Abstract
In prokaryotic and lower eukaryotic genomes, genes are often organized in a proximal pattern to ensure effective gene co-expression and co-regulation. Increasing evidences, however, suggest that in mammalian genomes some adjacent genes also tend to be in close physical vicinity. Two human brain disease-related genes, one coding for a serine protease inhibitor (SERPINI1) which is down-regulated in brain tumors, and the other coding for a programmed cell death gene (PDCD10) which is mutated in cerebral cavernous malformation (CCM), are adjacently located in a head-to-head configuration on chromosome 3q26.1. The two genes are only separated by as little as 851 bp. Unlike SERPINI1, which is expressed mainly in normal brain tissues but is down-regulated in brain tumors, PDCD10 is ubiquitously expressed in all normal tissues while its transcription becomes aberrant in different types of cancers. By performing the functional reporter gene analysis, a GC-rich 175-bp fragment within the intergenic region was shown to function as a bidirectional promoter to drive the transcription of the two flanking genes. In addition, we found that a non-canonical E-box element (5’-CATGCG-3’) identified within the minimal bidirectional promoter is crucial for the expression of these two genes. Deletions of this E-box either in the 175-bp minimal promoter or in the 851-bp full-length promoter of these two genes totally abolish the promoter activities in the functional reporter assays. Through the in vitro supershift experiment and the in vivo chromatin immunoprecipitation assay, we discovered that c-Myc transcription factor is able to bind to this E-box. The bidirectional promoter activities and mRNA expressions of the two genes were both affected while varying the amounts of cellular c-Myc protein. Furthermore, we discovered that the methylation of the specific C nucleotide within the E-box sequence (5’-CATGmCG-3’) would effectively interfere the binding of c-Myc to E-box. Taken together, we propose that c-Myc plays an important activator in turning on the transcription of SERPINI1 and PDCD10 and is possibly involved in differential expressions of these two flanking genes in brain tumors. On the other hand, one regulatory fragment which is from nt 176 to 473 of the intergenic region between SERPINI1 and PDCD10 possessing both the repressive activity for SERPINI1 and the enhancing activity for PDCD10 was found to coordinate the expression and regulation of both genes. Two CAAT boxes within this fragment seem to be involved in modulating the expression amount of two flanking genes. These data, along with a high degree of sequence conservation found in other species, suggest that a fine regulatory mechanism exists within this short 851-bp intergenic region. Such a mechanism should result in the asymmetrical gene expression pattern as well as the tissue specificity of SERPINI1 and PDCD10.