Regulation of the Human MSH6 Gene by the Sp1 Transcription Factor and Alteration of Promoter Activity and Expression by Polymorphisms

Abstract

Defects in human DNA mismatch repair have been reported to underlie a variety of hereditary and sporadic cancer cases. We characterized the structure of the MSH6 promoter region to examine the mechanisms of transcriptional regulation of the MSH6 gene. The 5-flanking region of the MSH6 gene was found to contain seven functional Sp1 transcription factor binding sites that each bind Sp1 and Sp3 and contribute to promoter activity. Transcription did not appear to require a TATA box and resulted in multiple start sites, including two major start sites and at least nine minor start sites. Three common polymorphisms were identified in the promoter region (؊557 T3G, ؊448 G3A, and ؊159 C3T): the latter two were always associated, and each of these functionally inactivated a different Sp1 site. The polymorphic allele ؊448 A ؊159 T was demonstrated to be a common Caucasian polymorphism found in 16% of Caucasians and resulted in a five-Sp1-site promoter that had 50% less promoter activity and was more sensitive to inactivation by DNA methylation than the more common seven Sp1 site promoter allele, which was only partially inactivated by DNA methylation. In cell lines, this five-Sp1-site polymorphism resulted in reduced MSH6 expression at both the mRNA and protein level. An additional 2% of Caucasians contained another polymorphism, ؊210 C3T, which inactivated a single Sp1 site that also contributes to promoter activity.The human DNA mismatch repair (MMR) system functions to repair mispaired bases in DNA that result from DNA replication errors and thereby prevents the accumulation of mutations due to such replication errors. Biochemical and genetic studies have identified a number of mismatch repair proteins involved in this system, including those encoded by the MSH2, MSH3, MSH6, MLH1, MLH3, PMS2, and EXO1 genes, as well as the replication proteins PCNA, RFC, RPA, and DNA polymerase delta (for reviews, see references 24 and 33). Loss of MMR function is associated with both inherited cancer susceptibility and the development of sporadic tumors. Inherited mutations in MSH2 and MLH1 are the most prevalent cause of hereditary nonpolyposis colorectal carcinoma (HNPCC) (for a review, see reference 52), and epigenetic silencing of MLH1 has been found to underlie most MMR defective sporadic cancer cases (14,25,31,46,47). Inherited mutations in MSH6 have been found in a small proportion (0 to 3%) of HNPCC families and appear to underlie a higher proportion for familial colorectal cancer cases that show later onset and a less pronounced family history than HNPCC (6,34,45,65,67,68). Mutations in PMS2 have been found in patients with Turcots syndrome but are only rarely found in patients with HNPCC (11, 39, 64, 66). Whether or not mutations in MLH3 or EXO1 underlie a significant proportion of HNPCC is unclear (2,29,38,69,70).In the human MMR system, two heterodimeric complexes-MSH2-MSH6 (MutS␣) MSH2-MSH3 (MutS␤)-function to recognize mispaired bases in DNA (1,19,21,49,61). MutS␣ appears to function in the repair of base-base and insert...

The MLH1 D132H variant is associated with susceptibility to sporadic colorectal cancer

“…Similarly, promoter polymorphisms that cause a partial reduction in MMR gene transcription may contribute to sporadic CRC 18 .…”

Novel Functional Single Nucleotide Polymorphisms in the Latent Transforming Growth Factor-β Binding Protein-1L Promoter

“…[35][36][37][38][39] Luciferase reporter assays revealed that the G-A haplotype caused transcriptional activation more efficiently than other haplotypes. The LTBP-1L promoter has a high GC content, but lacks a TATA box.…”

Association of MSH6 mutation with glioma susceptibility, drug resistance and progression

“…The MSH6 gene may be transcribed from a housekeeping gene promoter (10,12). The MSH6 gene contains no TATAA-or CAAT-boxes in its translation initiation site, which is surrounded by sequences containing CpG islands.…”

“…There are multiple start sites for transcription and copies of consensus-binding sequences of the transcription factors, including specificity protein (Sp)1, ETF, meta-regulatory transcription factor-1 and activator protein-1. The gene contains seven functional Sp1 transcription-factor binding sites in the 5'-flanking region, which bind Sp1 and Sp3 and regulate MSH6 promoter activity (12).…”

“…Such patients are also more susceptible to a wide spectrum of tumors, even more than to colon cancer, including tumors of the rectum, endometrium, small bowel, urinary tract and brain (17,18). Unlike early-onset HNPCC driven by MSH2 and MLH1 mutations, familial HNPCC driven by MSH6 mutations is associated with a later onset (9,10,12,19).…”