SYBR Safe DNA Gel Stain br of positive stained cells
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), SYBR Safe DNA Gel Stain 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.
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 . 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 , 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 . 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 . 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 . 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.