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  • br of positive stained cells E of SA

    2022-04-26


    of positive stained cells (E) of SA-β-Gal staining before and after knocking down of SMARCA5 by two shRNAs. *** represents p < 0.001 based on t-test with three independent countings.
    then influence senescence, we stably knocked down SMARCA5 by lentivirus mediated short hairpin RNA in A549 and U2OS cells, and reduced BAZ1A protein levels were found in SMARCA5-KD cells (Fig. 5A and Supplemental Fig. 15A), which also exhibited senescence-associated phenotypes, including decreased proliferation rate (Fig. 5B and Supplemental Fig. 15B), Veratridine arrest at G1 phase (Fig. 5C and Supplemental Fig. 15C), and increased percentage of positive SA-β-Gal stained cells (Fig. 5D–E and Supplemental Fig. 15D–E). Interestingly, SMARCA5 was also found downregulated in senescent MEFs (Supple-mental Fig. 16). These results suggested that in addition to BAZ1A, SMARCA5, the other subunit of ACF chromatin remodeling complex, can also regulate senescence-associated phenotypes. Further, SMARCA5-KD induced senescence was mediated, at least in part, through downregulated abundance of BAZ1A, in line with the stabili-zation function of SMARCA5 to BAZ1A.
    3. Discussion
    BAZ1A as one of the chromatin remodelers has emerged as an im-portant regulator of many biological processes, including neurodeve-lopment, DNA damage recovery and spermatogenesis [35,37,52]. However, whether BAZ1A plays a crucial role in cellular senescence is completely unknown. Cellular senescence has been widely accepted as one of the key cancer prevention mechanisms [53,54]. In this study, we were surprised to find that BAZ1A is prevalently downregulated in multiple senescence models. Knockdown of BAZ1A led to senescence-associated phenotypes in both normal and cancer cells. Conversely, elevated BAZ1A expression was observed in various types of tumors compared to the matched normal tissues (Supplemental Fig. 17) based on analysis of TCGA public datasets available in the GEPIA website [55]. Further, knockdown of BAZ1A in cancer cell line A549 exhibited reduced colony formation ability (Supplemental Fig. 18). As A549 is a widely used lung cancer cell line for senescence-associated tumor im-munotherapy study [56], we speculated that chromatin remodeler BAZ1A may be a novel regulator of cellular senescence and a novel target with potential implication in cancer treatment.
    Several studies have reported that BAZ1A (or ACF1) is a multi-do-main protein containing several conserved motifs for interaction with DNA, histones, and other members of the ACF complex [32,57]. Due to its ability of binding to DNA, BAZ1A acts partly as a transcription factor [58]. BAZ1A has been reported as a transcription repressor via orga-nizing other transcription factors to VDR (Vitamin D3 Receptor) gene and represses its transcription under the condition lack of vitamin D3 [44]. In the present study, we discovered that BAZ1A bound to the 
    promoter region of SMAD3 and inhibited its transcription. Meanwhile, it has been demonstrated that SMAD3 activates CDKN1A (coding for p21) transcription [47,48,59], which is a key mediator of cell senes-cence [9]. In line with this, our results also showed p21 upregulation upon BAZ1A knockdown (Fig. 2G–H and Supplemental Fig. 6G–H), which implied that BAZ1A-SMAD3-p21 is a new signal axis in pro-moting cellular senescence in human cancer cell lines (A549 and U2OS) (Fig. 6). Since p53-p21 is a classic signaling pathway functions in se-nescence, we further noticed SMAD3 has been reported interacting with p53 and bound to target gene promoters to regulate gene expression [60,61]. Interestingly, CDKN1A did not show significant changes after knockdown of BAZ1A in two cancer cell lines with TP53 genetic al-terations (MDA-MB-231 with TP53 point mutation and NCI-H1299 with
    Fig. 6. Working model for BAZ1A-SMAD3-CDKN1A signal axis in regulating cellular senescence in A549 and U2OS cells. Chromatin remodeling factor BAZ1A exhibits downregulated expression in multiple cellular senescence models. BAZ1A binds to the promoter region of SMAD3 to inhibit its tran-scription, thus the reduced BAZ1A level leads to increased expression of SMAD3, which may cooperate with p53 to regulate the expression of CDKN1A (coding for p21) [47,48,60,61]. SMAD3-induced CDKN1A upregulation ulti-mately promotes senescence-associated phenotypes.
    TP53 null mutation) (Supplemental Figs. 19F and 20F). These results further indicated that SMAD3 mediated CDKN1A upregulation required p53 in BAZ1A-KD cells, however, how they function cooperatively in BAZ1A-KD induced senescence reserves further study.
    In addition, BAZ1A-KD induced senescence-associated phenotypes in MDA-MB-231 and NCI-H1299 cells bypass the way of increasing CDKN1A (Supplemental Figs. 19 and 20) also hints that BAZ1A-SMAD3-p21 might not be the only signal axis functioning in BAZ1A-KD induced senescence. Actually, some results did indicate that other signaling pathways might also underlie BAZ1A-KD induced senescence. For ex-ample, genes involved in response to hydrogen peroxide were also found differentially expressed in BAZ1A-KD cells (Fig. 3B), which was further proved by elevated cellular superoxide level evaluated by DHE assay in BAZ1A-KD cells (Fig. 3C–D and Supplemental Fig. 11). Recent studies revealed that accumulated superoxide can cause increased mi-tochondrial DNA mutations and respiratory chain damage, as well as cellular macromolecular compound injury, which ultimately led to cellular senescence [62,63]. Therefore, superoxide mediated pathway could be another mechanism to explain BAZ1A-KD induced senescence, which warrants further investigation. In addition, FoxO signaling pathway, a conserved pathway mediating both senescence and in-dividual aging [64–66], was also enriched with differentially expressed genes upon BAZ1A-KD (Fig. 3A). Moreover, we looked into the pub-lished BAZ1A ChIP-seq results [58], and validated some reported target genes of BAZ1A through ChIP-qPCR in A549 cells, and found that BAZ1A enriched in the promoter region of FOXK1 and ZFP36L2 (Sup-plemental Fig. 21), which were responsible for cell proliferation and cell cycle control [67,68]. However, whether they contributed to BAZ1A-KD induced cellular senescence deserves further study.