Fig. 5: HNF4A recruits the MLL4 complex to establish H3K4me1.

a Heatmap showing the same clustering as in Fig. 4d with MLL4 CUT&Tag in control and EE cells in a 5 kb window around HNF4A bound regions. b Average profile plots for MLL4 in control (blue) and EE cells (green) across the 3 clusters. c Genome browser depiction of Apoc2 shows MLL4 binding at site of HNF4A binding. d Co-immunoprecipitation using antibodies for HNF4A, MLL4 and IgG (as negative control) in cells ectopically expressing HNF4A followed by western blot analysis with MLL4 complex proteins (UTX, ASH2L and RBBP5) or HNF4A shows interaction is occurring between HNF4A and MLL4 proteins in 3T3 cells. e Proximity ligation assay (PLA) in control and HNF4A EE 3T3 cells. Top two panels show negative controls while the bottom panel shows positive signals (red) identify interactions occurring between HNF4A and MLL4 (left) or HNF4A and ASH2L (right). DAPI stained nuclei are shown in blue. Scale bars, 10 µm; Ab, antibody. f–i 3T3 cells were transduced with control, Mll4, or Utx shRNA and after 24 h, the cells were transduced with HNF4A or empty construct. The cells were further incubated at 37 °C for 48 h before performing the ChIP-qPCR and RT-qPCR analyses. The ChIP-qPCR for H3K4me1 in control shRNA-transduced 3T3 cells show increased H3K4me1 at HBR regions linked to Apoc2 (f) and Aqp1 (g) when HNF4A was overexpressed. However, H3K4me1 enrichment levels were significantly reduced when either Mll4 or Utx was knocked down. A dotted line represents a fold enrichment of 1. Using RT-qPCR for Apoc2 (h) and Aqp1 (i), relative gene expression levels were measured. The bars display standard deviation (f, g) or standard error of mean (h, i). The statistically significant p-values were calculated by a t test. ***p < 0.001; **p < 0.05; ND, not detected; ns, not significant. n = 2 (f, g) or 3 (h, i) technical replicates.