Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: TIS7 epigenetically regulates MyoD expression. a TIS7 KO MSCs were transfected with indicated plasmids. Quantitative PCR analysis of myoD was normalized to GAPDH expression (n = 3 independent experiments). Values represent the mean ± SEM and resulting data were analyzed via two-tailed type 2 Student’s t-test, *** P < 0.001 and * P < 0.05. b Proliferating TIS7 KO MSCs were transfected with indicated plasmids and total cell lysates were probed with MyoD and control antibodies. Quantification (n = 3 biological replicates) was performed by normalization to the tubulin signal. Values represent the mean ± SD and resulting data were analyzed via two-tailed type 2 Student’s t-test, *** P < 0.001 and ** P < 0.01. Representative western blots are shown. ChIP analyses using chromatin from TIS7 WT or KO MSCs transfected with either GFP or GFP-TIS7 plasmid DNA. c Schematic outline of mouse myoD gene locus. Primer pairs used for ChIP assays are indicated by arrows. The alignment of human and mouse sequences amplified by the PCR is shown. Antisera directed against symmetrically di-methylated histone H3 at arginine 8 ( d ) or PRMT5 ( e ) were used for immunoprecipitation. Immunoprecipitated DNA was analyzed by qPCR in triplicates using primers specific for the mouse myoD regulatory region. ChIP with control rabbit IgG or 1.23 % of total chromatin (input) were used as controls. Signals were normalized to input chromatin and shown as percentage of input, ** P ≤ 0.001 and *** P < 0.00005

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: Expressing, Transfection, Real-time Polymerase Chain Reaction, Two Tailed Test, Western Blot, Plasmid Preparation, Amplification, Methylation, Immunoprecipitation

Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: ICln affects in vivo methylation of histone H3 and binding of PRMT5 to myoD locus, resulting in increased myoD RNA and protein levels. ChIP analysis using chromatin from TIS7 WT or KO MSCs transfected with either YFP or YFP-ICln plasmids. Antisera directed against symmetrically dimethylated histone H3 at arginine 8 ( a ) or PRMT5 ( b ) were used for immunoprecipitation. Immunoprecipitated DNA was analyzed in triplicate by qPCR using primers to amplify the mouse myoD regulatory region. ChIP with control rabbit IgG or 1.23 % of total chromatin (input) were used as controls. Signals were normalized to input chromatin and shown as percentage of input. ** P ≤ 0.001 and *** P < 0.00005. c TIS7 KO MSCs were transfected with indicated plasmids and total cell lysates were probed with anti-MyoD and anti-YFP antibodies. Quantification (n = 3 biological replicates) was performed by normalization to the tubulin signal. Representative immunoblots are shown. d TIS7 KO MSCs were transfected with the indicated plasmids. Quantitative PCR analysis of myoD was normalized to GAPDH expression (n = 3 biological replicates and ** P < 0.01). e PRMT5 knockdown significantly increased myoD RNA levels in TIS7 KO MSCs transduced with sh PRMT5-expressing lentivirus. myoD RNA levels were measured by qPCR and PRMT5 protein levels were documented by immunoblots with indicated antibodies

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: In Vivo, Methylation, Binding Assay, Transfection, Immunoprecipitation, Western Blot, Real-time Polymerase Chain Reaction, Expressing, Transduction

Journal: BMC Biology

Article Title: TIS7 induces transcriptional cascade of methylosome components required for muscle differentiation

doi: 10.1186/s12915-016-0318-6

Figure Lengend Snippet: Model of myogenesis regulation by the TIS7/ICln/PRMT5 pathway. In skeletal muscles, TIS7 transcriptionally regulates ICln. The accumulated ICln interacts with PRMT5 and prevents its recruitment to chromatin-bound complexes on the myoD gene. This regulates PRMT5-mediated symmetrical dimethylation of histone H3 Arg8 and myoD expression. The regulated myoD level affects the balance between proliferation and differentiation of MSCs

Article Snippet: Full length mouse TIS7 (mTIS7), human PRMT5 (hPRMT5, Origen, cat. no. ORIGSC116333), mouse myoD (CMV-myc-tagged myoD; Addgene plasmid 8399; principal investigator: Andrew Lassar), and human ICln (hICln) cDNA or plasmid DNA were PCR amplified and cloned in frame with the fluorescent proteins ECFP and EYFP into the pECFP-C1, pECFP-N1, pEYFP-C1, or pEYFP-N1 (Clontech) mammalian expression vectors.

Techniques: Expressing

Journal: Stem cells (Dayton, Ohio)

Article Title: Regulation of Myogenesis by a Na/K-ATPase α1 Caveolin Binding Motif

doi: 10.1093/stmcls/sxab012

Figure Lengend Snippet: Primer sequences

Article Snippet: Lentivirus expression vector for the mouse Myod1 gene (pLv-CMV-Myod) was from Addgene (Plasmid #26808).

Techniques: Sequencing

Journal: Stem cells (Dayton, Ohio)

Article Title: Regulation of Myogenesis by a Na/K-ATPase α1 Caveolin Binding Motif

doi: 10.1093/stmcls/sxab012

Figure Lengend Snippet: Rescue of Skm differentiation in mCBM cells with a mouse Myod1 transgene.

Article Snippet: Lentivirus expression vector for the mouse Myod1 gene (pLv-CMV-Myod) was from Addgene (Plasmid #26808).

Techniques:

Journal: Arteriosclerosis, Thrombosis, and Vascular Biology

Article Title: Direct Reprogramming of Fibroblasts Into Smooth Muscle-Like Cells With Defined Transcription Factors—Brief Report

doi: 10.1161/atvbaha.118.310870

Figure Lengend Snippet: Figure 1. Generation of induced smooth muscle-like cells (iSMCs) from mouse embryonic fibroblasts (MEFs). A, Schematic drawing of the differentiation protocol used to generate iSMCs. B, Induction of expression of the smooth muscle cell (SMC) marker Myh11 in MEFs treated with a combination of Myocd* (myocardin), Gata4, Mef2B*, and Mef2C* genes on day 5 post-transduction. C, Induction of Myh11 gene expression in MEFs treated with combination of Myocd*, Mef2C*, Gata4, and Gata6 genes and cultured for the 3, 4, and 5 d post-transduction. D, Downregulation of expression of fibroblast marker genes Fn-EDA and Thy1 in MEFs on day 5 post-transduction with the retrovirus encoding the reprogramming factors indicated in the figure. Normalized with empty vector virus control group=1.0 (B and C) or Myocd*, Mef2C*, and Gata6 group=1.0 (D). The data are shown as mean±SEM. n=3. FB indicates fibro- blasts; G4, Gata4; G5, Gata5; and G6, Gata6. *Potentiated genes with fusion of the M3 domain of human MyoD (myogenic differentiation 1). ****P<0.001, ***P<0.01, **P<0.05 compared with the control group.

Article Snippet: The human MyoD M3 domain (amino acids 1–62) was fused to fulllength cDNAs encoding human MEF2C at the carboxy-terminus to create the fusion gene MEF2C*. pLenti CMV Puro (kind gift from Drs Campeau and Kaufman, Addgene plasmid No. 17448) vector encoding the fusion gene MEF2* and pReceiver-Lv105 (GeneCopoeia) vector encoding human wild-type genes MYOCD, and GATA6, as well as the empty vector virus control, were separately cotransfected with packaging plasmids, psPAX2 (kind gift of Dr Trono, Addgene plasmid No. 12260) and envelope plasmid, pCMV-VSV-G (kind gift of Dr Weinberg, Addgene plasmid No. 8454) into 293T cells using Lipofectamine 2000 (Thermo Fisher Scientific).

Techniques: Expressing, Marker, Transduction, Gene Expression, Cell Culture, Plasmid Preparation, Virus, Control