Snail associates with EGR‐1 and SP‐1 to upregulate transcriptional activation of p15^INK4b

Abstract

Snail is a multifunctional transcriptional factor that has been described as a repressor in many different contexts. It is also proposed as an activator in a few cases relevant to tumor progression and cell‐cycle arrest. This study investigated the detailed mechanisms by which Snail upregulates gene expression of the CDK inhibitor p15INK4b in HepG2 induced by the tumor promoter tetradecanoyl phorbol acetate (TPA). Using deletion mapping, the TPA‐responsive element on the p15INK4b promoter was located between 77 and 228 bp upstream of the transcriptional initiation site, within which the putative binding regions of early growth response gene 1 (EGR‐1) and stimulatory protein 1 (SP‐1) were found. Gene expression of EGR‐1, Snail and SP‐1 can be induced by TPA within 0.5–6 h. In addition, basal levels of SP‐1, but not of the other two transcriptional factors, were observed. Blockade of TPA‐induced gene expression of Snail, EGR‐1 or SP‐1 suppressed activation of the p15–pro228 reporter plasmid harboring the TPA‐responsive element. More detailed deletion mapping and site‐directed mutagenesis further concluded that the overlapping EGR‐1/SP‐1‐binding site was required for TPA‐induced p15–pro228 activation. In an EMSA, a DNA–protein complex was elevated by TPA, which can be blocked by antibodies against EGR‐1, SP‐1 or Snail at 6 h. Immunoprecipitation/western blotting demonstrated that TPA could trigger the association of EGR‐1 with Snail or SP‐1. Furthermore, a double chromatin immunoprecipitation assay verified that EGR‐1 could form a complex with Snail or SP‐1 on the TPA‐responsive element after treatment with TPA for 2–6 h. Finally, we demonstrated a novel Snail‐target region which could be bound by Snail and was also required for TPA‐induced p15–pro228 activation. In conclusion, Snail associates with EGR‐1 and SP‐1 to mediate TPA‐induced transcriptional upregulation of p15INK4b in HepG2. Structured digital abstract http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7384899: Snail (uniprotkb:http://www.uniprot.org/uniprot/O95863?format=text&ascii) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with EGR‐1 (uniprotkb:http://www.uniprot.org/uniprot/P18146?format=text&ascii) by anti bait coimmunoprecipitation (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006) http://mint.bio.uniroma2.it/mint/search/interaction.do?interactionAc=MINT-7384908: SP‐1 (uniprotkb:http://www.uniprot.org/uniprot/P08047?format=text&ascii) physically interacts (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0915) with EGR‐1 (uniprotkb:http://www.uniprot.org/uniprot/P18146?format=text&ascii) by anti bait coimmunoprecipitation (http://www.ebi.ac.uk/ontology-lookup/?termId=MI:0006)

A conserved role for Snail as a potentiator of active transcription

“…Similar observations have been made in epithelial-mesenchymal transition-related processes, such as tumor formation or neural crest migration. In human HepG2 cells, Snail associates and acts in concert with EGR-1 and SP-1 to participate in p15 INK4b activation induced by tetradecanoyl phorbol acetate (TPA) (Hu et al 2010). A similar synergistic effect was reported for Snail2 and Sox9 in the autoactivation of Snail2 in quail (Sakai et al 2006).…”

“…Potential synergistic effects have not been analyzed in these studies. Snail is also sufficient for a weak activation of the p15 INK4b and Snail2 promoter (Sakai et al 2006;Hu et al 2010). Drosophila Snail instead acts in a synergistic manner for which the presence of all three factors-Twist, Snail, and the factor binding the Tlllike motif-are required for enhancer activation in vivo.…”

“…In summary, our study revealed a direct activator role for Drosophila Snail, a function that is seemingly conserved from flies to humans (Sakai et al 2006;Stemmer et al 2008;Reece-Hoyes et al 2009;Hu et al 2010;Tao et al 2011;Wels et al 2011;Hahn et al 2013) and places the Snail family of proteins in the category of dually acting TFs.…”

“…An activator role for Snail has been shown by genetic studies and reporter assays in Caenorhabditis elegans, mice, and quail-showing that Snail family members can activate B0507.1 (ReeceHoyes et al 2009), MMP15 (Tao et al 2011), and Snail2 (Sakai et al 2006)-and by in vitro studies of the p15 INK4b (Hu et al 2010) and ZNF281 genes (Hahn et al 2013). Furthermore, Snail can increase Wnt target gene expression in human cell lines independent of direct DNA binding but via physical interaction with b-catenin (Stemmer et al 2008).…”

Comparative and Evolutionary Studies of Vertebrate Extracellular Sulfatase Genes and Proteins: SULF1 and SULF2

“…The genes cover 194.3 and 130.5 kilobases in length, respectively, with 18 introns and 20 exons present for the mRNA transcripts. The human SULF1 gene promoter contained six predicted TFBS ( Figure 2 and Table 4), including 3 binding sites for FOXA1, encoding hepatocyte nuclear factor 3-alpha, which participates in embryonic development and directs tissue-specific gene expression [48]; 2 binding sites for TFA2PC, encoding transcription factor AP-2 gamma, which is involved in eye, face, body wall, limb and neural tube development [49]; and a binding site for EGR1, encoding early growth response protein 1, a gene regulator which regulates the transcription of several genes involved in early vertebrate development [50]. Three of these TFBS were also observed for the SULF2 promoter, including FOXA1, TFA2PC and EGR1, although three others were found in this region, including ESR1, encoding the estrogen receptor [51]; HNF4A, encoding hepatocyte nuclear factor 4-alpha, controlling several genes essential for the development of liver, intestine and kidney [52]; and CTCF, encoding CCCTC-binding factor, which is necessary for memory formation and for basal and experience-dependent gene regulation [53].…”