Molecular mechanisms of human single-minded 2 (SIM2) gene expression: identification of a promoter site in the SIM2 genomic sequence

We previously postulated that the single-minded 2 (SIM2) gene identified on the human chromosome 21q22.2 is a good candidate gene for the pathogenesis of mental retardation in Down syndrome because its mouse homolog exhibits preferential expression in the mouse diencephalon during early embryogenesi s. We analyzed the genomic sequence of the entire SIM2 gene which consists of ii exons and spans over 50 kb. As a step toward understanding the molecu lar mechanisms of SIM2 gene expression, we have analyzed the human SIM2 gen e expression in nine established human cell lines. Three transcripts of 3.6 , 4.4. and 6.0 kb were detected in the glioblastoma cell line, T98G, neurob lastoma cell line. TGW, and transformed embryonic kidney cell line, 293. Th e RACE analysis using SIM2-expressing human cell line T98G provided evidenc e for the transcription start site at similar to1.2 kb upstream of the tran slation initiation site. The transfection assay using various deletion cons tructs with reporter gene suggested the presence of a presumptive promoter region. Transient transfection assay in T98G cell line revealed a significa nt promoter activity located in the 60 bp sequence between nt - 1385 and - 1325 upstream region of the translation initiation site. This 60 bp sequenc e contains cis-elements for c-myb, E47 and E2F transcription factors. Moreo ver, the gel retardation assay using oligo-DNA of various cia-element seque nces indicated the presence of protein factor(s) which bind to the cis-elem ent for c-myb. These results suggested that binding of a protein transcript ion factor(s) such as c-myb or that alike regulates transcription of the SI M2 gene by binding to a small upstream region. (C) 2001 Elsevier Science B. V. All rights reserved.