The Mechanism Of Sal-like Protein 2 Regulates P16^INK4A Expression

BACKGROUND Sal-like protein 2(Sall2, also known as p150sal2 and hsal2) is the product of SALL2 gene. It is a zinc-finger transcription factor. Sall2 can inhibit cell growth and induce cell apoptosis by recognizing and binding to GC-box-like sequence of natural promoters of growth regulatory genes, including p21Cip1/Waf1 and BAX, in human ovarian tumor cells. Sal L2 binds to the GC-box like elements with a consensus sequence of GGG(T/C)GGG, placing Sall2 among the members of Sp1 family of transcription factors. Sp1 is known to positively regulate p16 INK4 A transcription upon binding to the corresponding GC-boxes in the p16 promoter. p16 INK4 A, also known as CDKN2 A, MTS1, p16, is a cyclin-dependent kinase inhibitor and an important tumor suppressor, which plays critical roles in cell cycle suppression, cellular senescence and the inhibition of human cancers. Cell-cycle arrest and apoptotic induction of p16 plays a primary role in its multiple anti-tumor functions, including inhibition of tumor cells proliferation, migration and tumorigenesis.OBJECTIVES The objectives are to identify additional Sall2 regulated target genes; to confirm that Sall2 regulation of the expression of p16 through the binding to the Sall2 consensus binding DNA sequence of p16 promoter; to investigated the biological function of Sall2 in regulating the p16 expression. Achieving these objectives will reveal new insights for further understanding of Sall2 gene regulation network and facilitating the identification of potential anticancer targets against Sall2 deregulation during cancer formation.METHODS We studied the downstream target genes regulated by Sal L2 expression through expression array analysis. In this analysis, c DNAs from SKOV3 cells transfected with either a Sal L2 expression plasmid or an empty vector as a control were tested in a bi-color array system from Affymatrix. Genes that were up- or down-regulated over 1.5 fold known to be associated with cancer promoter or repressor, such as p16, were selected for further analysis. We performed deletion analysis on p16 promoter, and later, identify Sall2 binding site on p16 promoter though luciferase assay. Using FACS, we confirmed that Sall2 inhibits tumor growth by hindering cell cycle though p16 up-regulation.RESULTS We identified 1616 Sall2-responsive genes through gene expression arrays that are either up or down-regulated by Sal L2 expression. Promoter-reporter assays of p16INK4 A and several other tumor-related genes indicated that the Sall2 regulation effects on these promoters was not significantly different between the expression of the two major forms of Sall2 with alternative exon1 or exon1 A. Additional analysis on p16-promoter-luciferase reporter assay showed that Sall2-induced p16 promoter activation was Sall2 dose-dependent. Systematic deletions and site-directed mutagenesis of the p16 promoter identified a consensus Sall2 binding site(GGGTGGG) located at the proximal region to the p16 transcription start site and was critical for p16 promoter activation. Finally, to confirm the significance of Sall2-activated p16 expression in cell cycle regulation, we co-transfected the SKOV3 cells with a Sall2 expression construct and a p16 minigene. We also co-transfected the ES-2 cells with a Sall2 expression construct and the si RNA against p16 for flow cytometry analysis. Our results showed that Sall2 enhanced the p16 minigene expression, thus blocking the cell cycle progression and p16 knockdown with si RNA abolished most of the Sall2 inhibition of cell cycle progression. These findings indicate that Sall2 targets multiple cell cycle regulators including p16, through binding to the relative promoters. We also analyzed the promoter region for conserved sequences according to the comparison in the promoter region of p16 using Encode program. According to this analysis, putative Sall2 binding sites and regulatory regions in humans and monkeys have high homologous as compared with other animals, indicating Sal L2 gene regulation is a recent development during evolutions.CONCLUSION The third zinc finger of Sall2 binds to a consensus(GGGTGGG) proximal to the p16 transcription start site and up regulates the p16 expression, thus inhibiting the cell cycle and tumor cell growth, adding knowledge to the understanding of Sall2 and p16 gene regulation, implying how Sall2 deregulation may promote cancer formation. Our research confirmed that p16 is a new important downstream target gene of Sal L2, prompting the fact that targeted Sall2 expression has important role as an anti-tumor agent, and presents a new direction to find new drug targets.