RESUMO
Expression of the mismatch repair gene MutL homolog 1 (MLH1) is silenced in a clinically important subgroup of sporadic colorectal cancers. These cancers exhibit hypermutability with microsatellite instability (MSI) and differ from microsatellite-stable (MSS) colorectal cancers in both prognosis and response to therapies. Loss of MLH1 is usually due to epigenetic silencing with associated promoter methylation; coding somatic mutations rarely occur. Here we use the presence of a colorectal cancer (CRC) risk variant (rs1800734) within the MLH1 promoter to investigate the poorly understood mechanisms of MLH1 promoter methylation and loss of expression. We confirm the association of rs1800734 with MSI+ but not MSS cancer risk in our own data and by meta-analysis. Using sensitive allele-specific detection methods, we demonstrate that MLH1 is the target gene for rs1800734 mediated cancer risk. In normal colon tissue, small allele-specific differences exist only in MLH1 promoter methylation, but not gene expression. In contrast, allele-specific differences in both MLH1 methylation and expression are present in MSI+ cancers. We show that MLH1 transcriptional repression is dependent on DNA methylation and can be reversed by a methylation inhibitor. The rs1800734 allele influences the rate of methylation loss and amount of re-expression. The transcription factor TFAP4 binds to the rs1800734 region but with much weaker binding to the risk than the protective allele. TFAP4 binding is absent on both alleles when promoter methylation is present. Thus we propose that TFAP4 binding shields the protective rs1800734 allele of the MLH1 promoter from BRAF induced DNA methylation more effectively than the risk allele.
Assuntos
Neoplasias Colorretais/genética , Proteínas de Ligação a DNA/metabolismo , Proteína 1 Homóloga a MutL/genética , Polimorfismo de Nucleotídeo Único , Regiões Promotoras Genéticas , Fatores de Transcrição/metabolismo , Alelos , Estudos de Casos e Controles , Ilhas de CpG , Metilação de DNA , Proteínas de Ligação a DNA/genética , Bases de Dados Factuais , Regulação Neoplásica da Expressão Gênica , Predisposição Genética para Doença , Humanos , Instabilidade de Microssatélites , Proteína 1 Homóloga a MutL/metabolismo , RNA Mensageiro/genética , Fatores de Transcrição/genéticaRESUMO
Members of the genus Neisseria have been isolated from or detected in a wide range of animals, from non-human primates and felids to a rodent, the guinea pig. By means of selective culture, biochemical testing, Gram staining and PCR screening for the Neisseria-specific internal transcribed spacer region of the rRNA operon, we isolated four strains of the genus Neisseria from the oral cavity of the wild house mouse, Mus musculus subsp. domesticus. The isolates are highly related and form a separate clade in the genus, as judged by tree analyses using either multi-locus sequence typing of ribosomal genes or core genes. One isolate, provisionally named Neisseria musculi sp. nov. (type strain AP2031T=DSM 101846T=CCUG 68283T=LMG 29261T), was studied further. Strain AP2031T/N. musculi grew well in vitro. It was naturally competent, taking up DNA in a DNA uptake sequence and pilT-dependent manner, and was amenable to genetic manipulation. These and other genomic attributes of N. musculi sp. nov. make it an ideal candidate for use in developing a mouse model for studying Neisseria-host interactions.
