Journal: Nature Communications

Article Title: Epigenetic interaction between UTX and DNMT1 regulates diet-induced myogenic remodeling in brown fat

doi: 10.1038/s41467-021-27141-7

Figure Lengend Snippet: A Pyrosequencing analysis of DNA methylation at Myod1 promoter in BAT1 adipocytes transfected with lentiviral vectors expressing dCas9-TET1CD along with lentiviral vectors expressing either Myod1 -targeting gRNA or scramble non-targeting gRNA ( n = 4/group). *indicates statistical significance between the two groups by two-tailed unpaired Student’s t -test. B Quantitative PCR analysis of Myod1 and BAT-specific gene expression in BAT1 adipocytes transfected with lentiviral vectors expressing dCas9-TET1CD along with lentiviral vectors expressing either Myod1 -targeting gRNA or scramble non-targeting gRNA ( n = 4/group). *indicates statistical significance between the two groups by two-tailed unpaired Student’s t -test. For ( A , B ), 4-day differentiated BAT brown adipocytes were transfected with lentiviral vectors FUW-dCas9-TET1CD along with lentiviral vectors pgRNA-mCherry encoding either scramble-gRNA or Myod1-targeting gRNA using Amaxa Nucleofector II Electroporator with an Amaxa cell line nucleofector kit L. Cells were harvested 2 days after for pyrosequencing ( A ) or gene expression ( B ) analysis. C , D Body weight ( C ) and Body composition ( D ) in mice with iBAT injection of lentiviruses expressing dCas9-TET1CD plus lentiviruses expressing either targeting Myod1-gRNA-mCherry or non-targeting scramble-gRNA-mCherry ( n = 4/group). *indicates statistical significance between the two groups by two-tailed unpaired Student’s t -test. E – G Methylation levels at Myod1 promoter ( E , n = 8/group), Myogenic marker gene expression (F, n = 7 for dCas9 + scramble, and 5 for dCas9 + Myod1 gRNA), and BAT-specific gene expression ( G , n = 7 for dCas9+scramble, and 6 for dCas9 + Myod1 gRNA) in iBAT of mice with iBAT injection of lentiviruses expressing dCas9-TET1CD plus lentiviruses expressing either targeting Myod1-gRNA-mCherry or non-targeting scramble-gRNA-mCherry. *Indicates statistical significance between the two groups by Mann–Whitney’s nonparametric U test in ( E ), ( F ) and ( G ). ( H ) Representative IHC staining of UCP1 (upper panel) and MyHC (lower panel) in iBAT of mice with iBAT injection of lentiviruses expressing dCas9-TET1CD plus lentiviruses expressing either targeting Myod1-gRNA-mCherry or non-targeting scramble-gRNA-mCherry ( n = 3 replicates). For ( C – H ), 3-month-old chow-fed male C57BL/6J mice were bilaterally injected with lentiviruses expressing dCas9-TET1CD plus lentiviruses expressing either targeting Myod1-gRNA-mCherry or non-targeting scramble-gRNA-mCherry into iBAT for up to 2 months. All data are expressed as mean ± SEM.

Article Snippet: For ( A , B ), 4-day differentiated BAT brown adipocytes were transfected with lentiviral vectors FUW-dCas9-TET1CD along with lentiviral vectors pgRNA-mCherry encoding either scramble-gRNA or Myod1-targeting gRNA using Amaxa Nucleofector II Electroporator with an Amaxa cell line nucleofector kit L. Cells were harvested 2 days after for pyrosequencing ( A ) or gene expression ( B ) analysis.

Techniques: DNA Methylation Assay, Transfection, Expressing, Two Tailed Test, Real-time Polymerase Chain Reaction, Injection, Methylation, Marker, Immunohistochemistry