Differential regulation of CpG island methylation within divergent and unidirectional promoters in colorectal cancer

The silencing of tumor suppressor genes by promoter CpG island (CGI) methylation isan important cause of oncogenesis. Silencing of MLH1 and BRCA1, two examples ofoncogenic events, results from promoter CGI methylation. Interestingly, both MLH1and BRCA1 have a divergent promoter, from which another gene on the oppositestrand is also transcribed. Although studies have shown that divergent transcriptionis an important factor in transcriptional regulation, little is known about its implicationin aberrant promoter methylation in cancer. In this study, we analyzed the methylationstatus of CGI in divergent promoters using a recently enriched transcriptomedatabase. We measured the extent of CGI methylation in 119 colorectal cancer (CRC)clinical samples (65 microsatellite instability high [MSI-H]CRC with CGI methylatorphenotype, 28 MSI-HCRC without CGI methylator phenotype and 26 microsatellitestable CRC) and 21 normal colorectal tissues using Infinium MethylationEPICBeadChip. We found that CGI within divergent promoters are less frequently methylatedthan CGI within unidirectional promoters in normal cells. In the genome of CRCcells, CGI within unidirectional promoters are more vulnerable to aberrant methylationthan CGI within divergent promoters. In addition, we identified three DNA sequencemotifs that correlate with methylated CGI. We also showed that methylatedCGI are associated with genes whose expression is low in normal cells. Thus, we hereprovide fundamental observations regarding the methylation of divergent promotersthat are essential for the understanding of carcinogenesis and development of cancerprevention strategies.

Identification of the binding site of the σ 54 hetero-oligomeric FleQ/FleT activator in the flagellar promoters of Rhodobacter sphaeroides

Expression of the flagellar genes in Rhodobacter sphaeroides is dependent on one of the four sigma-54 factors present in this bacterium and on the enhancer binding proteins (EBPs) FleQ and FleT. These proteins, in contrast to other well-characterized EBPs, carry out activation as a hetero-oligomeric complex. To further characterize the molecular properties of this complex we mapped the binding sites or upstream activation sequences (UASs) of six different flagellar promoters. In most cases the UASs were identified at approximately 100 bp upstream from the promoter. However, the activity of the divergent promoters flhAp-flgAp, which are separated by only 53 bp, is mainly dependent on a UAS located approximately 200 bp downstream from each promoter. Interestingly, a significant amount of activation mediated by the upstream or contralateral UAS was also detected, suggesting that the architecture of this region is important for the correct regulation of these promoters. Sequence analysis of the regions carrying the potential FleQ/FleT binding sites revealed a conserved motif. In vivo footprinting experiments with the motAp promoter allowed us to identify a protected region that overlaps with this motif. These results allow us to propose a consensus sequence that represents the binding site of the FleQ/FleT activating complex.

Analysis of the promoter activities of the genes encoding three quinoprotein alcohol dehydrogenases in Pseudomonas putida HK5

The transcriptional regulation of three distinct alcohol oxidation systems, alcohol dehydrogenase (ADH)-I, ADH-IIB and ADH-IIG, in Pseudomonas putida HK5 was investigated under various induction conditions. The promoter activities of the genes involved in alcohol oxidation were determined using a transcriptional lacZ fusion promoter-probe vector. Ethanol was the best inducer for the divergent promoters of qedA and qedC, encoding ADH-I and a cytochrome c, respectively. Primary and secondary C3 and C4 alcohols and butyraldehyde specifically induced the divergent promoters of qbdBA and aldA, encoding ADH-IIB and an NAD-dependent aldehyde dehydrogenase, respectively. The qgdA promoter of ADH-IIG responded well to (S)-(+)-1,2-propanediol induction. In addition, the roles of genes encoding the response regulators exaE and agmR, located downstream of qedA, were inferred from the properties of exaE- or agmR-disrupted mutants and gene complementation tests. The gene products of both exaE and agmR were strictly necessary for qedA transcription. The mutation and complementation studies also suggested a role for AgmR, but not ExaE, in the transcriptional regulation of qbdBA (ADH-IIB) and qgdA (AGH-IIG). A hypothetical scheme describing a regulatory network, which directs expression of the three distinct alcohol oxidation systems in P. putida HK5, was derived.

Regulation of the yjjQ-bglJ Operon, Encoding LuxR-Type Transcription Factors, and the Divergent yjjP Gene by H-NS and LeuO

ABSTRACT The yjjQ and bglJ genes encode LuxR-type transcription factors conserved in several enterobacterial species. YjjQ is a potential virulence factor in avian pathogenic Escherichia coli. BglJ counteracts the silencing of the bgl (β-glucoside) operon by H-NS in E. coli K-12. Here we show that yjjQ and bglJ form an operon carried by E. coli K-12, whose expression is repressed by the histone-like nucleoid structuring (H-NS) protein. The LysR-type transcription factor LeuO counteracts this repression. Furthermore, the yjjP gene, encoding a membrane protein of unknown function and located upstream in divergent orientation to the yjjQ-bglJ operon, is likewise repressed by H-NS. Mapping of the promoters as well as the H-NS and LeuO binding sites within the 555-bp intergenic region revealed that H-NS binds to the center of the AT-rich regulatory region and distal to the divergent promoters. LeuO sites map to the center and to positions distal to the yjjQ promoters, while one LeuO binding site overlaps with the divergent yjjP promoter. This latter LeuO site is required for full derepression of the yjjQ promoters. The arrangement of regulatory sites suggests that LeuO restructures the nucleoprotein complex formed by H-NS. Furthermore, the data support the conclusion that LeuO, whose expression is likewise repressed by H-NS and which is a virulence factor in Salmonella enterica, is a master regulator that among other loci, also controls the yjjQ-bglJ operon and thus indirectly the presumptive targets of YjjQ and BglJ.

RegA Control of Bacteriochlorophyll and Carotenoid Synthesis in Rhodobacter capsulatus

ABSTRACT We provide in vivo genetic and in vitro biochemical evidence that RegA directly regulates bacteriochlorophyll and carotenoid biosynthesis in Rhodobacter capsulatus. β-Galactosidase expression assays with a RegA-disrupted strain containing reporter plasmids for Mg-protoporphyrin IX monomethyl ester oxidative cyclase (bchE), Mg-protoporphyrin IX chelatase (bchD), and phytoene dehydrogenase (crtI) demonstrate RegA is responsible for fourfold anaerobic induction of bchE, threefold induction of bchD, and twofold induction of crtI. Promoter mapping studies, coupled with DNase I protection assays, map the region of RegA binding to three sites in the bchE promoter region. Similar studies at the crtA and crtI promoters indicate that RegA binds to a single region equidistant from these divergent promoters. These results demonstrate that RegA is directly responsible for anaerobic induction of bacteriochlorophyll biosynthesis genes bchE, bchD, bchJ, bchI, bchG, and bchP and carotenoid biosynthesis genes crtI, crtB, and crtA.