Journal: PLoS ONE

Article Title: A Novel YY1-miR-1 Regulatory Circuit in Skeletal Myogenesis Revealed by Genome-Wide Prediction of YY1-miRNA Network

doi: 10.1371/journal.pone.0027596

Figure Lengend Snippet: (A) Two conserved enhancers (E1 and E2) were identified in the promoter region and intragenic region of miR-1-2/miR-133a-1 cluster, respectively. Three putative YY1 binding sites, A, B and C, were identified. (B) One conserved enhancer (E3) was identified in between miR-1-1 and miR-133a-2 with a putative YY1 binding site, D, identified. (C) One conserved enhancer (E4) was identified upstream of miR-206 and miR-133b cluster with a putative YY1 binding site, E, identified. Binding sites for MyoD, MEF2 and SRF were also shown. (D) C2C12 cells were transfected with 250 ng of E1, E2, E3 or E4 reporter plasmid along with Renilla and control vector (YY1 0 ng) or 50 ng, 200 ng, 500 ng YY1 expressing plasmid. Cells were then cultured for 48 h at which time luciferase activities were determined and normalized to Renilla protein. The data represent the average of three independent experiments ± S.D. (E) C2C12 cells were transfected with 0.25 µg of E1, E2, E3 or E4 reporter plasmid along with Renilla luciferase vector and siYY1 or siNC oligos. Luciferase activity was determined as in (D). (F) Chromatins were harvested from C2C12 myoblasts growing in growth medium (GM) or myotubes maintained in differentiation medium (DM) and subjected to ChIP-PCR analysis. Primers were designed to amplify regions encompassing putative YY1 binding sites A, B, C, D, or E. MyHC and Tnni2 are known YY1 targets and used as positive controls. A genomic region that contains no YY1 binding sites was included as a negative control (NC). (G) Site A (Mut A) or both A and B (Mut A+B) were mutated in E1 luciferase reporter plasmid and luciferase reporter assay was performed to measure the response of mutants to YY1 over-expression as in (D). Relative luciferase unit (RLU) is shown with respect to Vector transfection where luciferase activities were set to a value of 1. (H) ChIP-PCR for Ezh2 or H3K27me3 was performed as in (F). The p value was determined by Student's T-test: *p<0.05, **p<0.01, ***p<0.001.

Article Snippet: ChIP assays were performed as previously described (Tong Ihn Lee, 2006) using 5 μg of antibodies against YY1 (rabbit polyclonal from Santa Cruz Biotechnology, Cat# SC-1703), Ezh2 (mouse monoclonal from Cell Signaling, Cat# AC22), trimethyl-histone H3-K27 (rabbit polyclonal from Milipore, Cat# 07-449), or without any antibody as a negative control.

Techniques: Binding Assay, Transfection, Plasmid Preparation, Control, Expressing, Cell Culture, Luciferase, Activity Assay, Negative Control, Reporter Assay, Over Expression

Journal: Theranostics

Article Title: HOXB13 networking with ABCG1/EZH2/Slug mediates metastasis and confers resistance to cisplatin in lung adenocarcinoma patients

doi: 10.7150/thno.29463

Figure Lengend Snippet: HOXB13 targets to and upregulates EZH2. (A) Upregulation of EZH2 by HOXB13 in lung adenocarcinoma cells. H1299 and A549 cells were transiently transfected by Flag-HOXB13 or GFP-HOBX13 separately, controlled by Flag or GFP. Left panel: Cell lysates were prepared and were subjected to Western blot analysis using anti-EZH2 antibody. Right panel: Transcriptional detection of HOXB13-upregulated EZH2 by qPCR. (B) Enrichment of HOXB13 on the EZH2 promoter analyzed by ChIP-seq database from prostate cancer . (C) HOXB13 targets EZH2 in lung adenocarcinoma cells. Upper panel: Diagram of the EZH2 promoter with potential HOXB13 binding sites (double arrow). Lower panel: ChIP analysis was performed using either an anti-HOXB13 ChIP-grade antibody or control IgG in H1299 Flag-HOXB13 cells. Sites 3, 4, and 5 in EZH2 promoter are enriched in a qPCR analysis with known target genes of HOXB13 including ORM1, NKX3.1 as positive controls, and actin as a negative control. Insert is the gel picture of ChIP analysis for HOXB13 targeting on EZH2 promoter. (D) EZH2 promoter-luciferase reporter construct map. Lower panel: Luciferase reporter constructs were co-transfected with vector or HOXB13, towards the identification of 1062-1875bp upstream region critical for HOXB13-directed enhancement (Unpaired Student's t -test, **p < 0.01) in H1299 (left panel) and in A549 cells (right panel). (E) Levels of HOXB13 and EZH2 in patients' tumor specimens were detected by immunohistochemical analyses using HOXB13 and EZH2 antibodies separately. Patients 1-3: HOXB13 and EZH2 were low in cisplatin- and paclitaxel-sensitive lung adenocarcinoma patients. Patients 4-6: HOXB13 and EZH2 were high in cisplatin- and paclitaxel-resistant lung adenocarcinoma patients. (F) Quantification for the levels of HOXB13 and EZH2 in cisplatin- and paclitaxel-sensitive (n=6) or resistant (n=9) lung adenocarcinoma patients (Unpaired Student's t -test, ** p<0.01).

Article Snippet: Briefly, deparaffinization and hydration were performed followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min, and then microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB13 antibody (Santa Cruz, SC-28333, USA) and EZH2 antibody (Cell Signaling Technology, 30233s, USA) were used at 2 μg/ml in all experiments, and incubated at 4 °C overnight followed by the PV-9000 2-step plus Poly-HRP anti-Mouse/Rabbit IgG Detection system (Zhong Shan Jin Qiao, China).

Techniques: Transfection, Western Blot, ChIP-sequencing, Binding Assay, Control, Negative Control, Luciferase, Construct, Plasmid Preparation, Immunohistochemical staining

Journal: Theranostics

Article Title: HOXB13 networking with ABCG1/EZH2/Slug mediates metastasis and confers resistance to cisplatin in lung adenocarcinoma patients

doi: 10.7150/thno.29463

Figure Lengend Snippet: Cisplatin induces expression of HOXB13. (A) HOXB13 and its target genes ABCG1and EZH2 were induced in cisplatin-resistant A549 cells (A549 DDP) at the protein (Upper) and transcriptional levels (Lower) determined by Western blot or qPCR analyses. All these drug resistance genes were significantly upregulated by cisplatin induction (**p<0.01). (B) HOXB13, EZH2, and ABCG1 were transiently induced in the presence of 5 μM or 10 μM cisplatin treatment at indicated time points in A549 and H1299 cells, as detected by Western blot analysis. (C) Quantification of the bands to show that cisplatin upregulates HOXB13 and its target protein expression. (Unpaired Student's t -test, *p<0.05, **p<0.01, ***p<0.001) (D) HOXB13 and EZH2 levels were detected in drug-sensitive and drug-resistant PDX samples with or without cisplatin treatment by IHC. Left were detected by HOXB13 antibody and right were detected by EZH2 antibody.

Article Snippet: Briefly, deparaffinization and hydration were performed followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min, and then microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB13 antibody (Santa Cruz, SC-28333, USA) and EZH2 antibody (Cell Signaling Technology, 30233s, USA) were used at 2 μg/ml in all experiments, and incubated at 4 °C overnight followed by the PV-9000 2-step plus Poly-HRP anti-Mouse/Rabbit IgG Detection system (Zhong Shan Jin Qiao, China).

Techniques: Expressing, Western Blot

Journal: Theranostics

Article Title: HOXB13 networking with ABCG1/EZH2/Slug mediates metastasis and confers resistance to cisplatin in lung adenocarcinoma patients

doi: 10.7150/thno.29463

Figure Lengend Snippet: Combination use of HOXB13 with ABCG1 and EZH2 gives high precision in predicting lung adenocarcinoma patients' outcome. (A) Combination of HOXB13 with its target gene expressions to predict lung adenocarcinoma prognosis. (B) Working model: HOXB13 induced by cisplatin confers lung adenocarcinoma patients' drug resistance by direct targeting to the newly identified drug resistance gene ABCG1 and also known drug resistance gene EZH2. Further, HOXB13 mediates metastasis of lung adenocarcinoma patients by direct targeting to EZH2 and Slug. Combination of HOXB13, ABCG1, EZH2 presents a better strategy to predict outcome or resistance to chemotherapy in lung adenocarcinoma patients.

Article Snippet: Briefly, deparaffinization and hydration were performed followed by abolishing endogenous peroxidase activity using 0.3% hydrogen peroxide for 30 min, and then microwaved for antigen retrieval in 10 mM sodium citrate buffer (pH 6.0) for 20 min. HOXB13 antibody (Santa Cruz, SC-28333, USA) and EZH2 antibody (Cell Signaling Technology, 30233s, USA) were used at 2 μg/ml in all experiments, and incubated at 4 °C overnight followed by the PV-9000 2-step plus Poly-HRP anti-Mouse/Rabbit IgG Detection system (Zhong Shan Jin Qiao, China).

Techniques:

Journal: Nature Communications

Article Title: HnRNP A1 and A2B1 enforce Ezh2 mRNA splicing to promote germinal center B cell responses

doi: 10.1038/s41467-026-74159-w

Figure Lengend Snippet: A Changes in five types of alternative splicing events upon hnRNP A1/A2B1 knockout. Scatter plots show exon inclusion level differences between Hnrnpa1 f/f Hnrnpa2b1 f/f (WT) and Hnrnpa1 f/f Hnrnpa2b1 f/f Cd19 Cre/+ (double knockout, DKO) naïve B cells. Each dot represents a significantly altered splicing event (|inclusion level difference| > 20%, P < 0.05). B Lollipop plot showing the top 10 most significant exon-skipping events (ranked by P -value) in naïve B cells from DKO mice. The height of each lollipop indicates the inclusion level difference, and the circle size reflects statistical significance. C RNA expression changes of the top 10 most significant exon-skipping target genes (ranked by P -value) in follicular and Germinal center (GC) B cells from WT mice. Circle size and color intensity represent changes in FPKM values. Data from GEO: GSE15907 . D–H Alternative splicing of Ezh2 following hnRNP A1/A2B1 deletion. D Sashimi plot illustrating exon 14 splicing changes in Ezh2 in WT and DKO naïve B cells, showing both the full-length isoform and the exon 14-skipped isoform ( Ezh2Δ14 ). E Quantification of splice junction reads spanning exons 13–14, 14–15, and 13–15 in WT and DKO naïve B cells ( n = 3), data in ( E ) are from a single experiment (RNA-seq). F Schematic and primer design for detecting full-length ( Ezh2-FL ) and exon 14-skipped ( Ezh2Δ14 ) isoforms. G Semi-quantitative PCR showing the effect of single and double hnRNP knockouts on Ezh2 exon 14 skipping in WT and DKO naïve B cells, with accompanying gel image and the ratio of Ezh2Δ14 to Ezh2-FL . The data shown are representative images from two independent experiments. H Quantification of Ezh2-FL and Ezh2Δ14 expression normalized to β-actin in WT and DKO naïve B cells ( n = 2), the data shown here represent one representative set of statistical results from two independent experiments. I , J Western blot analysis detecting the abundance of H3K27me3 in B cells from WT, A1-SKO, A2B1-SKO, and DKO mice after 48 h of stimulation with anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL). The data shown are representative images from two independent experiments. K Immunoblot analysis of H3K27me3 levels in WT and DKO B cells. Cells were pre-stimulated with anti-CD40 and anti-IgM and subsequently reconstituted with an empty vector (Vector), full-length EZH2 ( Ezh2 -FL), or the exon 14–skipped isoform ( Ezh2 Δ14). The data shown are representative images from two independent experiments. Each symbol represents an individual mouse ( E , H ). All data are shown as mean ± SEM. Results are representative of at least two independent experiments ( H ). Statistical significance was assessed using an unpaired two-tailed Student’s t -test ( E ).

Article Snippet: Primary antibodies used were: β-actin (1:5000, Proteintech, 20536-1-AP), hnRNP A1 (1:1000, Santa Cruz Biotechnology, sc-32301), hnRNP A2B1 (1:1000, Santa Cruz Biotechnology, sc-53531), EZH2 (1:1000, ABclonal, A5743; A19577), H3K27me3 (1:1000, ABclonal, A22396), CDKN1A (1:1000, Cell Signaling Technology, 2947).

Techniques: Alternative Splicing, Knock-Out, Double Knockout, RNA Expression, RNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Western Blot, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: HnRNP A1 and A2B1 enforce Ezh2 mRNA splicing to promote germinal center B cell responses

doi: 10.1038/s41467-026-74159-w

Figure Lengend Snippet: A Volcano plot showing differentially expressed genes identified by RNA-seq in Hnrnpa1 f/f Hnrnpa2b1 f/f (WT) and Hnrnpa1 f/f Hnrnpa2b1 f/f Cd19 Cre/+ (double knockout, DKO) B cells stimulated with anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL) for 48 h. Differentially expressed genes are defined as those with |log 2 FC| > 1 and p < 0.05. CDKN1A is significantly upregulated in DKO B cells. Differential gene expression analysis for RNA-seq data was performed using DESeq2, which models count data based on the negative binomial distribution. Statistical significance was assessed using two-sided tests. P -values were adjusted for multiple comparisons using the Benjamini–Hochberg method to control the false discovery rate (FDR). Genes with an adjusted P value (FDR) < 0.05 were considered significantly differentially expressed. B qRT-PCR analysis of CDKN1A expression in naïve B cells, B cells stimulated with anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL) for 48 h, and early Germinal center (GC) B cells isolated 5 days after NP-KLH/Alum immunization (naïve B : n = 6; anti-CD40 and anti-IgM: n = 6; early-GC: n = 5). C , D Western blot analysis of CDKN1A protein expression in unstimulated and 48-h-stimulated B cells ( n = 3). The data shown are represe n tative images from three independent experiments. E , F ChIP-qPCR analysis of Cdkn1a locus enriched by anti-H3K27me3 antibody in B cells from WT and DKO mice after 48 h of stimulation with anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL). E A schematic representation of the Cdkn1a gene showing exons and the putative H3K27me3-enriched regions, P1 and P2. F ChIP-qPCR analysis validated the enrichment of H3K27me3 at the Cdkn1a genomic loci in WT and DKO B cells. Data are presented as fold enrichment relative to the WT IgG control. p < 0.0001( p = 2.06e-6). G , H CFSE dilution assay assessing proliferation of WT and DKO B cells following 72 h of anti-CD40 (1 μg/mL) and anti-IgM (2 μg/mL) stimulation ( n = 4). G Representative flow cytometry histograms showing CFSE dilution. H Quantitative analysis of CFSE dilution reflecting proliferation capacity ( n = 4; two independent experiments). Each symbol represents an individual mouse ( B , D , F , H ). Data are shown as mean ± SEM. Results shown are representative of at least three independent experiments ( B , D , F , H ). Statistical significance was determined using an unpaired two-tailed Student’s t -test ( B , D , H ) or one-way ANOVA followed by Tukey’s multiple comparisons test ( F ).

Article Snippet: Primary antibodies used were: β-actin (1:5000, Proteintech, 20536-1-AP), hnRNP A1 (1:1000, Santa Cruz Biotechnology, sc-32301), hnRNP A2B1 (1:1000, Santa Cruz Biotechnology, sc-53531), EZH2 (1:1000, ABclonal, A5743; A19577), H3K27me3 (1:1000, ABclonal, A22396), CDKN1A (1:1000, Cell Signaling Technology, 2947).

Techniques: RNA Sequencing, Double Knockout, Gene Expression, Control, Quantitative RT-PCR, Expressing, Isolation, Western Blot, ChIP-qPCR, Dilution Assay, Flow Cytometry, Two Tailed Test

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 1 Epigenetic regulation of skeletal development of fetuses from HFD (high fat diet) rat dams. A, Representative images from Alizarin red/Alcian blue staining of E18.5 embryos from dams fed either control diet or HFD. Cartilage and calcification stained dark blue; arrows indicate differences of skeletal ossification in the head. B, Western blots for H3K27me3, Ezh2, p-Ezh2 and CDK1 in proteins from EOCCs (embryonic rat osteogenic calvarial cells) either from six HFD obese dams or six control diet dams. M, male; F, female. C, Real-time PCR for Ezh2 mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. D, and E, representing top genes and significantly H3K27me3 enriched or decreased gene body and gene promoter using Heat Map analysis of ChIP-seq data from EOCCs either from six HFD obese dams or six control diet dams (pooled)

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

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

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 2 Identification of epigenetic target genes affected by maternal obesity during fetal skeletal development. A, SATB2 was significantly enriched for H3K27me3 within the gene body, especially in the promoter region based on the detection of a peak (black and red arrow head) of enrichment by Illumina DNA sequencing (male and female are mixed, and samples were pooled to three per group). B, Real-time PCR for SATB2 and ALP mRNA expression in total RNA from EOCCs either from six HFD obese dams or six control diet dams. *P < .05 by t-test. C, ChIP of rat SATB2 enhancer elements by specific anti H3K27me3 and Ezh2 antibodies. D, ChIP of enrichment of SATB2 after IP with H3K27me3 and Ezh2 antibodies. Fold enrichment relative to IgG. *P < .05 by t-test EOCCs from control vs EOCCs from HFD obese dams. E, Control EOCCs were treated with vehicle (control), NEFA mixture 400 µM with Palmitic and Oleic acid 2:1 mixture, Sigma-Aldrich), Palmitic acid (270 µM) or Oleic acid (130 µM) for 48 hours: Western blots for H3K27me3, Ezh2, p-Ezh2, SATB2 and Col 1 (collagen 1). n = 3/treatment

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: DNA Sequencing, Real-time Polymerase Chain Reaction, Expressing, Control, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 4 Increased Ezh2/H3K27me3 but decreased SATB2 expression in human UC MSCs from obese mothers. A, UC MSCs from either lean or obese mothers were cultured, passage 2 cells were immune-stained with anti-Ezh2 antibody (red, white arrows) or anti-SATB2 antibody (green, yellow arrows). B, and C, Real-time PCR (Box & Whiskers graphs) and Western blots (under Box & Whiskers graphs) of Ezh2 and SATB2 mRNA and protein expression in UC MSCs either from lean or obese mothers. *P < .05 vs lean, t-test. D, ChIP of human Ezh2, SATB2 and GAPDH enhancer elements by specific anti H3K27me3 antibody, and E, ChIP of enrichment of human Ezh2, SATB2 and GAPDH after IP with H3K27me3 antibody, fold enrichment relative to IgG. *P < .01 by t-test vs lean

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Expressing, Cell Culture, Staining, Real-time Polymerase Chain Reaction, Western Blot

Journal: The FASEB Journal

Article Title: Maternal regulation of SATB2 in osteo‐progeniters impairs skeletal development in offspring

doi: 10.1096/fj.201901901r

Figure Lengend Snippet: FIGURE 6 Increased trabecular bone mineral density in Ezh2 osteoblastic cell specific deletion male mice. A, Sagittal views of total, trabecular and cortical bone mineral density, and Strength Strain Index (SSI) from a represented mouse from cko and their respective control mice. B, Total bone mineral content in all female mice. B, Tibia pQCT parameters, TOT-BMC (total bone mineral content), P = .0499 by one-way ANOVA; TOT-BMD (total bone mineral density), P = .0365 by one-way ANOVA; TRAB-BMD (trabecular bone mineral density), P = .0188 by one-way ANOVA; CRT-BMD (cortical bone mineral density), P = .0620 by one-way ANOVA in male cko mice compared to their respective genotypic control mice, followed by Tukey's post hoc test comparing cko with its respective control genotype group, *means P < .05 significantly different, n = 4

Article Snippet: Ezh2 overexpression plasmid (Ezh2, #28060, Addgene) and shRNA Ezh2 (Ezh2-sh, #TG509722, Origene) were used.

Techniques: Control