Journal: Journal of molecular and cellular cardiology

Article Title: Transcriptional Control of a Novel Long Noncoding RNA Mymsl in Smooth Muscle Cells by a Single Cis-Element and its Initial Functional Characterization in Vessels

doi: 10.1016/j.yjmcc.2019.11.148

Figure Lengend Snippet: A. The schematic of luciferase reporter of the −400 bp of Mymsl putative promoter (CArG WT) with the predicted CArG sequence. 10T1/2 cells were co-transfected with WT Mymsl or SM22 (positive control) and either SRF-VP16 or MYOCD expression plasmids versus their individual control vectors for 36 hrs before assessment of luciferase activity. Luciferase activity was normalized to the internal control reporter renilla. SRF and MYOCD-dependent activation of the Mymsl promoter was defined as fold increase over its individual vector control group (set to 1). Values are means± SEM from one experiment with 3 biological replicates. Two separate experiments were performed. B. The indicated Mymsl CArG WT or CArG mutant reporters were transfected for luciferase assay as in (A). Data are means ± SEM from 3 separate experiments. C. Chromatin Immunoprecipitation assays (ChIPs) were carried out in growing MASMCs for the analysis of SRF binding to the putative CArG box. Signal of amplified DNA was normalized to the input control. Relative enrichment of the CArG box containing fragment was expressed as fold increase over the IgG control (set to 1). Primers to CArG in the intron1 of mouse Cnn1 was used as positive control. Data are means ± SEM from 4 separate experiments. *P < 0.05.

Article Snippet: Chromatin complexes were precipitated with either antibody to SRF (Cat. # D7A9, Cell Signaling for bladder; Santa Cruz, G-20, sc-335 X for MASMC), H3K79me2 (Cat. # ab3594, Abcam) or rabbit negative IgG control (Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab171870","term_id":"90078505","term_text":"AB171870"}} Ab171870 ).

Techniques: Luciferase, Sequencing, Transfection, Positive Control, Expressing, Control, Activity Assay, Activation Assay, Plasmid Preparation, Mutagenesis, Chromatin Immunoprecipitation, Binding Assay, Amplification

Journal: Journal of molecular and cellular cardiology

Article Title: Transcriptional Control of a Novel Long Noncoding RNA Mymsl in Smooth Muscle Cells by a Single Cis-Element and its Initial Functional Characterization in Vessels

doi: 10.1016/j.yjmcc.2019.11.148

Figure Lengend Snippet: A. qRT-PCR analysis of Mymsl and Myh11 in cultured MASMCs isolated from WT versus CArG mutants transduced with Ad-MYOCD or Ad-empty control virus. Experiments were done with 2 MASMC isolates. Values are means± SEM from one experiment with 3 biological replicates. *P < 0.05. B. Mymsl RNA levels in medial SMC layers of aortas (differentiated/contractile phenotype) versus cultured MASMCs (dedifferentiated/synthetic phenotype) from WT and CArG mutant mice. Two separate experiments were performed. Values are means± SEM from one experiment with 3 biological replicates. *P < 0.05. C. RNA levels of Mymsl and the indicated SMC-marker genes in uninjured versus injured carotid arteries from WT and CArG mutants at 1 week post injury. Values are shown as means ± SEM (n ≥ 5). *P < 0.05. D. qPCR for in vivo ChIPs in bladders from WT versus CArG mutants for SRF binding to the promoter region of Mymsl flanking the above CArG element. The enrichment of intronic CArG1 of Cnn1 was included as a negative control. Values are presented as enrichment ratio of mutant to WT (set to 1) (n=3). E. qPCR analysis of the enrichment of H3K79Me2 to CArG chromatin in Mymsl promoter in bladders from both WT and CArG mutants. Values are presented as enrichment ratio of mutant to WT (set to 1) (n=5).

Article Snippet: Chromatin complexes were precipitated with either antibody to SRF (Cat. # D7A9, Cell Signaling for bladder; Santa Cruz, G-20, sc-335 X for MASMC), H3K79me2 (Cat. # ab3594, Abcam) or rabbit negative IgG control (Abcam, {"type":"entrez-nucleotide","attrs":{"text":"Ab171870","term_id":"90078505","term_text":"AB171870"}} Ab171870 ).

Techniques: Quantitative RT-PCR, Cell Culture, Isolation, Transduction, Control, Virus, Mutagenesis, Marker, In Vivo, Binding Assay, Negative Control

Journal: Nature Communications

Article Title: MKL1-actin pathway restricts chromatin accessibility and prevents mature pluripotency activation

doi: 10.1038/s41467-019-09636-6

Figure Lengend Snippet: MKL1 activity results in reduced chromatin accessibility. a Quantification of the size distribution of DNase I digested DNA fragments from control iPS cells and caMKL1-blocked cells. n = 3 for each genotype. b FRAP analysis of the dynamic movement of histone H1 0 -mCherry in control iPS cell nuclei or caMKL1-blocked cell nuclei. **** P < 0.0001. n denotes the number of analyzed nuclei. Statistics were performed using two-tailed paired t -test. Error bars denote standard deviation. c , d Quantification of the size distribution of DNase I digested DNA fragments from WT and caMKL1-overexpressing ES cells ( c ) and SRF f/f iPS cells and SRF Δ/Δ iPS cells ( d ). n = 3 for each genotype. e , f MA plots of differential ATAC-seq peaks in WT vs caMKL1-expressing ES cells ( e ) and SRF f/f vs SRF Δ/Δ iPS cells ( f ). Pink dots indicate regions of significant differences in chromatin accessibility (left). The width of boxplots (right) is proportional to the number of regions of differential chromatin accessibility, showing overall lower accessibility in caMKL1-expressing ESCs ( e ), and overall increased accessibility in SRF Δ/Δ iPS cells ( f ). The center line of boxplots indicate the median value; the edges of the boxplots are at the 25th and 75th percentile; the whiskers extend to 1.5 times the interquartile range past the box. Points that are outside whiskers are shown individually. g Quantification of the size distribution of DNase I digested DNA fragments from caMKL1-blocked cells treated with or without UNC0642. n = 3 for each genotype. h Percentage of Oct4:GFP+ cells emerging from blocked cells following treatment with UNC0642. GFP+ cells were determined by FACS. All data shown are representative of three independent experiments

Article Snippet: For immunoprecipitation, Oct4 antibody (Cell Signaling, 5677S) and SRF antibody (Active Motif, 61385, Santa Cruz, sc-335) were used at 6 μg/ChIP sample, H3K4me3 antibody (Millipore, 07-473), H3K27ac antibody (Abcam, Ab4729), and H3K27me3 antibody (Millipore, 07-449) were used at 1 μg/ChIP sample.

Techniques: Activity Assay, Two Tailed Test, Standard Deviation, Expressing

Journal: The Journal of Biological Chemistry

Article Title: The Smooth Muscle Cell-restricted KCNMB1 Ion Channel Subunit Is a Direct Transcriptional Target of Serum Response Factor and Myocardin *

doi: 10.1074/jbc.M109.050419

Figure Lengend Snippet: SRF binds conserved CArG elements in KCNMB1. A, 32P-labeled double-stranded oligonucleotides containing CArG boxes from the human KCNMB1 promoter (C3) or intron 1 (C5) were incubated with the in vitro translated SRF in the absence or presence of SRF or MEF2A antibody or a 100-fold molar excess of cold, wild type (WT), or mutant oligonucleotides. SRF-CArG nucleoprotein complex and supershifted complexes (SS) are indicated with arrows. NS, nonspecific nucleoprotein complex. B, ChIP assay from growing HCASMCs. Equal aliquots of chromatin lysates were immunoprecipitated with antibodies to either SRF or a control rabbit IgG. DNA associated with the immunoprecipitates was isolated and used as templates for PCR amplification of either KCNMB1 promoter (C3) or intron 1 region (C5). As a control, primers to exon 4 of KCNMB1 (without CArG boxes) were used following SRF immunoprecipitation. Results were repeated in two independent studies.

Article Snippet: Supershift assays were conducted by a 20-min incubation with either rabbit anti-SRF (sc-335, Santa Cruz Biotechnology, Inc.) or control rabbit anti-MEF2A (sc-313, Santa Cruz Biotechnology, Inc.) following incubation of probe and SRF in binding buffer.

Techniques: Labeling, Incubation, In Vitro, Mutagenesis, Immunoprecipitation, Isolation, Amplification