Kynurenine 3-Monooxygenase Gene Associated With Nicotine Initiation and Addiction: Analysis of Novel Regulatory Features at 5' and 3'-Regions.

Tobacco smoking is widespread behavior in Qatar and worldwide and is considered one of the major preventable causes of ill health and death. Nicotine is part of tobacco smoke that causes numerous health risks and is incredibly addictive; it binds to the α7 nicotinic acetylcholine receptor (α7nAChR) in the brain. Recent studies showed α7nAChR involvement in the initiation and addiction of smoking. Kynurenic acid (KA), a significant tryptophan metabolite, is an antagonist of α7nAChR. Inhibition of kynurenine 3-monooxygenase enzyme encoded by KMO enhances the KA levels. Modulating KMO gene expression could be a useful tactic for the treatment of tobacco initiation and dependence. Since KMO regulation is still poorly understood, we aimed to investigate the 5' and 3'-regulatory factors of KMO gene to advance our knowledge to modulate KMO gene expression. In this study, bioinformatics methods were used to identify the regulatory sequences associated with expression of KMO. The displayed differential expression of KMO mRNA in the same tissue and different tissues suggested the specific usage of the KMO multiple alternative promoters. Eleven KMO alternative promoters identified at 5'-regulatory region contain TATA-Box, lack CpG Island (CGI) and showed dinucleotide base-stacking energy values specific to transcription factor binding sites (TFBSs). The structural features of regulatory sequences can influence the transcription process and cell type-specific expression. The uncharacterized LOC105373233 locus coding for non-coding RNA (ncRNA) located on the reverse strand in a convergent manner at the 3'-side of KMO locus. The two genes likely expressed by a promoter that lacks TATA-Box harbor CGI and two TFBSs linked to the bidirectional transcription, the NRF1, and ZNF14 motifs. We identified two types of microRNA (miR) in the uncharacterized LOC105373233 ncRNA, which are like hsa-miR-5096 and hsa-miR-1285-3p and can target the miR recognition element (MRE) in the KMO mRNA. Pairwise sequence alignment identified 52 nucleotides sequence hosting MRE in the KMO 3' UTR untranslated region complementary to the ncRNA LOC105373233 sequence. We speculate that the identified miRs can modulate the KMO expression and together with alternative promoters at the 5'-regulatory region of KMO might contribute to the development of novel diagnostic and therapeutic algorithm for tobacco smoking.

Multifaceted roles of 5'-regulatory region of the cancer associated gene B4GALT1 and its comparison with the gene family.

ß1,4-Galactosylransferases are a family of enzymes encoded by seven B4GALT genes and are involved in the development of anticancer drug resistance and metastasis. Among these genes, the B4GALT1 shows significant variations in the transcript origination sites in different cell types/tissues and encodes an interesting dually partitioning ß-1, 4-galactosyltransferase protein. We identified at 5'-end of B4GALT1 a 1.454 kb sequence forming a transcription regulatory region, referred to by us as the TR1-PE1, had all characteristics of a bidirectional promoter directing the transcription of B4GALT1 in a divergent manner along with its long non-coding RNA (lncRNA) antisense counterpart B4GALT1-AS1. The TR1-PE1 showed unique dinucleotide base-stacking energy values specific to transcription factor binding sites (TFBSs), INR and BRE, and harbored CpG Island (CGI) that showed GC skew with potential for R-loop formation at the transcription starting sites (TSSs). The 5'-regulatory axis of B4GALT1 also included five more novel TFBSs for CTCF, GLI1, TCF7L2, GATA3 and SOX5, in addition to unique (TG)18 repeats in conjunction with 22 nucleotide TG-associated sequence (TGAS). The five lncRNA B4GALT1-AS1 transcripts showed significant complementarity with B4GALT1 mRNA. In contrast, the rest of B4GALT genes showed fewer lncRNAs, and all lacked the (TG)18 and TGAS. Our results are strongly supported by the FANTOM5 study which showed tissue-specific variations in transcript origination sites for this gene. We suggest that the unique expression patterns for the B4GALT1 in normal and malignant tissues are controlled by a differential usage of 5'-B4GALT1 regulatory units along with a post-transcriptional regulation by the antisense RNA, which in turn govern the cell-matrix interactions, neoplastic progression, anticancer drug sensitivity, and could be utilized in personalized therapy.

Characterization of regulatory sequences in alternative promoters of hypermethylated genes associated with tumor resistance to cisplatin.

The development of cisplatin resistance in human cancers is controlled by multiple genes and leads to therapeutic failure. Hypermethylation of specific gene promoters is a key event in clinical resistance to cisplatin. Although the usage of multiple promoters is frequent in the transcription of human genes, the role of alternative promoters and their regulatory sequences have not yet been investigated in cisplatin resistance genes. In a new approach, we hypothesized that human cancers exploit the specific transcription factor-binding sites (TFBS) and CpG islands (CGIs) located in the alternative promoters of certain genes to acquire platinum drug resistance. To provide a useful resource of regulatory elements associated with cisplatin resistance, we investigated the TFBS and CGIs in 48 alternative promoters of 14 hypermethylated cisplatin resistance genes previously reported. CGIs prone to methylation were identified in 28 alternative promoters of 11 hypermethylated genes. The majority of alternative promoters harboring CGIs (93%) were clustered in one phylogenetic subclass, whereas the ones lacking CGIs were distributed in two unrelated subclasses. Regulatory sequences, initiator and TATA-532 prevailed over TATA-8 and were found in all the promoters. B recognition element (BRE) sequences were present only in alternative promoters harboring CGIs, but CCAAT and TAACC were found in both types of alternative promoters, whereas downstream promoter element sequences were significantly less frequent. Therefore, it was hypothesized that BRE and CGI sequences co-localized in alternative promoters of cisplatin resistance genes may be used to design molecular markers for drug resistance. A more extensive knowledge of alternative promoters and their regulatory elements in clinical resistance to cisplatin is likely to usher novel avenues for sensitizing human cancers to treatment.

Unconventional P-35S sequence identified in genetically modified maize.

The Cauliflower Mosaic Virus 35S promoter sequence, CaMV P-35S, is one of several commonly used genetic targets to detect genetically modified maize and is found in most GMOs. In this research we report the finding of an alternative P-35S sequence and its incidence in GM maize marketed in Jordan. The primer pair normally used to amplify a 123 bp DNA fragment of the CaMV P-35S promoter in GMOs also amplified a previously undetected alternative sequence of CaMV P-35S in GM maize samples which we term V3. The amplified V3 sequence comprises 386 base pairs and was not found in the standard wild-type maize, MON810 and MON 863 GM maize. The identified GM maize samples carrying the V3 sequence were found free of CaMV when compared with CaMV infected brown mustard sample. The data of sequence alignment analysis of the V3 genetic element showed 90% similarity with the matching P-35S sequence of the cauliflower mosaic virus isolate CabbB-JI and 99% similarity with matching P-35S sequences found in several binary plant vectors, of which the binary vector locus JQ693018 is one example. The current study showed an increase of 44% in the incidence of the identified 386 bp sequence in GM maize sold in Jordan's markets during the period 2009 and 2012.