Genomic structure, transcriptional control, and tissue distribution of HERG1 and KCNQ1 genes

The long QT syndrome genes human ether-a-go-go-related gene ( HERG1) and voltage-gated K+ channel, KQT-like subfamily, member 1, gene ( KCNQ1), encoding K+ channels critical to the repolarization rate and repolarization reserve in cardiac cells, and thereby the likelihood of arrhythmias, are both composed of two isoforms: HERG1a and HERG1b and KCNQ1a and KCNQ1b, respectively. Expression of these genes is dynamic, depending on the differentiation status and disease states. We identified their core promoter regions and transcription start sites. Our data suggest that HERG1a and HERG1b, and KCNQ1a and KCNQ1b, represent independent transcripts instead of being alternatively spliced variants of the same gene, for they each have their own transcription start sites and their own promoter regions. We obtained data pointing to the potential role of stimulating protein 1 (Sp1) in the transactivation of these genes. We compared expression profiling of these genes across a variety of human tissues. Consistent with the general lack of cis elements for cardiac-specific transcription factors and the presence of multiple sites for ubiquitous Sp1 sites in the core promoter regions of HERG1a/HERG1b and KCNQ1a/KCNQ1b genes, the transcripts demonstrated widespread distribution across a variety of human tissues. We further revealed that the mRNA levels of all HERG1 and KCNQ1 isoforms were asymmetrically distributed within the heart, being more abundant in the right atria and ventricles relative to the left atria and ventricles. These findings open up an opportunity for studying interventricular gradients of slow and rapid delayed rectifier K+ current and of cardiac repolarization as well. Our study might help us understand the molecular mechanisms for arrhythmias since heterogeneity of ion channel activities is an important substrate for arrhythmogenesis.

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SFPQ, a multifunctional nuclear protein, regulates the transcription of PDE3A

Bioscience Reports ◽

10.1042/bsr20170975 ◽

2017 ◽

Vol 37 (4) ◽

Cited By ~ 2

Author(s):

Dong Keun Rhee ◽

Steven C. Hockman ◽

Sunkyung Choi ◽

Yong-Eun Kim ◽

Chungoo Park ◽

...

Keyword(s):

Hela Cells ◽

Molecular Mechanisms ◽

Regulatory Region ◽

Mrna Levels ◽

Transcription Start ◽

Transcription Start Sites ◽

Chip Sequencing ◽

Variable Expression ◽

Adenocarcinoma Cells ◽

Rapid Amplification

Phosphodiesterase 3A (PDE3A), a member of the cGMP-inhibited cyclic nucleotide phosphodiesterase (PDE) family, plays important roles in oocyte maturation and vascular smooth muscle cell proliferation. However, the molecular mechanisms that regulate PDE3A gene expression remain largely unknown. In the present study, we investigated the transcriptional regulation of PDE3A, and found that the splicing factor proline- and glutamine-rich (SFPQ) protein modulated PDE3A mRNA levels. Multiple transcription start sites (TSS1, 2, and 3) were identified within the first exon of PDE3A using 5′-rapid amplification of cDNA ends (RACE). Variable expression levels of three PDE3A variants were also observed in human tissues and HeLa cells. Several putative SFPQ-binding sites were identified upstream of the regulatory region of PDE3A-TSSs using ChIP sequencing (ChIP-seq). Serum-induced PDE3A expression was affected by increasing the amount of SFPQ binding to the upstream regulatory region of PDE3A. In addition, transcription of PDE3A was lower in human cervical adenocarcinoma cells compared with normal cervical tissue. Furthermore, overexpression of PDE3A induced sensitivity to anticancer therapeutic agent, 6-(4-(diethylamino)-3-nitrophenyl)-5-methyl-4,5-dihydropyridazin-3(2H)-one (DNMDP), in HeLa cells. Taken together, these results suggest that SFPQ functions as a transcriptional activator of PDE3A, which is involved in the regulation of DNMDP sensitivity, offering a novel molecular target for the development of anticancer therapies.

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Global analysis of transcription start sites in the new ovine reference genome (Oar rambouillet v1.0)

10.1101/2020.07.06.189480 ◽

2020 ◽

Cited By ~ 2

Author(s):

Mazdak Salavati ◽

Alex Caulton ◽

Richard Clark ◽

Iveta Gazova ◽

Timothy P. L. Smith ◽

...

Keyword(s):

Reference Genome ◽

Expression Profiles ◽

Global Analysis ◽

Integrative Genomics ◽

Sequencing Data ◽

Transcription Start ◽

Promoter Regions ◽

Tissue Samples ◽

Transcription Start Sites ◽

Transcript Regulation

AbstractThe overall aim of the Ovine FAANG project is to provide a comprehensive annotation of the new highly contiguous sheep reference genome sequence (Oar rambouillet v1.0). Mapping of transcription start sites (TSS) is a key first step in understanding transcript regulation and diversity. Using 56 tissue samples collected from the reference ewe Benz2616 we have performed a global analysis of TSS and TSS- Enhancer clusters using Cap Analysis Gene Expression (CAGE) sequencing. CAGE measures RNA expression by 5’ cap-trapping and has been specifically designed to allow the characterization of TSS within promoters to single-nucleotide resolution. We have adapted an analysis pipeline that uses TagDust2 for clean-up and trimming, Bowtie2 for mapping, CAGEfightR for clustering and the Integrative Genomics Viewer (IGV) for visualization. Mapping of CAGE tags indicated that the expression levels of CAGE tag clusters varied across tissues. Expression profiles across tissues were validated using corresponding polyA+ mRNA-Seq data from the same samples. After removal of CAGE tags with < 10 read counts, 39.3% of TSS overlapped with 5’ ends of 31,113 transcripts that had been previously annotated by NCBI (out of a total of 56,308 from the NCBI annotation). For 25,195 of the transcripts, previously annotated by NCBI, no TSS meeting stringent criteria were identified. A further 14.7% of TSS mapped to within 50bp of annotated promoter regions. Intersecting these predicted TSS regions with annotated promoter regions (±50bp) revealed 46% of the predicted TSS were ‘novel’ and previously un-annotated. Using whole genome bisulphite sequencing data from the same tissues we were able to determine that a proportion of these ‘novel’ TSS were hypo-methylated (32.2%) indicating that they are likely to be reproducible rather than ‘noise’. This global analysis of TSS in sheep will significantly enhance the annotation of gene models in the new ovine reference assembly. Our analyses provide one of the highest resolution annotations of transcript regulation and diversity in a livestock species to date.

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Multiple transcription start sites, DNase I-hypersensitive sites, and an opposite-strand exon in the 5' region of the CHO dhfr gene

Molecular and Cellular Biology ◽

10.1128/mcb.6.2.425-440.1986 ◽

1986 ◽

Vol 6 (2) ◽

pp. 425-440

Author(s):

P J Mitchell ◽

A M Carothers ◽

J H Han ◽

J D Harding ◽

E Kas ◽

...

Keyword(s):

Cho Cells ◽

Dnase I ◽

Mrna Levels ◽

Dhfr Gene ◽

Transcription Start ◽

Transcription Start Sites ◽

Dnase I Hypersensitive Sites ◽

Translation Start ◽

Hypersensitive Sites ◽

Opposite Strand

Transcription of the 26-kilobase (kb) dihydrofolate reductase (dhfr) gene in CHO cells is initiated at two sites: a major site (approximately 85% of the dhfr mRNA) at -63 relative to the translation start and a minor site (approximately 15%) at -107. Transcription also occurs from the opposite DNA strand in the dhfr 5' region, with a probable initiation site at approximately -195 relative to the dhfr translation start. A 4-kb polyadenylated RNA that is derived from the opposite-strand transcription increases threefold in abundance after serum starvation of CHO cells for 24 h. dhfr mRNA levels do not change during this time. The first dhfr exon lies within a 1-kb genomic region marked by exceptionally high G + C content and lack of DNA methylation. This region also includes a 214-base-pair (bp) exon for the opposite-strand transcript and five of the six DNase I-hypersensitive sites identified at the dhfr locus. Analysis of the DNA sequences of hamster, human (M. Chen, T. Shimada, A. D. Moulton, A. Cline, R. K. Humphries, J. Maizel, and A. W. Nienhuis, J. Biol. Chem. 259:3933-3943, 1984), and mouse (M. McGrogan, C. C. Simonsen, D. T. Smouse, P. J. Farnham, and R. T. Schimke, J. Biol. Chem. 260:2307-2314, 1985) dhfr genes reveals the presence of a 29-bp unit that is conserved 45 to 49 bp upstream of major and minor dhfr transcription start sites. This unit follows the consensus: GRGGCGGTGGCCTNNNNTGTCRCAARTRGGTR. The 5' part of the 29-bp unit contains a GC box that agrees with the GGGCGG consensus-binding site for the RNA polymerase II transcription factor Sp1 (D. Gidoni, W. A. Dynan, and R. Tjian, Nature (London) 312:409-413, 1984). Each of the three mammalian dhfr genes has several G-rich GC boxes proximal to the major dhfr transcription start site and several GC boxes of the opposite orientation (C rich) in a distal region about 500 bp upstream.

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Species-specific 5′-genomic structure and multiple transcription start sites in the chicken Pax5 gene

Gene ◽

10.1016/j.gene.2011.01.008 ◽

2011 ◽

Vol 477 (1-2) ◽

pp. 24-31 ◽

Cited By ~ 8

Author(s):

Toshitsugu Fujita ◽

Hodaka Fujii

Keyword(s):

Genomic Structure ◽

Transcription Start ◽

Transcription Start Sites ◽

Species Specific ◽

Pax5 Gene

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Molecular mechanisms underlying K+ current downregulation in canine tachycardia-induced heart failure

AJP Heart and Circulatory Physiology ◽

10.1152/ajpheart.00320.2004 ◽

2005 ◽

Vol 288 (6) ◽

pp. H2887-H2896 ◽

Cited By ~ 63

Author(s):

Fadi G. Akar ◽

Richard C. Wu ◽

George J. Juang ◽

Yanli Tian ◽

Mirka Burysek ◽

...

Keyword(s):

Heart Failure ◽

Molecular Mechanisms ◽

Canine Model ◽

Delayed Rectifier ◽

Interacting Protein ◽

Altered Expression ◽

Protein Levels ◽

Delayed Rectifier Current ◽

Repolarization Reserve ◽

Genes Encoding

Heart failure (HF) is characterized by marked prolongation of action potential duration and reduction in cellular repolarization reserve. These changes are caused in large part by HF-induced K+ current downregulation. Molecular mechanisms underlying these changes remain unclear. We determined whether downregulation of K+ currents in a canine model of tachycardia-induced HF is caused by altered expression of underlying K+ channel α- and β-subunits encoding these currents. K+ channel subunit expression was quantified in normal and failing dogs at the mRNA and protein levels in epicardial (Epi), midmyocardial (Mid), and endocardial (Endo) layers of left ventricle. Analysis of mRNA and protein levels of candidate genes encoding the transient outward K+ current ( Ito) revealed marked reductions in canine cKv4.3 expression in HF in Epi (44% mRNA, 39% protein), Mid (52% mRNA, 34% protein), and Endo (49% mRNA, 73% protein) layers and a paradoxical enhancement (41% Epi, 97% Mid, 113% Endo) in cKv1.4 protein levels, without significant changes in Kv channel-interacting protein cKChIP2 expression. Expression of cKir2.1, the gene underlying inward rectifier K+ current ( IK1), was unaffected by HF at mRNA and protein levels despite significant reduction in IK1, whereas canine ether-à-go-go-related gene (cERG), which encodes the rapidly activating component of the delayed rectifier current ( IK), exhibited increased protein expression. HF was not accompanied by significant changes in cKvLQT1 or cMinK mRNA and protein levels. These data indicate that 1) downregulation of Ito in HF is associated with decreased cKv4.3 and not cKv1.4 or cKChIP2, and 2) alterations in both the rapidly activating and slowly activating components of IK as well as IK1 in nonischemic dilated cardiomyopathy are not caused by changes in either transcript or immunoreactive protein levels of relevant channel subunits, which suggests posttranslational modification of these currents by HF.

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Genomic structure and promoter characterization of the human Sprouty4 gene, a novel regulator of lung morphogenesis

AJP Lung Cellular and Molecular Physiology ◽

10.1152/ajplung.00430.2003 ◽

2004 ◽

Vol 287 (1) ◽

pp. L52-L59 ◽

Cited By ~ 13

Author(s):

Wei Ding ◽

Saverio Bellusci ◽

Wei Shi ◽

David Warburton

Keyword(s):

Promoter Activity ◽

Molecular Mechanisms ◽

Core Promoter ◽

Genomic Structure ◽

Inducible Promoter ◽

Luciferase Reporter ◽

Luciferase Reporter Gene ◽

Significant Similarity ◽

The Core ◽

Flanking Region

The expression of Sprouty4 ( Spry4), an intracellular FGF receptor antagonist, shows a temporally and spatially restricted pattern in embryonic lung and is induced by ERK signaling. To clarify the molecular mechanisms regulating Spry4 transcription, the genomic structure of the human Sprouty4 ( hSpry4) gene was first determined by using the GenomeWalker kit. The hSpry4 gene spans > 14 kb and is organized in three exons and two introns. Multiple transcription start sites were subsequently mapped by 5′-rapid amplification of cDNA ends. Analysis of up to 4 kb of sequence in the 5′-flanking region of the gene showed the presence of multiple potential transcription factor binding sites but no TATA or CAAT boxes. Transient transfection using luciferase reporter gene constructs with progressive deletions of the hSpry4 5′-flanking region revealed that the core promoter activity is located within the proximal 0.4-kb region, whereas the minimal ERK-inducible promoter activity is between −69 and −31. Homology analysis further showed that the core promoter region of the hSpry4 gene exhibits significant similarity to the 5′-flanking region of the mouse gene.

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Principles for RNA metabolism and alternative transcription initiation within closely spaced promoters

10.1101/055731 ◽

2016 ◽

Author(s):

Yun Chen ◽

Athma A. Pai ◽

Jan Herudek ◽

Michal Lubas ◽

Nicola Meola ◽

...

Keyword(s):

Transcription Initiation ◽

Core Promoter ◽

Building Blocks ◽

Gene Promoters ◽

Mrna Transcription ◽

Transcription Start ◽

Transcription Start Sites ◽

Alternative Transcription ◽

Mammalian Genomes ◽

Polyadenylation Sites

AbstractMammalian transcriptomes are complex and formed by extensive promoter activity. In addition, gene promoters are largely divergent and initiate transcription of reverse-oriented promoter upstream transcripts (PROMPTs). Although PROMPTs are commonly terminated early, influenced by polyadenylation sites, promoters often cluster so that the divergent activity of one might impact another. Here, we find that the distance between promoters strongly correlates with the expression, stability and length of their associated PROMPTs. Adjacent promoters driving divergent mRNA transcription support PROMPT formation, but due to polyadenylation site constraints, these transcripts tend to spread into the neighboring mRNA on the same strand. This mechanism to derive new alternative mRNA transcription start sites (TSSs) is also evident at closely spaced promoters supporting convergent mRNA transcription. We suggest that basic building blocks of divergently transcribed core promoter pairs, in combination with the wealth of TSSs in mammalian genomes, provides a framework with which evolution shapes transcriptomes.

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Linking FANTOM5 CAGE peaks to annotations with CAGEscan

10.1101/126474 ◽

2017 ◽

Author(s):

Nicolas Bertin ◽

Mickaël Mendez ◽

Akira Hasegawa ◽

Marina Lizio ◽

Imad Abugessaisa ◽

...

Keyword(s):

Quantitative Measurement ◽

Core Promoter ◽

Promoter Regions ◽

Single Nucleotide ◽

Transcription Start Sites ◽

Human Primary Cells ◽

Expression Atlas ◽

Transcriptional Units ◽

Nucleotide Resolution ◽

Single Nucleotide Resolution

AbstractThe FANTOM5 expression atlas is a quantitative measurement of the activity of nearly 200,000 promoter regions across nearly 2,000 different human primary cells, tissue types and cell lines. Generation of this atlas was made possible by the use of CAGE, an experimental approach to localise transcription start sites at single-nucleotide resolution by sequencing the 5′ ends of capped RNAs after their conversion to cDNAs. While 50% of CAGE-defined promoter regions could be confidently associated to adjacent transcriptional units, nearly 100,000 promoter regions remained gene-orphan. To address this, we used the CAGEscan method, in which random-primed 5′-cDNAs are paired-end sequenced. Pairs starting in the same region are assembled in transcript models called CAGEscan clusters. Here, we present the production and quality control of CAGEscan libraries from 56 FANTOM5 RNA sources, which enhances the FANTOM5 expression atlas by providing experimental evidence associating core promoter regions with their cognate transcripts.

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Ssl2/TFIIH function in transcription start site scanning by RNA Polymerase II in Saccharomyces cerevisiae

eLife ◽

10.7554/elife.71013 ◽

2021 ◽

Vol 10 ◽

Author(s):

Tingting Zhao ◽

Irina O Vvedenskaya ◽

William KM Lai ◽

Shrabani Basu ◽

B Franklin Pugh ◽

...

Keyword(s):

Saccharomyces Cerevisiae ◽

Rna Polymerase ◽

Rna Polymerase Ii ◽

Core Promoter ◽

Interaction Network ◽

Transcription Start ◽

Pol Ii ◽

Transcription Start Sites ◽

Scanning Rate ◽

Residue Interaction

In Saccharomyces cerevisiae, RNA Polymerase II (Pol II) selects transcription start sites (TSS) by a unidirectional scanning process. During scanning, a preinitiation complex (PIC) assembled at an upstream core promoter initiates at select positions within a window ~40-120 basepairs downstream. Several lines of evidence indicate that Ssl2, the yeast homolog of XPB and an essential and conserved subunit of the general transcription factor (GTF) TFIIH, drives scanning through its DNA-dependent ATPase activity, therefore potentially controlling both scanning rate and scanning extent (processivity). To address questions of how Ssl2 functions in promoter scanning and interacts with other initiation activities, we leveraged distinct initiation-sensitive reporters to identify novel ssl2 alleles. These ssl2 alleles, many of which alter residues conserved from yeast to human, confer either upstream or downstream TSS shifts at the model promoter ADH1 and genome-wide. Specifically, tested ssl2 alleles alter TSS selection by increasing or narrowing the distribution of TSSs used at individual promoters. Genetic interactions of ssl2 alleles with other initiation factors are consistent with ssl2 allele classes functioning through increasing or decreasing scanning processivity but not necessarily scanning rate. These alleles underpin a residue interaction network that likely modulates Ssl2 activity and TFIIH function in promoter scanning. We propose that the outcome of promoter scanning is determined by two functional networks, the first being Pol II activity and factors that modulate it to determine initiation efficiency within a scanning window, and the second being Ssl2/TFIIH and factors that modulate scanning processivity to determine the width of the scanning widow.

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