Journal: Nature Communications
Article Title: MyoD induced enhancer RNA interacts with hnRNPL to activate target gene transcription during myogenic differentiation
Figure Lengend Snippet: seRNA-1 modulates hnRNPL, RNA Pol II and H3K36me3 at Mb locus. a Western blot analysis of hnRNPL cellular distribution in differentiating C2C12 untreated or treated with RNaseA. Whole cell extracts (WCE), nuclei (N). Relative levels of hnRNPL were normalized with H3K36me3 and measured by Image J. b qPCR quantification of RNA (left) and DNA (right) recovered after lacZ ChIRP or seRNA-1 ChIRP with two different biotinylated probe sets (even and odd) in C2C12 MT. P1, P3 and P4 are three different primer pairs for detecting seRNA-1 RNA. Mb, seRNA-1 and Atp1a1 indicate the primers corresponding to the promoter regions. c ChIP-PCR of hnRNPL, CCNT1, CDK9, Pol II, H3K36me3, and KMT3a at regions (1, 2, 3, and 4) across Mb locus in MT vs MB. d Co-IP assay was performed using antibodies against hnRNPL or CCNT1 in C2C12 MT and the interaction between endogenous hnRNPL and CCNT1 or CDK9 was detected. * IgG light chain. e ChIP-PCR of hnRNPL, Pol II, and H3K36me3 at the above Mb loci in control or hnRNPL knockdown cells (#1 or #2). f ChIP-PCR of hnRNPL, CDK9, CCNT1 and H3K36me3 at the above Mb loci in control or seRNA-1 knockdown cells (#1 or #2). g ChIP-PCR of hnRNPL at the above Mb loci in the two CAAA KO cell lines compared to WT control. h Overexpression of hnRNPL in C2C12 cells decreased the expression of seRNA-1 and Mb but not Apol6. i Schematic illustration of dCas9 mediated tethering of seRNA-1 wild type or the CAAA KO mutant (ΔCAAA) to the seRNA-1 TSS or Mb promoter. j qRT-PCR detection of seRNA-1 and Mb in the above cells. k ChIP-PCR of hnRNPL binding at the indicated seRNA-1 or Mb promoter following the above tethering. Data represent the average of three independent experiments ± s.d. Statistical analysis was done by two-tailed unpaired Student’s t -test ( b , c , e – h , j , k ). n.s., not significant, * P
Article Snippet: The resultant RNA was reverse transcribed with RP1 primer (5′-AATGATACGGCGACCACCGAGATCTACACGTTCAGAGTTCTACAGTCCGA-3′) (IDT) using Superscript III RT enzyme (Thermo Fisher Scientific).
Techniques: Western Blot, Real-time Polymerase Chain Reaction, Chromatin Immunoprecipitation, Polymerase Chain Reaction, Co-Immunoprecipitation Assay, Over Expression, Expressing, Mutagenesis, Quantitative RT-PCR, Binding Assay, Two Tailed Test