Journal: Nucleic Acids Research

Article Title: Histone chaperone FACT represses retrotransposon MERVL and MERVL-derived cryptic promoters

doi: 10.1093/nar/gkaa732

Figure Lengend Snippet: FACT complex represses ERVs. ( A ) qPCR analysis of the expression of Ssrp1 and retrotransposons after Ssrp1 depletion in E14 ESCs. Data represent mean ± s.e.m., n = 3 biological replicates. ( B ) The expression levels of Supt16 and retrotransposons after Supt16 depletion in E14 ESCs, as measured by qPCR and normalized to Gapdh levels. Biological triplicate data ( n = 3 dishes) are presented as mean ± s.e.m. ( C ) DNA sequencing results of mutation sites in two Ssrp1 −/− ESC lines. ( D ) RT–qPCR analysis of the expression of Ssrp1 using specific primers located near the sgRNA sites in WT and Ssrp1 −/− ESCs. Biological triplicate data ( n = 3 dishes) are presented as mean ± s.e.m. ( E ) Western blot analysis of Ssrp1 protein in WT and Ssrp1 −/− ESCs. Gapdh was used as a loading control. ( F ) Immunofluorescence staining of Ssrp1 protein in WT ESC and Ssrp1 −/− ESCs. Ssrp1 was stained in green. DNA was stained by Hoechst 33342. Scale bar, 50 μm. ( G ) qPCR analysis of pluripotent genes ( Oct4 , Sox2 and Nanog ) expression in WT ESCs and Ssrp1 −/− ESCs. ( H ) qPCR analysis of expression of Ssrp1 and retrotransposons in WT ESCs and Ssrp1 −/− ESCs. The results were presented as mean ± s.e.m from three biological replicates. ( I ) Western blot analysis of Ssrp1, Supt16 and Gapdh protein levels in WT ESCs and Ssrp1 −/− ESCs. ( J ) Western blot analysis of Supt16 and Ssrp1 protein levels in ESCs expressing Supt16 shRNA or control shRNA. Gapdh was included as a loading control.

Article Snippet: Primary antibodies used are anti-Flag (F1804, Sigma), anti-HA (sc7392, Santa Cruz), anti-Gapdh (KM9002, Sungene), anti-H3 (17168–1-AP, Proteintech), anti-Ssrp1 (sc-74536, Santa Cruz) and anti-Supt16 (#12191, Cell Signaling Technology).

Techniques: Expressing, DNA Sequencing, Mutagenesis, Quantitative RT-PCR, Western Blot, Immunofluorescence, Staining, shRNA

Journal: Nucleic Acids Research

Article Title: Histone chaperone FACT represses retrotransposon MERVL and MERVL-derived cryptic promoters

doi: 10.1093/nar/gkaa732

Figure Lengend Snippet: Restoration of MERVL expression by the introduction of Ssrp1 or Supt16. ( A and B ) Immunoblot analysis of the expression of Ssrp1 (A) or Supt16 (B) after overexpression of Ssrp1 or Supt16 in Ssrp1 −/− ESCs. Gapdh was included as a loading control. OE, overexpression. Ctrl, control vector. ( C ) qPCR analysis of MERVL expression in Ssrp1 −/− ESC after overexpression of Ssrp1 . Biological triplicate data ( n = 3 dishes) are presented as mean ± s.e.m. Significant differences were determined by Student's t -test and defined as *** P < 0.001. ( D ) qPCR analysis of MERVL expression in Ssrp1 −/− ESC after overexpression of Supt16 . Ctrl, control vector overexpression. Biological triplicate data ( n = 3 dishes) are presented as mean ± s.e.m. Significant differences were determined by Student's t -test and defined as *** P < 0.001. ns, non-significant. ( E ) A schematic summary of Ssrp1 mutants used for functional rescue. The length of each mutant form is indicated at the right in amino acids (AA). Δ, deletion. ( F – H ) qPCR analysis of MERVL expression after overexpression of Ssrp1 ΔHMG (F), Ssrp1 ΔRtt106 (G), or Ssrp1ΔSSrecog (H) in Ssrp1 −/− ESCs. qPCR results are normalized to Gapdh. Biological triplicate data ( n = 3 dishes) are presented as mean ± s.e.m. Ctrl, control vector overexpression. Significant differences were determined by Student's t -test and defined as ** P < 0.01 or *** P < 0.001.

Article Snippet: Primary antibodies used are anti-Flag (F1804, Sigma), anti-HA (sc7392, Santa Cruz), anti-Gapdh (KM9002, Sungene), anti-H3 (17168–1-AP, Proteintech), anti-Ssrp1 (sc-74536, Santa Cruz) and anti-Supt16 (#12191, Cell Signaling Technology).

Techniques: Expressing, Western Blot, Over Expression, Plasmid Preparation, Functional Assay, Mutagenesis

Journal: Nucleic Acids Research

Article Title: Histone chaperone FACT represses retrotransposon MERVL and MERVL-derived cryptic promoters

doi: 10.1093/nar/gkaa732

Figure Lengend Snippet: FACT complex genome-wide regulates TE transcription. ( A ) The volcano plot of gene expression in Ssrp1 −/− ESCs versus WT ESCs. Significantly upregulated genes were labeled in red and significantly downregulated genes were labeled in blue. Horizontal red dash line marked adjusted P -value (Wald test) 0.05 and vertical lines marked expression fold change 1.5. ( B and C ) KEGG analysis of pathways related to downregulated genes (B) and upregulated genes (C) after Ssrp1 knockout in ESCs. The analysis was done in DAVID. Color gradient indicated significance in −log10 ( P -value) and dot size indicated the number of genes in the corresponding pathway. ( D ) A scatter diagram shows a transcriptome analysis of TE expression after Ssrp1 knockout. The result from Squire was used to plot the diagram. Colored dots indicate TE with significant expression change ( P < 0.05, Wald test). Triangles represent TEs with log2 (fold change) > 4. ( E and F ) The top 10 TEs with the highest number of loci upregulated (E) or downregulated (F) after Ssrp1 loss. The subfamily type of each TE was labeled in brackets. ( G ) Locations of Ssrp1 peaks relative to the nearest transcription units (Promoter, 2 kb around transcriptional start sites; 5’ proximal, 2–10 kb upstream of the gene; 5’ distal, 10–100 kb upstream of the gene; 3’ proximal, 0–10 kb downstream of the gene; 3’ distal, 10–100 kb downstream of the gene; Gene desert, >100 kb away from the nearest gene). ( H ) Ssrp1 binding profile around the center of MERVL locus. The ChIP-seq signal was calculated as the log2 ratio of the normalized number of reads relative to the input. ( I ) ChIP-qPCR analysis of Ssrp1 binding on different retrotransposons. ChIP-qPCR data were normalized to input and Gapdh. Biological triplicate data ( n = 3 extracts) are presented as mean ± s.e.m.

Article Snippet: Primary antibodies used are anti-Flag (F1804, Sigma), anti-HA (sc7392, Santa Cruz), anti-Gapdh (KM9002, Sungene), anti-H3 (17168–1-AP, Proteintech), anti-Ssrp1 (sc-74536, Santa Cruz) and anti-Supt16 (#12191, Cell Signaling Technology).

Techniques: Genome Wide, Expressing, Labeling, Knock-Out, Binding Assay, ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: Histone chaperone FACT represses retrotransposon MERVL and MERVL-derived cryptic promoters

doi: 10.1093/nar/gkaa732

Figure Lengend Snippet: Ssrp1 recruits Usp7 to repress MERVL expression. ( A ) Mass spectrometry analysis of Ssrp1-associated proteins after Ssrp1 co-IP. Y -axis indicates the transcriptional level of corresponding proteins and the X -axis indicates the binding strength of proteins to Ssrp1. ( B ) Correlation heatmap of Ssrp1 duplicate binding profile and Usp7 binding profile together with known histone marks/regulators of ERVs. Pearson's correlation coefficient was used to estimate the strength of the correlation. The ChIP-seq signal was calculated as the log2 ratio of normalized reads relative to the input. ( C ) Western blot analysis of Ssrp1-HA/Usp7 co-immunoprecipitation in ESCs overexpressing control empty vector or HA-tagged Ssrp1. IP was done with anti-HA magnetic beads. 1.25% input was loaded as control. ( D ) Western blot analysis of Usp7-Flag/Ssrp1 co-immunoprecipitation in ESCs overexpressing control empty vector or Flag-tagged Usp7. IP was done with anti-Flag magnetic beads. 1.25% input was loaded as control. IP: immunoprecipitation; OE: overexpression. ( E ) RT-qPCR analysis of the expression of Usp7 and MERVL after Usp7 depletion in ESCs. Data are shown as mean ± s.e.m. ( n = 3). ( F ) Scatter plot of TE expression after Usp7 depletion. Squire results were used to plot the diagram. Colored dots indicate TEs with significant expression change ( P < 0.05, Wald test). Triangles represent TEs with log2 (fold change) > 4. ( G ) GSEA analysis of enrichment of genes repressed by Ssrp1 in the upregulated transcriptome of ESCs with Usp7 depleted. Red, upregulated genes; blue, downregulated genes. NES: normalized enrichment scores; FDR: false discovery rate. The Kolmogorov–Smirnov statistic was used for calculation of P -value. ( H ) Enrichment heatmap of Ssrp1 binding in WT ESCs, Usp7 binding in WT ESCs and Usp7 binding in Ssrp1 −/− ESCs around binding regions of Ssrp1. The regions are sorted by the Ssrp1 binding strength. The ChIP-seq signal was calculated as the log2 ratio of normalized reads relative to the input. ( I ) ChIP-qPCR analysis of Usp7 enrichment on MERVL in WT ESCs and Ssrp1 −/− ESCs. ChIP-qPCR data were normalized to input and Gapdh locus. Biological triplicate data ( n = 3 extracts) are presented as mean ± s.e.m. ( J ) Usp7 binding profile around the center of MERVL locus in WT ESCs and Ssrp1 −/− ESCs. The ChIP-seq signal was calculated as the log2 ratio of the normalized number of reads relative to the input. ( K ) ChIP-qPCR analysis of Usp7 enrichment on MERVL in Ssrp1 −/− ESCs after overexpression of Supt16. Biological triplicate data ( n = 3 dishes) are presented as mean ± s.e.m. Ctrl: control vector; OE: overexpression.

Article Snippet: Primary antibodies used are anti-Flag (F1804, Sigma), anti-HA (sc7392, Santa Cruz), anti-Gapdh (KM9002, Sungene), anti-H3 (17168–1-AP, Proteintech), anti-Ssrp1 (sc-74536, Santa Cruz) and anti-Supt16 (#12191, Cell Signaling Technology).

Techniques: Expressing, Mass Spectrometry, Co-Immunoprecipitation Assay, Binding Assay, ChIP-sequencing, Western Blot, Immunoprecipitation, Plasmid Preparation, Magnetic Beads, Over Expression, Quantitative RT-PCR

Journal: Nature Communications

Article Title: BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis

doi: 10.1038/ncomms14182

Figure Lengend Snippet: ( a ) Immunofluorescence (IF) staining of BCAS2 in the paraffin sections of testes from E15.5 to P14 mice. The DNA was stained with Hoechst 33342. Scale bar, 50 μm. ( b ) Real-time PCR analysis of Bcas2 expression in the fraction of spermatogenic cells (FSPCs) and the fraction of somatic cells (FSCs) enriched from P9 testes. Gapdh was used as the internal control for normalization ( n =4). Error bars represent s.e.m. ( c ) Western blotting analysis of BCAS2 expression in the fraction of spermatogenic cells (FSPCs) and the fraction of somatic cells (FSCs) enriched from P9 testes. Germ cell markers (DAZL and MVH) were used as the indicator of the enrichment efficiency and α-tubulin was used as the loading control. ( d ) Paraffin sections of P8 testes were co-stained with rabbit anti-BCAS2 and mouse anti-PLZF antibodies. The DNA was stained with Hoechst 33342. Scale bar, 20 μm.

Article Snippet: The primary antibodies used were as follows: rabbit anti-BCAS2 polyclonal antibody (10414-1-AP, Proteintech, 1:1000); rabbit anti-PLZF antibody (sc22839, Santa Cruz, 1:1,000); rabbit anti-DDX4/MVH polyclonal antibody (ab13840, Abcam, 1:1,000); mouse phosphor-Histone H2A.X (Ser139/Tyr142) antibody (#5438, CST, 1:2,000); rabbit anti-H2AX (phospho S139) antibody (ab22551, Abcam, 1:500); rabbit anti-SCP3 antibody (ab15093, Abcam, 1:1,000); rabbit anti-Stra8 polyclonal antibody (ab49405, Abcam, 1:500); rabbit anti-α-Tubulin antibody (#2144, CST, 1:1,000); anti-GAPDH (1C4) mouse mAb (KM9002, Sungene Biotech, 1:3,000); and anti-DAZL mouse antibody (MCA2336, AbD Serotec, 1:1,000).

Techniques: Immunofluorescence, Staining, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: Nature Communications

Article Title: BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis

doi: 10.1038/ncomms14182

Figure Lengend Snippet: ( a ) Western blotting analysis of MVH in control and Bcas2 F/ − ;Vasa-Cre testes at P10, P12 and P15. α-tubulin was used as the loading control. ( b ) IF staining of MVH in control and Bcas2 F/ − ;Vasa-Cre testes at P10, P12 and P15. The DNA was stained with Hoechst 33342. Scale bar, 50 μm. ( c ) Western blotting analyses of SCP3 and γH2AX in control and Bcas2 F/ − ;Vasa-Cre testes at P10 and P12. α-tubulin was used as the loading control. ( d – i ) Hematoxylin and eosin (H&E) staining of control ( d – f ) and Bcas2 F/ − ;Vasa-Cre testes ( g – i ) at P10, P12 and P15. Spermatogenic cells were shown in cross-sections of seminiferous tubules from control and Bcas2 F/ − ;Vasa-Cre testes. Scale bar, 15 μm. Black arrows indicate the representative stages of the spermatocytes. L, leptotene; eP, early pachytene spermatocytes; Z, zygotene spermatocytes; P, pachytene spermatocytes; PL, pre-leptotene spermatocytes; red arrows, apoptotic cells. ( j ) Real-time RT-PCR analysis of marker gene expression in pre-meiotic testes from control and Bcas2 F/ − ;Vasa-Cre males at P9 with Gapdh as the internal control. (* P <0.05; ** P <0.01; *** P <0.001, n =5). Error bars represent s.e.m.

Article Snippet: The primary antibodies used were as follows: rabbit anti-BCAS2 polyclonal antibody (10414-1-AP, Proteintech, 1:1000); rabbit anti-PLZF antibody (sc22839, Santa Cruz, 1:1,000); rabbit anti-DDX4/MVH polyclonal antibody (ab13840, Abcam, 1:1,000); mouse phosphor-Histone H2A.X (Ser139/Tyr142) antibody (#5438, CST, 1:2,000); rabbit anti-H2AX (phospho S139) antibody (ab22551, Abcam, 1:500); rabbit anti-SCP3 antibody (ab15093, Abcam, 1:1,000); rabbit anti-Stra8 polyclonal antibody (ab49405, Abcam, 1:500); rabbit anti-α-Tubulin antibody (#2144, CST, 1:1,000); anti-GAPDH (1C4) mouse mAb (KM9002, Sungene Biotech, 1:3,000); and anti-DAZL mouse antibody (MCA2336, AbD Serotec, 1:1,000).

Techniques: Western Blot, Control, Staining, Quantitative RT-PCR, Marker, Gene Expression

Journal: Nature Communications

Article Title: BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis

doi: 10.1038/ncomms14182

Figure Lengend Snippet: ( a ) Scatter plot of significantly differentially expressed transcripts in Bcas2 F/ − ;Vasa-Cre testes compared with the controls. Blue dots represent significantly down-regulated transcripts, while red dots show significantly up-regulated transcripts ( P <0.05, fold-change of RPKM>1.5). Grey dots illustrated unchanged transcripts. ( b ) Real-time RT-PCR analysis of Pre-mRNA and mature (m) mRNA expression level of Tuba3a , Tuba3b and Tubb4b in control and Bcas2 F/ − ;Vasa-Cre testes of P9 mice with Gapdh as the internal control (* P <0.05, n =5). Error bars represent s.e.m. The pre-mRNA primers were designed in one of the exons and the adjacent intron and the mature mRNA primers were designed to span an exon-exon junction. ( c ) Seven AS events significantly affected by depletion of BCAS2 in the testes at P9. The simple diagrams of seven AS events recognized by ASD software and splicing events affected by depletion of BCAS2 analysing the RNA-seq data using ASD software ( P <0.05). ( d ) GO term enrichment analysis of genes with significantly affected AS events.

Article Snippet: The primary antibodies used were as follows: rabbit anti-BCAS2 polyclonal antibody (10414-1-AP, Proteintech, 1:1000); rabbit anti-PLZF antibody (sc22839, Santa Cruz, 1:1,000); rabbit anti-DDX4/MVH polyclonal antibody (ab13840, Abcam, 1:1,000); mouse phosphor-Histone H2A.X (Ser139/Tyr142) antibody (#5438, CST, 1:2,000); rabbit anti-H2AX (phospho S139) antibody (ab22551, Abcam, 1:500); rabbit anti-SCP3 antibody (ab15093, Abcam, 1:1,000); rabbit anti-Stra8 polyclonal antibody (ab49405, Abcam, 1:500); rabbit anti-α-Tubulin antibody (#2144, CST, 1:1,000); anti-GAPDH (1C4) mouse mAb (KM9002, Sungene Biotech, 1:3,000); and anti-DAZL mouse antibody (MCA2336, AbD Serotec, 1:1,000).

Techniques: Quantitative RT-PCR, Expressing, Control, Software, RNA Sequencing

Journal: Nature Communications

Article Title: BCAS2 is involved in alternative mRNA splicing in spermatogonia and the transition to meiosis

doi: 10.1038/ncomms14182

Figure Lengend Snippet: ( a ) RNA-seq results of alternative sites in genes related to spermatogenesis using IGV software. Red arrowheads indicate splicing sites. ( b ) RT-PCR analysis of alterative splicing patterns of the changed splicing genes in control and Bcas2 F/ − ;Vasa-Cre testes of P9 mice with Gapdh as the internal control. RT-PCR was performed with specific primers in three independent experiments. ( c ) Real-time RT-RCR verified the changed splicing genes with Hprt as the internal control. Dazl- FL and Ehmt2 -FL represent the full-length isoform and Dazl -Δ8 and Ehmt2 -Δ10 denote the short form that lacked exon 8 and exon 10, respectively. An alternative first exon of Hmga1 was shown by Hmga1 -E1. Dazl +7 primers specifically recognized intron 7 of Dazl . Dazl , Ehmt2 and Hmga1 were detected using primers at the common region (except for the splicing site) of both isoforms (* P <0.05; ** P <0.01; *** P <0.001, n >5). Error bars represent s.e.m. ( d ) Western blotting analysis of the expression of two isoforms of DAZL in control and Bcas2 F/ − ;Vasa-Cre testes of P9 and P12 mice with GAPDH as the loading control. ( e and f ) Relative abundances of DAZL-FL ( e ) and DAZL-total ( f ) in control and Bcas2 F/ − ;Vasa-Cre testes from P9 and P12 mice were determined by Western blotting analyses of four independent experiments. Error bars represent s.e.m.

Article Snippet: The primary antibodies used were as follows: rabbit anti-BCAS2 polyclonal antibody (10414-1-AP, Proteintech, 1:1000); rabbit anti-PLZF antibody (sc22839, Santa Cruz, 1:1,000); rabbit anti-DDX4/MVH polyclonal antibody (ab13840, Abcam, 1:1,000); mouse phosphor-Histone H2A.X (Ser139/Tyr142) antibody (#5438, CST, 1:2,000); rabbit anti-H2AX (phospho S139) antibody (ab22551, Abcam, 1:500); rabbit anti-SCP3 antibody (ab15093, Abcam, 1:1,000); rabbit anti-Stra8 polyclonal antibody (ab49405, Abcam, 1:500); rabbit anti-α-Tubulin antibody (#2144, CST, 1:1,000); anti-GAPDH (1C4) mouse mAb (KM9002, Sungene Biotech, 1:3,000); and anti-DAZL mouse antibody (MCA2336, AbD Serotec, 1:1,000).

Techniques: RNA Sequencing, Software, Reverse Transcription Polymerase Chain Reaction, Control, Western Blot, Expressing

Journal: Journal of Biological Chemistry

Article Title: Adipose-specific BMP and activin membrane-bound inhibitor (BAMBI) deletion promotes adipogenesis by accelerating ROS production

doi: 10.1074/jbc.ra120.014793

Figure Lengend Snippet: Figure 2 Adipose-specific BAMBI AKO mice have increased fat deposition and reduced heat production with high-fat diet. (A) Representative image of H&E-stained sections of iWAT tissues from BAMBI Flox and BAMBI AKO mice with HFD diet, Scale bar: 50 μm. (B) Adipocyte area statistics of iWAT from HFD-fed BAMBI Flox and AKO mice (n=10). (C) Comparison of mRNA expression levels of PPARγ, C/EBPβ, aP2 in iWAT of BAMBI-Flox and AKO mice, β-actin as a correction (n=7), Scale bar: 50 μm. (D) Western blot analysis of PPARγ, aP2, ATGL, HSL, and BAMBI expression in iWAT of BAMBI Flox and AKO mice and quantification, with β-Tubulin as a control (n=4). (E) Representative image of H&E-stained sections of BAT tissues from BAMBI Flox and AKO mice

Article Snippet: Western blot analysis Briefly, 15 μg samples of total lysates from tissues or cells were run on a 10% SDS-PAGE gel and immunoblotted with the primary antibodies (1:1000) to BAMBI (ThermoFisher, PA5-38027), β-Tubulin (Sungene Biotech, KM9003), PPARγ (Abcam, ab3442), aP2 (Santa, Sc-271529), ATGL (CST, 2138), HSL (CST, 4107), Akt (CST, 4691), p-Akt (ser473,CST, 4060), UCP1 (Abcam, ab10983), PGC1α (CST, 2178), C/EBPβ (Santa, sc-7962), Oxidative phosphorylation (OXPHOS) (Abcam, ab110413), and Nox4 (ABclonal, A11274).

Techniques: Staining, Comparison, Expressing, Western Blot, Control