Multiple Alternative Promoters Research Articles

Multiple Alternative Promoters and Alternative Splicing Enable Universal Transcription-Based Logic Computation in Mammalian Cells

Multi-input logic gene circuits can enable sophisticated control of cell function, yet large-scale synthetic circuitry in mammalian cells has relied on post-transcriptional regulation or recombinase-triggered state transitions. Large-scale transcriptional logic, on the other hand, has been challenging to implement. Inspired by a naturally found regulatory strategy of using multiple alternative promoters, followed by alternative splicing, we developed a scalable and compact platform for transcriptional OR logic using inputs to those promoters. The platform is extended to implement disjunctive normal form (DNF) computations capable of implementing arbitrary logic rules. Specifically, AND logic is implemented at individual promoters using synergistic transcriptional inputs, and NOT logic via microRNA inputs targeting unique exon sequences driven by those promoters. Together, these regulatory programs result in DNF-like logic control of output gene expression. The approach offers flexibility for building complex logic programs in mammalian cells.

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.

Selective Activation of Alternative MYC Core Promoters by Wnt-Responsive Enhancers.

In Metazoans, transcription of most genes is driven by the use of multiple alternative promoters. Although the precise regulation of alternative promoters is important for proper gene expression, the mechanisms that mediates their differential utilization remains unclear. Here, we investigate how the two alternative promoters (P1, P2) that drive MYC expression are regulated. We find that P1 and P2 can be differentially regulated across cell-types and that their selective usage is largely mediated by distal regulatory sequences. Moreover, we show that in colon carcinoma cells, Wnt-responsive enhancers preferentially upregulate transcription from the P1 promoter using reporter assays and in the context of the endogenous Wnt induction. In addition, multiple enhancer deletions using CRISPR/Cas9 corroborate the regulatory specificity of P1. Finally, we show that preferential activation between Wnt-responsive enhancers and the P1 promoter is influenced by the distinct core promoter elements that are present in the MYC promoters. Taken together, our results provide new insight into how enhancers can specifically target alternative promoters and suggest that formation of these selective interactions could allow more precise combinatorial regulation of transcription initiation.

Molecular, Enzymatic, and Cellular Characterization of Soluble Adenylyl Cyclase From Aquatic Animals.

The enzyme soluble adenylyl cyclase (sAC) is the most recently identified source of the messenger molecule cyclic adenosine monophosphate. sAC is evolutionarily conserved from cyanobacteria to human, is directly stimulated by [Formula: see text] ions, and can act as a sensor of environmental and metabolic CO2, pH, and [Formula: see text] levels. sAC genes tend to have multiple alternative promoters, undergo extensive alternative splicing, be translated into low mRNA levels, and the numerous sAC protein isoforms may be present in various subcellular localizations. In aquatic organisms, sAC has been shown to mediate various functions including intracellular pH regulation in coral, blood acid/base regulation in shark, heart beat rate in hagfish, and NaCl absorption in fish intestine. Furthermore, sAC is present in multiple other species and tissues, and sAC protein and enzymatic activity have been reported in the cytoplasm, the nucleus, and other subcellular compartments, suggesting even more diverse physiological roles. Although the methods and experimental tools used to study sAC are conventional, the complexity of sAC genes and proteins requires special considerations that are discussed in this chapter.

Abstract 1075: Multiple alternative promoters and a long non-coding RNA constitute the complex regulatory region of the cancer and drug resistance associated gene B4GALT1

Abstract Background: The B4GALT1 (β-1,4-galactosyltransferase) gene, overexpressed in human cancers is highly implicated in tumor progression and metastasis. Used as a prototype in genomic analyses, it shows significant variations in the transcript origination sites in normal and cancer tissues. Little is known on the regulation of B4GALT1 transcription and regulatory regions involved. Objectives: To characterize the alternative promoters and the regulatory elements of B4GALT1 and gain further insight into its cancer and tissue-specific transcription. Methods: The genomic criteria of the bidirectional loci, B4GALT1 and B4GALT1-AS1 (encoding a long non-coding antisense RNA; lncRNA) and their promoters were searched in the NCBI-GenBank, UCSC Genome Bioinformatics, Ensembl, TRED, EDP, PEDB and MPromDb databases to specify the map locations and identify the transcription factor binding sites (TSS), namely the TATA-8 (TATAWAWR) and TATA-532 (HWHWWWWR, excluding: HTYTTTWR, CAYTTTWR, MAMAAAAR, CTYAAAAR) elements, INR (YYANWYY), CCAAT and its inverted sequence, BRE (SSRCGCC) and DPE (RGWCGTG) sequences. Several bioinformatic tools were used to retrieve the promoter sequences, identify and flip the forward or reverse strands in search for specific sequences and quantitate the dinucleotide base-stacking energy values (Kcal/mol/dimer) indicative of the helix stability and strength of protein binding. Results: 13 alternative promoters were identified in the databases B4GALT1. Six of these resided within 1.454 kb forming a complex transcriptional regulatory unit we refer to as the TR1-PE1; this unit had all characteristics known for bidirectional transcription of divergent genes, B4GALT1 and B4GALT1-AS1. The TR1-PE1 sequence harbored CpG Island and several types of TFBSs, INR, BRE, ERE, TCCAA and TATA-532. The dinucleotide base-stacking energy along TR1-PE1 showed variable values, indicating a differential stability along the sequence. The INR sequences were found mainly along sequences with lower dinucleotide base-stacking energy values, whereas BRE sequences were observed in regions with higher values. Two highly conserved (TG)18 repeats were identified in non-overlapping B4ALT1 promoter located 9.744 kb from 3′ TR1-PE1 side, which enhance transcription especially when they are located closer to the promoter. The transcripts of B4GALT1 and B4GALT1-AS1 are of different lengths, one of B4GALT1-AS1 transcripts showed a 66% complementarity with the primary B4GALT1 transcript. Conclusions: The unique expression of the B4GALT1 in malignant and non-malignant tissues is controlled by a differential usage of alternative and bidirectional promoters along with post-transcriptional regulation by the antisense lncRNA and possible involvement of (TG)18 repeats, which in turn govern the cell-matrix interactions, malignant progression and anticancer sensitivity. Citation Format: MOHAMMED A. AlOBAIDE, Hytham Alobydi, Abdelsalam Abdelsalam, Ruiwen Zhang, Kalkunte S. Srivenugopal. Multiple alternative promoters and a long non-coding RNA constitute the complex regulatory region of the cancer and drug resistance associated gene B4GALT1. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1075. doi:10.1158/1538-7445.AM2015-1075

Evolutionarily Dynamic Alternative Splicing of GPR56 Regulates Regional Cerebral Cortical Patterning

The human neocortex has numerous specialized functional areas whose formation is poorly understood. Here, we describe a 15-base pair deletion mutation in a regulatory element of GPR56 that selectively disrupts human cortex surrounding the Sylvian fissure bilaterally including "Broca's area," the primary language area, by disrupting regional GPR56 expression and blocking RFX transcription factor binding. GPR56 encodes a heterotrimeric guanine nucleotide-binding protein (G protein)-coupled receptor required for normal cortical development and is expressed in cortical progenitor cells. GPR56 expression levels regulate progenitor proliferation. GPR56 splice forms are highly variable between mice and humans, and the regulatory element of gyrencephalic mammals directs restricted lateral cortical expression. Our data reveal a mechanism by which control of GPR56 expression pattern by multiple alternative promoters can influence stem cell proliferation, gyral patterning, and, potentially, neocortex evolution.

Identification of Novel Pretranslational Regulatory Mechanisms for NF-κB Activation

NF-κB-controlled transcriptional regulation plays a central role in inflammatory and immune responses. Currently, understanding about NF-κB activation mechanism emphasizes IκB-tethered complex inactivation in the cytoplasm. In the case of NF-κB activation, IκB phosphorylation leads to its degradation, followed by NF-κB relocation to the nucleus and trans-activation of NF-κB-targeted genes. Pretranslational mechanism mediated NF-κB activation remains poorly understood. In this study, we investigated NF-κB pretranslational regulation by performing a series of database mining analyses and using six large national experimental databases (National Center of Biotechnology Information UniGene expressed sequence tag profile database, Gene Expression Omnibus database, Transcription Element Search System database, AceView database, and Epigenomics database) and TargetScan software. We reported the following findings: 1) NF-κB-signaling genes are differentially expressed in human and mouse tissues; 2) heart and vessels are the inflammation-privileged tissues and less easy to be inflamed because lacking in key NF-κB-signaling molecular expression; 3) NF-κB-signaling genes are induced by cardiovascular disease risk factors oxidized phospholipids and proinflammatory cytokines in endothelial cells; 4) transcription factors CCAAT/enhancer-binding proteins and NF-κB have higher binding site frequencies in the promoters of proinflammatory cytokine-induced NF-κB genes; 5) most NF-κB-signaling genes have multiple alternative promoters and alternatively spliced isoforms; 6) NF-κB family genes can be regulated by DNA methylation; and 7) 27 of 38 NF-κB-signaling genes can be regulated by microRNAs. Our findings provide important insight into the mechanism of NF-κB activation, which may contribute to cardiovascular disease, inflammatory diseases, and immunological disorders.

High-fidelity promoter profiling reveals widespread alternative promoter usage and transposon-driven developmental gene expression

Many eukaryotic genes possess multiple alternative promoters with distinct expression specificities. Therefore, comprehensively annotating promoters and deciphering their individual regulatory dynamics is critical for gene expression profiling applications and for our understanding of regulatory complexity. We introduce RAMPAGE, a novel promoter activity profiling approach that combines extremely specific 5′-complete cDNA sequencing with an integrated data analysis workflow, to address the limitations of current techniques. RAMPAGE features a streamlined protocol for fast and easy generation of highly multiplexed sequencing libraries, offers very high transcription start site specificity, generates accurate and reproducible promoter expression measurements, and yields extensive transcript connectivity information through paired-end cDNA sequencing. We used RAMPAGE in a genome-wide study of promoter activity throughout 36 stages of the life cycle of Drosophila melanogaster, and describe here a comprehensive data set that represents the first available developmental time-course of promoter usage. We found that >40% of developmentally expressed genes have at least two promoters and that alternative promoters generally implement distinct regulatory programs. Transposable elements, long proposed to play a central role in the evolution of their host genomes through their ability to regulate gene expression, contribute at least 1300 promoters shaping the developmental transcriptome of D. melanogaster. Hundreds of these promoters drive the expression of annotated genes, and transposons often impart their own expression specificity upon the genes they regulate. These observations provide support for the theory that transposons may drive regulatory innovation through the distribution of stereotyped cis-regulatory modules throughout their host genomes.

From gene expression patterns to cis-regulatory elements: the cREMaG database

Event Abstract Back to Event From gene expression patterns to cis-regulatory elements: the cREMaG database Marcin Piechota1*, Michal Korostynski1 and Ryszard Przewlocki1 1 Institute of Pharmacology PAS, Poland A growing number of brain derived gene expression profiling data provides a reliable source of information about gene co-expression. The analysis of the common properties of the promoters of co-expressed genes facilitates identification of transcription factors (TFs) involved in their co-regulation. We introduce the cREMaG (cis-Regulatory Elements in the Mammalian Genome) system designed for in silico studies of the promoter properties of co-regulated mammalian genes. The cREMaG system offers an analysis of data obtained from human, mouse and rat brain derived gene expression profiling studies. The system allows for the identification of over-represented transcription factor binding sites in the promoter regions of genes from queried gene-sets. The unique features of cREMaG include: multiple alternative promoters selection for analysis, equal number of TFBSs per gene, optimized level of restrictiveness of transcription factor binding site matrix score threshold, three distinct pre-compiled backgrounds and CPG islands analysis. To demonstrate the functionalities of cREMaG we utilized the results from gene expression profiling in the striatum of mice lacking CREB in the forebrain, accompanied by deletion of CREM, treated with cocaine. The cREMaG constitutes a valuable resource for all researchers working with gene expression data. The resource is freely available at http://bioputer.mimuw.edu.pl/~if-pan/cREMaG/. Conference: Neuroinformatics 2009, Pilsen, Czechia, 6 Sep - 8 Sep, 2009. Presentation Type: Oral Presentation Topic: Genomics and genetics Citation: Piechota M, Korostynski M and Przewlocki R (2019). From gene expression patterns to cis-regulatory elements: the cREMaG database. Front. Neuroinform. Conference Abstract: Neuroinformatics 2009. doi: 10.3389/conf.neuro.11.2009.08.038 Copyright: The abstracts in this collection have not been subject to any Frontiers peer review or checks, and are not endorsed by Frontiers. They are made available through the Frontiers publishing platform as a service to conference organizers and presenters. The copyright in the individual abstracts is owned by the author of each abstract or his/her employer unless otherwise stated. Each abstract, as well as the collection of abstracts, are published under a Creative Commons CC-BY 4.0 (attribution) licence (https://creativecommons.org/licenses/by/4.0/) and may thus be reproduced, translated, adapted and be the subject of derivative works provided the authors and Frontiers are attributed. For Frontiers’ terms and conditions please see https://www.frontiersin.org/legal/terms-and-conditions. Received: 21 May 2009; Published Online: 09 May 2019. * Correspondence: Marcin Piechota, Institute of Pharmacology PAS, Cracow, Poland, marpiech@if-pan.krakow.pl Login Required This action requires you to be registered with Frontiers and logged in. To register or login click here. Abstract Info Abstract The Authors in Frontiers Marcin Piechota Michal Korostynski Ryszard Przewlocki Google Marcin Piechota Michal Korostynski Ryszard Przewlocki Google Scholar Marcin Piechota Michal Korostynski Ryszard Przewlocki PubMed Marcin Piechota Michal Korostynski Ryszard Przewlocki Related Article in Frontiers Google Scholar PubMed Abstract Close Back to top Javascript is disabled. Please enable Javascript in your browser settings in order to see all the content on this page.

Alternative promoter usage and alternative splicing contribute to mRNA heterogeneity of mouse monocarboxylate transporter 2

Expression patterns of monocarboxylate transporter 2 (MCT2) display mRNA diversity in a tissue-specific fashion. We cloned and characterized multiple mct2 5'-cDNA ends from the mouse and determined the structural organization of the mct2 gene. We found that transcription of this gene was initiated from five independent genomic regions that spanned >80 kb on chromosome 10, resulting in five unique exon 1 variants (exons 1a, 1b, 1c, 1d, and 1e) that were then spliced to the common exon 2. Alternative splicing of four internal exons (exons AS1, AS2, AS3, and exon 3) greatly increased the complexity of mRNA diversity. While exon 1c was relatively commonly used for transcription initiation in various tissues, other exon 1 variants were used in a tissue-specific fashion, especially exons 1b and 1d that were used exclusively for testis-specific expression. Sequence analysis of 5'-flanking regions upstream of exons 1a, 1b, and 1c revealed the presence of numerous potential binding sites for ubiquitous transcription factors in all three regions and for transcription factors implicated in testis-specific or hypoxia-induced gene expression in the 1b region. Transient transfection assays demonstrated that each of the three regions contained a functional promoter and that the in vitro, cell type-specific activities of these promoters were consistent with the tissue-specific expression pattern of the mct2 gene in vivo. These results indicate that tissue-specific expression of the mct2 gene is controlled by multiple alternative promoters and that both alternative promoter usage and alternative splicing contribute to the remarkable mRNA diversity of the gene.

OPOSSUM: integrated tools for analysis of regulatory motif over-representation

The identification of over-represented transcription factor binding sites from sets of co-expressed genes provides insights into the mechanisms of regulation for diverse biological contexts. oPOSSUM, an internet-based system for such studies of regulation, has been improved and expanded in this new release. New features include a worm-specific version for investigating binding sites conserved between Caenorhabditis elegans and C. briggsae, as well as a yeast-specific version for the analysis of co-expressed sets of Saccharomyces cerevisiae genes. The human and mouse applications feature improvements in ortholog mapping, sequence alignments and the delineation of multiple alternative promoters. oPOSSUM2, introduced for the analysis of over-represented combinations of motifs in human and mouse genes, has been integrated with the original oPOSSUM system. Analysis using user-defined background gene sets is now supported. The transcription factor binding site models have been updated to include new profiles from the JASPAR database. oPOSSUM is available at http://www.cisreg.ca/oPOSSUM/