pet-15b vector plasmid  (Promega)

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A ) The constructs showing the dead Cas9 (dCas9) fused with p300core HAT domain (dCas9-p300 C ) and expressed from cytomegalovirus (CMV) promoter. The small guide RNA (sgRNA) designed for hs1.2 and hs4 enhancers were expressed from the U6 promoter. The cotransfection of dCas9-p300 C and either sgRNAs (hs1.2/hs4) in CH12-F3-2A cells showing site-specific enhancer activation at 3′RR. ( B ) The ChIP-qPCR showing the enrichment of H3K27ac histone in dual-transfected dCas9-p300 C with either sgRNAs in Med12 KD cells. ( C ) Corresponding RT-qPCR data showing the enhancer transcripts level. ( D ) The bar plot showing the IgA rescue efficiency by dCas9-p300 C –mediated enhancer activation in Med12 KD CH12F3-2A cells. The black bar and gray bar showing the control samples. The left blue bars showing the rescue efficiency in WT R cells. The right two blue bars showing the dual-transfected dCas9-p300 C with either sgRNAs (hs1.2 and hs4) in Med12 KD cells. ( E ) LM-PCR assay estimating DNA break rescue in indicated samples as described above. Right: The ImageJ analysis showing the band intensities in the respective samples. ( F and G ) The ChIP assay was performed using the indicated antibodies, followed by qPCR showing the enrichment at S regions in dual-transfected dCas9-p300 C with sgRNAs (hs1.2 and hs4) in Med12 KD cells. ( H ) The 3C-PCR assay showing long-range interaction (LRI) in dual-transfected dCas9-p300 C with Med12 KD cells. Bottom: The ImageJ analysis showing the band intensities in the respective samples. ( I ) The ChIP-qPCR showing the AID enrichment in dual-transfected dCas9-p300 C with sgRNAs in Med12 KD cells. The result summarizes the means ± SD of three independents experiments, and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05).

Article Snippet: The vector dCas9-KRAB-MeCP2 was purchased from Addgene (no. 11082).

Techniques: Construct, Cotransfection, Activation Assay, Transfection, Quantitative RT-PCR, Two Tailed Test

Journal: Science Advances

Article Title: XLID syndrome gene Med12 promotes Ig isotype switching through chromatin modification and enhancer RNA regulation

doi: 10.1126/sciadv.add1466

Figure Lengend Snippet: ( A and B ) The ChIP-qPCR showing the occupancy of Jmjd6 at 3′RR in control and either Jmjd6- or Med12-depleted cells. ( C ) Top: The FACS data showing the effect of Jmjd6 and Carm1 KD by respective siRNAs. Bottom left: The RT-qPCR data showing the KD efficiency by respective samples, and the data were normalized with β2m abundance. ( D ) The bar plot showing the effect of Jmjd6 and Carm1 KD on 3′RR transcripts. ( E and F ) The ChIP assay was performed by indicated the antibodies, followed by qPCR in control and Jmjd6 KD cells. ( G ) The bar plot showing the IgA rescue efficiency in dual-transfected dCas9-p300 C with hs4 sgRNAs in Jmjd6 and/or Carm1 KD cells. ( H ) The FACS analysis showing the IgA rescue efficiency in methylation-defective Med12 mutants in Med12 KD cells. The position of the mutations at PQL domain is shown at the right. ( I ) The RT-qPCR analysis showing the rescue of hs1.2 and hs4 transcripts in WT R and ΔPQL Med12 mutant in Med12 KD cells. ( J ) The schematic representation showing the sequential steps of Med12 workflow in CSR. Med12 is methylated by Jmjd6/Carm1 complex (magenta) at different positions (black boll). Methylation recruits p300 protein to 3′RR, which marks histone H3K27 acetylation and activate the enhancers. Activated enhancers were transcribed into eRNA, which regulates H3K4me3 at S region and recruits DNA break and repair complex for CSR. The result summarizes the means ± SD of three independents experiments and the statistical significance was determined by two-tailed Student’s t test ( P > 0.05). NHEJ, nonhomologous end joining.

Article Snippet: The vector dCas9-KRAB-MeCP2 was purchased from Addgene (no. 11082).

Techniques: Quantitative RT-PCR, Transfection, Methylation, Mutagenesis, Two Tailed Test