa Cruz Biotechnology , SCBT, Cat# sc-398400; RRID: AB_2938698.

Techniques: Virus, Bacteria, Recombinant, Western Blot, Protease Inhibitor, Proximity Ligation Assay, Luciferase, Reporter Assay, Bicinchoninic Acid Protein Assay, CCK-8 Assay, Small Interfering RNA, Plasmid Preparation, Software