Journal: Nucleic Acids Research

Article Title: Ablation of PRMT6 reveals a role as a negative transcriptional regulator of the p53 tumor suppressor

doi: 10.1093/nar/gks764

Figure Lengend Snippet: PRMT6 −/− MEFs harbor H3R2me2 hypomethylation and display growth defects. ( A ) Total cellular RNA was isolated from primary MEFs of the indicated genotype and subjected to semi-quantitative RT–PCR. The migration of the DNA fragments for PRMT6 and GAPDH is shown. The molecular weight marker is the 1 kb ladder and the gel was visualized using ethidium bromide. No RT denotes the absence of reverse transcriptase and is a control used to show that RNA was indeed amplified and not DNA. ( B ) Total cellular proteins from PRMT6 +/ + and PRMT6 −/− MEFs were separated by SDS–PAGE and subsequently immunoblotted with anti-PRMT6, anti-H3R2(me2a), anti-H3 and anti-α-Tubulin antibodies. ( C ) PRMT6 +/ + and PRMT6 −/− MEFs growing in log phase were trypsinized, fixed in 75% MeOH, stained with PI and analyzed by flow cytometry.

Article Snippet: Immunoblotting was performed as described previously ( ) and antibodies used were the following: α-PRMT6 (A300-929 A, Bethyl laboratories, 1:1000); α-tubulin (B-5-1-2, Sigma, 1:50 000); α-H3R2(me2a) (NB21-1002, Novus Biologicals, 1:2000); α-H3 (ab1791, Abcam, 1:1000); α-Cyclin D1 (06-137, Millipore, 1:2000); α-p53 (2524, Cell Signaling, 1:1000); α-PML(36.1-104, Millipore, 1:1000); and α-p21 (05-345, Millipore, 1:1000).

Techniques: Isolation, Quantitative RT-PCR, Migration, Molecular Weight, Marker, Reverse Transcription, Control, Amplification, SDS Page, Staining, Flow Cytometry

Journal: Nucleic Acids Research

Article Title: Ablation of PRMT6 reveals a role as a negative transcriptional regulator of the p53 tumor suppressor

doi: 10.1093/nar/gks764

Figure Lengend Snippet: PRMT6 associates with the promoter region of Trp53 to regulate H3R2 methylation. ( A ) A schematic representation of the murine Trp53 gene with the primers used for ChIP analysis. The transcription start site is shown with the numbering upstream of this site. ChIP-qPCR analysis was performed using wild-type MEFs and the immunoprecipitated DNA enriched using anti-PRMT6, -PRMT1, -CARM1 or -PRMT5 antibodies was normalized with an internal IgG control. ( B ) ChIP-qPCR analysis using anti-PRMT6 antibodies from PRMT6 +/ + and PRMT6 −/− primary MEFs at −1322 of the Trp 53 promoter is shown. PRMT6 occupancy was normalized to an IgG control. ( C ) The presence of H3R2(me2a), H3K4(me3), H3R26(me2a) and H3R17(me2a) at the −1322 upstream region of Trp 53 promoter was determined using ChIP-qPCR analysis. The values for the histone marks were normalized to total histone H3 levels. ( D ) SA-β-Gal staining of PRMT6 −/− , p53 −/− , PRMT6 −/− ; p53 −/− and wild-type primary MEFs is shown. The numbers represent the percentage of SA-β-Gal positive cells. ( E ) Whole-cell extracts obtained from PRMT6 −/− ; p53 −/− MEFs or controls were analyzed by immunoblotting using anti-p53, -PML, -PRMT6 antibodies. Anti-α-tubulin was used as a loading control. Molecular mass markers for PML isoforms are shown on the right.

Article Snippet: Immunoblotting was performed as described previously ( ) and antibodies used were the following: α-PRMT6 (A300-929 A, Bethyl laboratories, 1:1000); α-tubulin (B-5-1-2, Sigma, 1:50 000); α-H3R2(me2a) (NB21-1002, Novus Biologicals, 1:2000); α-H3 (ab1791, Abcam, 1:1000); α-Cyclin D1 (06-137, Millipore, 1:2000); α-p53 (2524, Cell Signaling, 1:1000); α-PML(36.1-104, Millipore, 1:1000); and α-p21 (05-345, Millipore, 1:1000).

Techniques: Methylation, ChIP-qPCR, Immunoprecipitation, Control, Staining, Western Blot

Journal: Genome Biology

Article Title: Genome-wide mapping of FOXM1 binding reveals co-binding with estrogen receptor alpha in breast cancer cells

doi: 10.1186/gb-2013-14-1-r6

Figure Lengend Snippet: FOXM1 binding at ERα co-bound sites is dependent on ERα . (a) FOXM1 binding was assessed following depletion of ER by fulvestrant (10 nM) treatment for 3 h. Western blot showing depletion of ERα whilst FOXM1 protein levels are unchanged. (b) ChIP for FOXM1 was followed by qPCR to assess binding at ERα co-bound sites and FOXM1-only binding sites. ERα binding was assessed following depletion of FOXM1 by siRNA treatment for 48 h. (c) Western blot showing depletion of FOXM1 whilst ERα protein levels are not significantly changed. (d) ChIP for ER followed by qPCR to detect binding at FOXM1 co-bound regions. Depletion of FOXM1 affects ERα-regulated gene expression. (e) qPCR of ERα-regulated gene expression in MCF7 cells treated with siRNA for siControl or FOXM1 for 48 h. Nascent and total mRNA levels were measured. FOXM1 interacts directly with the co-activator CARM1 and regulates CARM1-mediated histone H3 arginine methylation. (f) Co-immunoprecipitation showing pull-down of CARM1 with FOXM1 antibody using either low binding (LB) or high binding (HB) immunoprecipitation buffers additionally supplemented with dithiothreitol (DTT). (g) ChIP for methylated arginine 17 on histone H3 followed by qPCR for regions of FOXM1 and ER co-binding. Data are normalized to total H3 in MCF7 transfected with siRNA for 48 h (siControl or FOXM1). (h) Proposed simplified model for FOXM1/ER/CARM1 complex in transcription regulation at enhancer regions. Data representative of triplicate experiments ± standard deviation. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Antibodies used were anti-FOXM1 (Santa Cruz sc-502, Genetex GTX1000276, Genetex GTX102170 [GeneTex, Irvine, California, USA]) and anti-ER (Santa Cruz sc-543), anti-histone H3 (Abcam ab1791) and anti-histone H3 (asym-dimethyl Arg17; Novus NB21-1132 (Novus Biologicals, Cambridge, Cambridgeshire, UK)).

Techniques: Binding Assay, Western Blot, Expressing, Methylation, Immunoprecipitation, Transfection, Standard Deviation