Abstract
SNA is one of the essential EMT transcriptional factors capable of suppressing epithelial maker while upregulating mesenchymal markers. However, the mechanisms for SNA to transactivate mesenchymal markers was not well elucidated. Recently, we demonstrated that SNA collaborates with EGR1 and SP1 to directly upregulate MMP9 and ZEB1. Remarkably, a SNA-binding motif (TCACA) upstream of EGR/SP1 overlapping region on promoters was identified. Herein, we examined whether four other mesenchymal markers, lymphoid enhancer-binding factor (LEF), fibronectin (FN), cyclooxygenase 2 (COX2), and collagen type alpha I (COL1A1) are upregulated by SNA in a similar fashion. Expectedly, SNA is essential for expression of these mesenchymal genes. By deletion mapping and site directed mutagenesis coupled with dual luciferase promoter assay, SNA-binding motif and EGR1/SP1 overlapping region are required for TPA-induced transcription of LEF, FN, COX2 and COL1A1. Consistently, TPA induced binding of SNA and EGR1/SP1 on relevant promoter regions of these mesenchymal genes using ChIP and EMSA. Thus far, we found six of the mesenchymal genes are transcriptionally upregulated by SNA in the same fashion. Moreover, comprehensive screening revealed similar sequence architectures on promoter regions of other SNA-upregulated mesenchymal markers, suggesting that a general model for SNA-upregulated mesenchymal genes can be established.
Highlights
It is tempting to investigate whether SNA can directly activate transcriptional upregulation of other epithelial mesenchymal transition (EMT)-related genes involved in hepatocellular carcinoma (HCC) progression such as FN [19], lymphoid enhancer-binding factor (LEF) [20], cyclooxygenase 2 (COX2) [21–23], and COL1A1 [24–26] in a similar fashion as observed in matrix metalloproteinases 9 (MMP9) and zinc finger E-box binding homeobox 1 (ZEB1)
By gene expression and promoter analysis coupled with protein–DNA interaction assay in HCC, we did find that FN, LEF, COX2, and COL1A1 were transcriptionally upregulated by SNA via binding to the proposed SNA binding motifs, which are close to the EGR-1/SP1 overlapping regions
The time courses of the tetradecanoyl-phorbol 13-acetate (TPA)-induced expression of these genes were quantitatively analyzed in HCC340, a patient-derived HCC cell lines used for studying the SNA-upregulated MMP9 and ZEB1 transcription [17]
Summary
A specific putative SNA binding motif “TCACA”, required for activating their promoters was identified [17] This is similar to those observed in the transcription of cyclin-dependent kinase 4/6 (CDK4/6), p15INK4b, a cell cycle-related gene upregulated by SNA [18]. After examining sequences of the other known SNA upregulated mesenchymal markers, including vimentin, TWIST1, vitronectin, α2 smooth muscle actin (ACTA2), fibronectin (FN), and lymphoid enhancer-binding factor (LEF), we found similar sequence architecture, i.e., the SNA motif TCACA coupled with neighboring EGR1 and SP1 region, exists in their promoters around 300–2000 bp upstream of the translational initiation sites. It is tempting to investigate whether SNA can directly activate transcriptional upregulation of other EMT-related genes involved in HCC progression such as FN [19], LEF [20], COX2 [21–23], and COL1A1 [24–26] in a similar fashion as observed in MMP9 and ZEB1. By gene expression and promoter analysis coupled with protein–DNA interaction assay in HCC, we did find that FN, LEF, COX2, and COL1A1 were transcriptionally upregulated by SNA via binding to the proposed SNA binding motifs, which are close to the EGR-1/SP1 overlapping regions
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