STK11 Core Promoter Activity, Identification Of Cis-element, And The Positive-feedback Between P53 And STK11

Peutz-Jeghers syndrome (PJS) is an autosomal dominant inheriteddisease. Patients with PJS develop benign hamartomatous polyps,especially in the gastrointestinal tract, as well as marked cutaneouspigmentation of the mucous membranes. Another key feature of PJS is agreatly increased risk of developing malignant tumors in multiple tissues.STK11/LKB1-the responsible gene of PJS was cloned in 1998. Recentworks indicate that STK11 be a master kinase associated in the regulationof p53 pathway, Wnt and AMPK signaling, which are involved in theregulation of very important cellular vital movements.STK11 was found ubiquitously expressed in human normal tissuesby Northern hybridization. Researches on model animals showed that theexpression level of STK11 is totally different. XEEK1, xenopus' Homologof STK11 was found to express only in the early stage of development,while the expression level of Mouse STK11 gene differs in differenttissues and stages in their individual developments. For example, theexpression of STK11 shows no difference at 7-11 weeks, then centralizesin tract and testis at 15-19 weeks. Further study on human tissue showsthat the expression of STK11 in embryo tissue is higher than adult tissue.There are hardly any reports referring the transcriptional controlmechanisms of STK11 up to present. However, to study the transcriptionalregulation mechanism of such important gene, which is involved in somany vital signaling pathway, is essential.We identified the STK11 core promoter previously, which is locatedwithin-1322 bp/-1160bp (define translation code as+1). To identifypotential transcription factor binding sites on STK11 promoter region, wefirst analyzed this sequence using Matlnspector2.2 software. Then, we performed site-mutated, EMSA, and over-expression assay to learn howSTK11 is transcriptionally regulated. We hope that those results willelucidate the mechanism of transcriptional regulation of STK11.Our results show that: 1. STK11 promoter could drive theexpression of GFP: The fragment-1425/-1160 could drive theexpression of GFP in HeLa cells, HUH-7 cells, HT-29 cells, L02 cells, andHEK293 cells 2. P53 has the transregulationai effect on STK11promoter: EMSA confirms that the predicted "p53 binding site" doeshave the capability to bind related nuclear protein. The Luc activity assayof mutants shows remarkably decrease compared with wild type.Theover-expression of p53 in vivo resulted in elevated level of STK11 both inmRNA and protein in L02 cells. Our results demonstrate that p53 play asignificant role in the transcriptional activation of the STK11 geneexpression. 3. Core promoter of STK11 has two Spl binding site:Computer analysis shows that the 163bp promoter of human STK11contains neither canonical TATA/CAAT box nor initiator core element.The GC-rich promoter is located in the predicted CpG island and havetwo tightly aligned "Spl-binding site". The first one was confirmed byEMSA.4. STK11 promoter has a novel transcription factor bindingsite, and that may be related to PJS: we identified a variation (G/T) inSTK11 promoter region, which is located at-1275 bp just two basepairspart from the core binding site of transcription factor Y. The frequency ofgenotype GG and TT among patients was significantly higher than thatamong the normal individuals. In present study, we confirmed that thelateral sequence aligned to variation has the capability to bind relatednuclear protein, and the binding intensity of genotype GG is higher thanthat of genotype TT. To our surprise, the complex formation betweenprobe and protein was dramatically inhibited by competitive binding ofthe wild type and mutant. And the super-shift of the DNA-protein complexes was not observed by the addition of NFY polyclonal antibody.These results suggested the promoter of STK11 have a novel cis-element,and the variation (G/T) may be related to PJ syndrome.In summary, we have characterized the promoter of human STK11gene.The results establish the basis for further research on thetranscription regulation mechanism of STK11 and the other tumorsuppressor.