Fig. 7: MLL4 is required for BAF binding on C/EBPβ-activated enhancers.

Mll3^‒/‒Mll4^f/f brown preadipocytes were infected with retroviruses expressing C/EBPβ or vector (Vec), and then infected with adenoviruses expressing GFP or Cre. Sub-confluent cells were collected without inducing adipogenesis for Western blot and ChIP-Seq analyses. Published ChIP-Seq data sets for C/EBPβ, MLL4, H3K27ac were used (GSE50466)¹⁴. a Western blot analyses of BAF subunits (SMARCA4, SS18, and ARID1A) and C/EBPβ expression in control (GFP) and Mll4 KO (Cre) preadipocytes. n = 2 biological replicates. b Among the 571 C/EBPβ-activated enhancers in preadipocytes, BAF (SMARCA4 and ARID1A) binding was induced on 324 AEs only after ectopic expression of C/EBPβ (BAF-de novo), while 247 AEs were already bound by BAF before ectopic expression of C/EBPβ in preadipocytes (BAF-prebound). c Motif analysis of BAF-de novo and BAF-prebound AEs using SeqPos motif tool. d–f Deletion of Mll4 reduces BAF binding on C/EBPβ-activated enhancers. Heat maps (d) and average profiles (e) around C/EBPβ-activated enhancers are shown. Enhancers shown in the heat maps were ranked by the intensity of C/EBPβ at the center in control cells expressing ectopic C/EBPβ. f Fold changes of intensities between control and Mll4 KO (Cre) cells on BAF-de novo (n = 324) and BAF-prebound (n = 247) C/EBPβ-activated enhancers are shown in box plots. The box represents the first and third quartiles with the horizontal line showing the median, and whiskers indicating minimum and maximum in all box plots. Outliers were not included. Statistical significance levels are as follows (Wilcoxon signed rank test, one-sided): BAF-de novo C/EBPβ (p = 1.3E-19); BAF-de novo SMARCA4 (p = 1.1E-51); BAF-de novo ARID1A (p = 4.1E-50); BAF-de novo H3K27ac (p = 1.1E-48); BAF-prebound C/EBPβ (p = 2.9E-15); BAF-prebound SMARCA4 (p = 1.0E-32); BAF-prebound ARID1A (p = 3.4E-27); BAF-prebound H3K27ac (p = 5.6E-34).