Figure 8. ZFP423 induces a NFκB/p65 co-regulator switch.

a) Schematic of ZFP423: ZFP423 contains 30 C2H2 zinc-fingers (ZF) clustered into five domains. Amino acids 2–13 represent a defined NuRD-interaction domain that resides within numerous zinc-finger proteins that interact directly with the NuRD co-repressor complex.

b) Western blot of indicated protein expression in FIPs whole cell lysates (left) and p65 immunoprecipitates (right) from primary FIPs transduced with control retrovirus or retrovirus expressing FLAG-tagged ZFP423 (FLAG-ZFP423). Cells were treated with LPS (100ng/ml) or vehicle for 2 hours prior to harvest.

c) Western blot of indicated protein expression in FIPs whole cell lysates (top) and p65 immunoprecipitates (bottom) from primary FIPs transduced with control retrovirus or retrovirus expressing FLAG-tagged ZFP423 (FL-ZFP423) or FLAG-tagged ZFP423 lacking the NuRD interaction domain (NID) (FL-ZFP423^ΔNID). Cells were treated with LPS (100ng/ml) or vehicle for 2 hours prior to harvest.

d) Western blot of indicated protein expression within whole cell lysates of FIPs transduced with control retrovirus, wild-type FLAG-tagged ZFP423 (FLAG-ZFP423^WT), or retrovirus expressing FLAG-ZFP423 lacking the 12 amino acid NuRD-interaction domain (NID) (FLAG-ZFP423^ΔNID) and treated with LPS (100ng/ml) or vehicle. β-actin expression was used as loading control.

e) NFκB-dependent gene reporter activity in 293-TLR4 cells co-transfected with control, FLAG-ZFP423^WT, or FLAG-ZFP423^ΔNID plasmids. Cells were treated with LPS (100ng/ml) (n=10 per group) or vehicle (PBS) (n=3 per group) for 8 hours prior to harvest. Bars represent mean ± s.e.m., ** denotes p<0.01 by two-way ANOVA.

f) mRNA levels of indicated genes in FIPs transduced with control, FLAG-ZFP423^WT, or FLAG-ZFP423^ΔNID, retroviruses and treated with vehicle (PBS) (n=3 per group) or 100 ng/ml LPS (n=4 per group) for 2 hours. * denotes p<0.05, ** denotes p<0.01 by two-way ANOVA.

g) Proposed model: The nuclear p65 subunit of NFκB is acetylated (Ac) and activated by its interaction with CBP/p300. The presence of ZFP423 triggers a transcriptional co-regulator switch; ZFP423 facilitates the recruitment of the NuRD co-repressor complex to p65, thereby excluding CBP/p300 association and p65 acetylation.

Exact p values, numbers of repetitions, and uncropped western blots can be found in Source Data Figure 8.