Co-expression of IGFs and GH receptors (GHRs) in gilthead sea bream (Sparus aurata L.): sequence analysis of the GHR-flanking region

The tissue-specific expression of IGFs and GH receptors (GHRs) was analyzed in gilthead sea bream (Sparus aurata L.) as an attempt to understand the functional partitioning of duplicated GHRs on the regulation offish growth by season and aging. Gene transcripts were measured in liver, muscle, and adipose tissue by means of quantitative real-time PCR assays. In juvenile fish, concurrent increases in circulating levels of GH and IGF-I and hepatic mRNA levels of IGF-I and GHR-I were evidenced with the summer growth spurt. Conversely, muscle and adipose tissue expression of GHR-I and IGF-II were significantly upregulated by over wintering. The aging decrease of growth rates was accompanied by a reduced activity of the liver GH/IGF axis, and parallel increases in muscle IGF expression would be dictated at the local tissue level by the enhanced expression of GHR-I. Extra-hepatic expression of IGFs and GHR-II did not correlate seasonally in juvenile fish, and nonsignificant effects of aging were found on the summer expression of GHR-II in any analyzed tissue. One transcription start site was identified by RLM-RACE in GHR-I and GHR-II. Sequence analyses indicated that both genes have TATA-less promoters containing consensus initiator sequences and downstream promoter elements surrounding the transcription start site. Conserved CCAAT-boxes and GC-rich regions were retrieved in the GHR-I promoter, whereas stress- and redox-sequence elements (cAMP-responsive element-binding protein, activator proteins; AP-1, and AP-4) were characteristic features of GHR-II. All this supports the functional partitioning of fish GHRs regardless of fish species differences.

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Sp1 and Sp3 regulate glucokinase gene transcription in the liver of gilthead sea bream (Sparus aurata)

Journal of Molecular Endocrinology ◽

10.1677/jme.1.02176 ◽

2007 ◽

Vol 38 (4) ◽

pp. 481-492 ◽

Cited By ~ 11

Author(s):

M Egea ◽

I Metón ◽

I V Baanante

Keyword(s):

Binding Site ◽

Transcription Start Site ◽

Hepg2 Cells ◽

Sparus Aurata ◽

Gilthead Sea Bream ◽

Sea Bream ◽

Proximal Promoter ◽

Start Site ◽

Transcription Start ◽

Mobility Shift

To better understand the transcriptional machinery that governs glucokinase (GCK) expression, we have cloned and characterized the proximal promoter region of GCK from gilthead sea bream (Sparus aurata). The 5′-flanking region of GCK was isolated by chromosome walking. SMART RACE-PCR allowed us to locate the transcription start site 98 bp (bp) upstream from the translational start. Transfection analysis in HepG2 cells revealed the presence of a functional promoter in the 1397 bp 5′-flanking isolated fragment (positions −1321 to +76 relative to the transcription start site). Sequential 5′-deletion analysis indicated a core functional promoter for basal transcription within the 288 bp upstream from the transcription start site. Transient transfection experiments performed in HepG2 cells and electrophoretic mobility shift assays denoted that Sp1 binds and transactivates GCK promoter, whereas Sp3 repressed Sp1-mediated activation of GCK by competing for the same binding site. Mutations in the Sp binding site completely abolished the enhancing effect of Sp1. Treatment with insulin stimulated GCK expression, and increased Sp1 levels in S. aurata liver. We propose a new mechanism that involves Sp1 and Sp3 to mediate insulin activation of GCK transcription.

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Abstract 51: LincAQPEP, an Adipocyte-Specific Intergenic Noncoding RNA, Modulates Lipid Metabolism in Human Adipocytes

Arteriosclerosis Thrombosis and Vascular Biology ◽

10.1161/atvb.35.suppl_1.51 ◽

2015 ◽

Vol 35 (suppl_1) ◽

Author(s):

Xuan Zhang ◽

Chenyi Xue ◽

Yumiao Han ◽

Benjamin Garcia ◽

Raymond Soccio ◽

...

Keyword(s):

Gene Expression ◽

Adipose Tissue ◽

Transcription Start Site ◽

Molecular Mechanisms ◽

Ppar Gamma ◽

Regulatory Function ◽

Binding Motif ◽

Start Site ◽

Human Adipocytes ◽

Transcription Start

Long intergenic noncoding RNAs (lincRNAs) have emerged as key mediators of cellular functions and are increasingly implicated in human diseases. Recently a set of lincRNAs was identified to regulate adipogenesis in mice and humans. Here we report that lincAQPEP, a lincRNA predominantly expressed in adipose tissue, is one of the most abundant lincRNAs detected in human fat by RNA sequencing. It is highly expressed in mature adipocytes but not detected in pre-adipocytes, monocytes or macrophages, indicating its adipocyte specificity. Interestingly, no syntenic or conserved RNA transcript was detected in mouse genome for lincAQPEP, suggesting it might be a human specific lincRNA. A binding motif for PPAR gamma (PPARγ), a critical transcription factor in adipogenesis, was found in the promoter region of lincAQPEP (-612 to -596 bp relative to transcription start site). In addition, chromatin immunoprecipitation sequencing confirmed high occupancy of PPARγ around lincAQPEP transcription start site in human adipose tissue and cultured adipocytes, suggesting PPARγ may mediate adipocyte-specific lincAQPEP transcription. LincAQPEP knockdown (~70%) by lentiviral-based short hairpin RNAs resulted in ~35% decrease in triglyceride content as well as 30-70% reduction of lipogenic gene expression (e.g. SREBP1, FASN, FABP4) in human adipocytes, suggesting a modulatory role of lincAQPEP in adipocyte function. To investigate the molecular mechanisms of its regulatory function, we performed RNA pulldown assays with biotinylated lincAQPEP and lincAQPEP antisense transcript (negative control), followed by mass spectrometry to discover potential lincAQPEP-interacting proteins. IGF2BP3 (Insulin-like growth factor 2 mRNA-binding protein 3), implicated in post-transcriptional regulation of gene expression, was identified as one of top candidates to physically bind lincAQPEP. Interestingly, lincAQPEP knockdown resulted in ~30% reduction in IGF2BP3 mRNA abundance. These data suggested that lincAQPEP may impact mRNA stability and translation of specific genes by interacting with IGF2BP3. In summary, our data indicate that lincAQPEP, an adipose-specific lincRNA, plays important roles in adipocyte biology likely through interaction with IGF2BP3.

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Transcriptional regulation of glucose-6-phosphatase catalytic subunit promoter by insulin and glucose in the carnivorous fish, Sparus aurata

Journal of Molecular Endocrinology ◽

10.1677/jme.1.01552 ◽

2004 ◽

Vol 33 (3) ◽

pp. 783-795 ◽

Cited By ~ 17

Author(s):

M C Salgado ◽

I Metón ◽

M Egea ◽

I V Baanante

Keyword(s):

Transcription Start Site ◽

Hepg2 Cells ◽

Sparus Aurata ◽

Catalytic Subunit ◽

Mrna Levels ◽

Start Site ◽

Transcription Start ◽

Carnivorous Fish ◽

Repressive Effect ◽

Insulin Dependent

Increase in glucose-6-phosphatase catalytic subunit (G6Pase, G6pc) transcription enhances hepatic glucose production in non-insulin-dependent diabetes mellitus (NIDDM). The fact that carnivorous fish is an alternative model to study NIDDM led us to clone and characterise the first G6pc promoter region reported for fish and non-mammalian animals. The 5′-flanking region of G6pc from gilthead sea bream (Sparus aurata) was isolated by chromosome walking. With SMART RACE-PCR, the transcription start site was located 106 base pairs (bp) upstream of the translational start. Transfection analysis in HepG2 cells located a functional promoter in the 850 bp 5′-flanking isolated fragment (positions −770 to +80 relative to the transcription start). Sequential 5′-deletion analysis of the promoter fragment revealed that a core functional promoter for basal transcription is comprised within the 190 bp upstream of the transcription start site. In vivo, glucose and insulin reduced G6Pase mRNA levels in the fish liver. Transfection experiments in HepG2 cells showed that insulin repressed S. aurata G6pc under high-glucose conditions. Synergistic activation of piscine G6pc promoter was induced by cotransfection with expression plasmids for hepatocyte nuclear factor-4α (HNF-4α) and peroxisome proliferator-activated receptor-γ coactivator-1 (PGC-1α). No direct relationship was found between PGC-1α coactivation of HNF-4α transactivation and the repressive effect of insulin. Interestingly, insulin hardly affected G6pc promoter activity in the absence of glucose, suggesting that a reduced capacity of insulin-dependent repression of piscine G6pc may lead to insulin resistance in carnivorous fish.

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The Role of XPB/Ssl2 dsDNA Translocase Processivity in Transcription Start-site Scanning

Journal of Molecular Biology ◽

10.1016/j.jmb.2021.166813 ◽

2021 ◽

pp. 166813

Author(s):

Eric J. Tomko ◽

Olivia Luyties ◽

Jenna K. Rimel ◽

Chi-Lin Tsai ◽

Jill O. Fuss ◽

...

Keyword(s):

Transcription Start Site ◽

Start Site ◽

Transcription Start

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Growth hormone (GH) and insulin-like growth factor-I (IGF-I) treatment of the GH-deficient dwarf rat: differential effects on IGF-I transcription start site expression in hepatic and extrahepatic tissues and lack of effect on type I IGF receptor mRNA expression

Molecular and Cellular Endocrinology ◽

10.1016/0303-7207(94)90249-6 ◽

1994 ◽

Vol 101 (1-2) ◽

pp. 321-330 ◽

Cited By ~ 11

Author(s):

A.A Butler ◽

G.R Ambler ◽

B.H Breier ◽

D LeRoith ◽

C.T Roberts ◽

...

Keyword(s):

Growth Hormone ◽

Type I ◽

Start Site ◽

Transcription Start ◽

Factor I ◽

Receptor Mrna ◽

Type I Igf Receptor ◽

Igf I ◽

Extrahepatic Tissues ◽

Igf Receptor

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Interactions between RNA polymerase and the core recognition element are a determinant of transcription start site selection

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1603271113 ◽

2016 ◽

Vol 113 (21) ◽

pp. E2899-E2905 ◽

Cited By ~ 23

Author(s):

Irina O. Vvedenskaya ◽

Hanif Vahedian-Movahed ◽

Yuanchao Zhang ◽

Deanne M. Taylor ◽

Richard H. Ebright ◽

...

Keyword(s):

Rna Polymerase ◽

Transcription Start Site ◽

Transcription Initiation ◽

Specific Protein ◽

Start Site ◽

Transcription Start ◽

Transcription Bubble ◽

Recognition Element

During transcription initiation, RNA polymerase (RNAP) holoenzyme unwinds ∼13 bp of promoter DNA, forming an RNAP-promoter open complex (RPo) containing a single-stranded transcription bubble, and selects a template-strand nucleotide to serve as the transcription start site (TSS). In RPo, RNAP core enzyme makes sequence-specific protein–DNA interactions with the downstream part of the nontemplate strand of the transcription bubble (“core recognition element,” CRE). Here, we investigated whether sequence-specific RNAP–CRE interactions affect TSS selection. To do this, we used two next-generation sequencing-based approaches to compare the TSS profile of WT RNAP to that of an RNAP derivative defective in sequence-specific RNAP–CRE interactions. First, using massively systematic transcript end readout, MASTER, we assessed effects of RNAP–CRE interactions on TSS selection in vitro and in vivo for a library of 47 (∼16,000) consensus promoters containing different TSS region sequences, and we observed that the TSS profile of the RNAP derivative defective in RNAP–CRE interactions differed from that of WT RNAP, in a manner that correlated with the presence of consensus CRE sequences in the TSS region. Second, using 5′ merodiploid native-elongating-transcript sequencing, 5′ mNET-seq, we assessed effects of RNAP–CRE interactions at natural promoters in Escherichia coli, and we identified 39 promoters at which RNAP–CRE interactions determine TSS selection. Our findings establish RNAP–CRE interactions are a functional determinant of TSS selection. We propose that RNAP–CRE interactions modulate the position of the downstream end of the transcription bubble in RPo, and thereby modulate TSS selection, which involves transcription bubble expansion or transcription bubble contraction (scrunching or antiscrunching).

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