Chromatin-Directed Proteomics Identifies ZNF84 as a p53-Independent Regulator of p21 in Genotoxic Stress Response

SIMPLE SUMMARY: Chemotherapy is a commonly applied anticancer treatment, however therapy-induced senescent growth arrest has been associated with aggressive disease recurrence. The p21 protein, encoded by CDKN1A, plays a vital role in the induction of senescence. Its transcriptional control by p53 is well-established. However, in many cancers where TP53 is mutated, p21 expression must be triggered by p53-independent mechanisms. We here used a chromatin-directed proteomic approach and identified ZNF84 as a regulator of CDKN1A gene expression in various p53-deficient cell lines. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin and facilitated senescence bypass. Intriguingly, ZNF84 depletion diminished genotoxic burden evoked by doxorubicin. Clinical data association studies indicated the relevance of ZNF84 expression for patient survival. Collectively, we identified ZNF84 as a critical regulator of senescence-proliferation outcome of chemotherapy, opening possibilities for its targeting in novel anti-cancer therapies of p53-mutated tumours. ABSTRACT: The p21(WAF1/Cip1) protein, encoded by CDKN1A, plays a vital role in senescence, and its transcriptional control by the tumour suppressor p53 is well-established. However, p21 can also be regulated in a p53-independent manner, by mechanisms that still remain less understood. We aimed to expand the knowledge about p53-independent senescence by looking for novel players involved in CDKN1A regulation. We used a chromatin-directed proteomic approach and identified ZNF84 as a novel regulator of p21 in various p53-deficient cell lines treated with cytostatic dose of doxorubicin. Knock-down of ZNF84, an as-yet un-characterized protein, inhibited p21 gene and protein expression in response to doxorubicin, it attenuated senescence and was associated with enhanced proliferation, indicating that ZNF84-deficiency can favor senescence bypass. ZNF84 deficiency was also associated with transcriptomic changes in genes governing various cancer-relevant processes e.g., mitosis. In cells with ZNF84 knock-down we discovered significantly lower level of H2AX Ser139 phosphorylation (γH2AX), which is triggered by DNA double strand breaks. Intriguingly, we observed a reverse correlation between the level of ZNF84 expression and survival rate of colon cancer patients. In conclusion, ZNF84, whose function was previously not recognized, was identified here as a critical p53-independent regulator of senescence, opening possibilities for its targeting in novel therapies of p53-null cancers.