Fig. 2. ZNF354A controls the expression of stress-induced ZDHHC20L.

a Overview of ZDHHC20 locus (intron, exon, 5′UTR; hg19). Tracks: transcription start codons (ATGs), transcript annotations (GENCODE), ChIP-seq peaks (ENCODE: SETDB1/KAP1, ATF2, SP1, FOXA1), ZNF354A and KZFP peaks from HEK293T cells (squish view,) from KRABopedia⁵⁸,(https://tronoapps.epfl.ch/web/krabopedia/); b Western blot (WB) of ZDHHC20 isoforms (Short-S, 20L) and GAPDH (loading control) in U2OS cells depleted of SETDB1, KAP1, or indicated KZFPs. c Immunoprecipitation (IP) of endogenous KAP1 in U2OS cells overexpressing HA-tagged KZFPs, untreated or treated with proaerolysin toxin (10 ng/ml, 1 h at 37 °C, washed, incubated further 8 h). Blots: co-IP fractions and total cell extracts (TCE). d IP-KAP1 as in (c) from siControl (Ctrl) or siATF2 U2OS cells expressing ZNF354A-FLAG complemented with indicated ATF2 constructs (WT, 6D, 6 A; 24 h). Results mean ± SEM; dots represent independent experiments (n  =  3); P values vs. untreated, two-way ANOVA, Tukey’s correction. e WB as (b) in human primary lung epithelial cells infected with SARS-CoV-2 (MOI 0.1). f IP-KAP1 in siKAP1 U2OS cells expressing KAP1-WT or S473A mutant, treated with aerolysin as in (c). Blots show KAP1 and phospho-Ser/Thr proteins in IP fractions. g WB of TCEs as (b) in siKAP1 U2OS cells expressing KAP1 WT, S473A (S/A), or S473D (S/D). h IP-ZNF354A from Calu-3 cells infected with SARS-CoV-2 (24 h), aerolysin-treated U2OS cells (as in c), or siCtrl/siZNF354A-depleted U2OS cells. Blots show ZNF354A and phospho-Ser/Thr proteins (IP fractions) and ZNF354A and GAPDH (TCE). i IP-ZNF354A -HA from U2OS cells expressing indicated ZNF354A-FLAG and ATF2 constructs, aerolysin-treated as (c). Blots: IP fractions (KAP1, ATF2, phospho-Ser/Thr); TCE (total/pS473-KAP1, ATF2-HA). j Model of ATF2-KAP1-ZNF354A repressive complex before and after stress. Source data and entire blots provided as a Source Data file and supplementary information.