Exon 1U Research Articles

Proximal Promoter of the Cytochrome P450 Oxidoreductase Gene: Identification of Microdeletions Involving the Untranslated Exon 1 and Critical Function of the SP1 Binding Sites

POR (cytochrome P450 oxidoreductase) is a ubiquitously expressed gene encoding an electron donor to all microsomal P450 enzymes and several non-P450 enzymes. POR mutations cause an autosomal recessive disorder characterized by skeletal dysplasia, adrenal dysfunction, and disorders of sex development. Although recent studies have indicated the presence of a CpG-rich region characteristic of housekeeping genes around the untranslated exon 1 (exon 1U) and a tropic effect of thyroid hormone on POR expression via thyroid hormone receptor-β, detailed regulatory mechanisms for the POR expression remain to be clarified. Our objective was to report a pivotal element of the proximal promoter of POR. We first studied three patients (cases 1-3) with POR deficiency due to compound heterozygosity with an p.R457H mutation and transcription failure of an apparently normal allele, by oligoarray comparative genomic hybridization and serial direct sequencing of the deletion fusion points. Consequently, a 2,487-bp microdeletion involving exon 1U was identified in case 1 and an identical 49,604-bp deletion involving exon 1U and exon 1 was found in cases 2 and 3. We next analyzed the 2,487-bp region commonly deleted in cases 1-3 by in silico analysis, DNA binding analysis, luciferase assays, and methylation analysis. The results showed a critical function of the evolutionally conserved SP1 binding sites just upstream of exon 1U, especially the binding site at the position -26/-17, in the transcription of POR. The results suggest that the SP1 binding sites constitute an essential element of the POR proximal promoter.

Identification of the new polymorphisms in the promoter region of the CAST gene in cattle

Calpastatin ( CAST) is a specific inhibitor of the ubiquitous calcium-dependent proteases – μ-calpain and m-calpain. This proteolytic system plays a key role in the tenderization process that occurs during postmortem storage of meat under refrigerated conditions. In the present study using comparative sequencing seven novel polymorphisms located within P3 promoter region for exon 1u of the bovine CAST gene: −357 (C/G), −556 (G/T), −557 (A/G), −580 (G/C), −750 (T/C), and two InDel at position −890 (A/−) and (GTT/−) at position −353/−351 were found. This region directs the expression of type III calpastatin mRNA, encoding the prototypical calpastatin. The genotype frequencies and haplotypes distribution were studied in 191 bulls belonging to six cattle breeds. All genotypes were distributed according to the HWE test and two major combined haplotypes were identified. The frequency of the haplotype1 varied from 0.45 in Aberdeen Angus to 0.82 in Simmental.

Apparent Manifesting Heterozygosity in P450 Oxidoreductase Deficiency and Its Effect on Coexisting 21-Hydroxylase Deficiency

P450 oxidoreductase (POR) deficiency is a disorder of steroidogenesis affecting the microsomal P450 enzymes that use POR as an electron donor. The clinical presentation is variable; patients can be asymptomatic or can present with genital anomalies and the Antley-Bixler syndrome, characterized by craniosynostosis and other bony anomalies. Obligately heterozygous parents are normal. Combined POR and 21-hydroxylase deficiencies have not been reported. The aim was to explore the manifestations of combined deficiencies of 21-hydroxylase and POR and to search for lesions in apparent manifesting POR heterozygotes. A newborn female had craniosynostosis, severe salt wasting, minimal virilization, grossly elevated 17OH-progesterone, and minimally elevated androgens. DNA encoding 21-hydroxylase, POR, and fibroblast growth factor receptor 2 was sequenced. For POR, the first untranslated exon (exon 1U), 5' flanking DNA, and most introns were sequenced in five apparent manifesting POR heterozygotes. CYP21B mutations were found on both alleles, proving classical 21-hydroxylase deficiency. Fibroblast growth factor receptor 2 exons 8 and 10 were normal. A POR mutation, A287P, was found only on the maternal allele. Five previously reported patients had POR mutations found on only one allele, but their clinical characteristics were indistinguishable from patients with mutations on both alleles. Sequencing of exon 1U, 274 bp of POR 5' flanking DNA, and 12 of the 15 POR introns did not identify additional mutations affecting gene expression or splicing. Manifesting heterozygosity is a possible feature of POR deficiency and may ameliorate the findings in coexisting 21-hydroxylase deficiency.

Effect of anabolic agents on calpastatin promoters in porcine skeletal muscle and their responsiveness to cyclic adenosine monophosphate-and calcium-related stimuli1

The calpain proteinases and their specific inhibitor calpastatin have been proposed to influence both the rates of myofibrillar protein turnover in vivo and meat tenderization postmortem. Elevated calpastatin concentrations in particular are associated with certain forms of hypertrophic growth and meat toughness. In the 5'region of the porcine calpastatin gene, there are 3 calpastatin promoters upstream of exons 1xa, 1xb, and 1u, respectively, each of which contain transcription factor-binding motifs, suggesting sensitivity to a variety of growth-promoting stimuli. This study examined the effect of the beta-adrenergic agonist clenbuterol and porcine ST (pST) treatment on calpastatin promoter usage in porcine LM in vivo using real-time PCR and also the responsiveness of transfected calpastatin promoter sequences to cyclic adenosine monophosphate (cAMP) and calcium (Ca2+)-related stimuli in reporter gene systems in cell studies. The effect of clenbuterol and pST on potential signaling pathways in vivo was also assessed by monitoring protein phosphatase 2B (calcineurin), NFATc3, calpain 3, IkappaB alpha, and NFkappaB by quantitative immunoblotting. Total calpastatin mRNA was increased by 52% (P < 0.05) after treatment with clenbuterol for 1 d and reduced by 35% (P < 0.01) after pST treatment for 7 d. Whereas clenbuterol had no significant differential effects on individual mRNA transcripts (types 1 to 3) derived from the 3 upstream promoters, pST significantly reduced all of these by 51, 39, and 40% (P < 0.001, 0.05, and 0.05), respectively. Promoter activity was increased in rat L6G8 cells transfected with a construct derived from exon 1u after treatment with dibutyryl cAMP (68%, P < 0.05) or forskolin (43%, P < 0.05), whereas 1xa activity was reduced by both of these agents (47 and 33%, respectively, P < 0.05). Treatment of cells with the calcium ionophore calcimycin reduced the activity of the 1u promoter by 40% (P < 0.01), with no effect on the other promoter constructs. Cyclosporin A had no effect on any promoter construct. The only signaling pathway component to be significantly altered by the in vivo treatments was calcineurin, which was decreased by 24% (P < 0.05) in clenbuterol-treated animals. In conclusion, 2 types of growth promoter in pigs had contrasting effects on calpastatin expression in LM. Transfected calpastatin promoters were differentially sensitive to cAMP- and Ca2+-related stimuli, in agreement with the proposed mode of action of the 2 growth promoters.

Autoregulation of Oct-1 gene expression is mediated by two octa-sites in alternative promoter

Transcription of the oct-1 gene is regulated by two alternative promoters: U promoter and L promoter located upstream of the exons 1U and 1L, respectively. The L promoter contains two octamer sequences of opposite orientation: proximal (ATTTGCAT) and distal (ATGCAAAT), showing high affinity toward the Oct proteins. Binding of the Oct-1 protein to the octa-sites located in the L promoter region has been confirmed in footprinting experiments. Dual luciferase assay using wild-type and mutated promoters have indicated that mutations in the proximal octa-site resulted in significant transcription enhancement both in myeloma cell line NS/0 and in fibroblast cell line 3T3 (about twofold and fivefold, respectively), whereas mutations in the distal site decreased the promoter activity (about 10% and 40%, respectively). Mutations in both octa-sites enhanced the effect and increased transcription to about fourfold in myeloma cell line NS/0 and about sixfold in fibroblast cell line 3T3. These results demonstrate that transcription of the oct-1 gene may be autoregulated by two octa-sites within the L promoter. Different function and interactive tissue-specific effect of distal and proximal octamer sequences can be suggested.

Alternative Promoters and Tissue-Specific Regulation of Mouse oct-1 Gene Transcription

The gene encoding mouse transcription factor Oct-1 contains two exons, 1U and 1L, at the 5′ end. Oct-1 mRNA from all tissues contains exon 1U, whereas exon 1L is found only in Oct-1 mRNA from mouse and human lymphoid cells. Upstream of 1U and 1L exons, the respective specific promoters, U and L, are located at a considerable distance from each other (67 kb in the mouse otf-1 locus). These regions differ in their structure. The region upstream from exon 1U contains numerous Sp1 sites, whereas the region upstream from exon 1L contains homeospecific NTAATNN sites and two octa sites ATGCAAAT recognized by transcription factors Oct-1, Oct-2, and by other POU domain-containing proteins. The octa and homeospecific sites promote autoregulation of the oct-1 gene. Transfection of U and L promoter fragments within pGL3 Basic plasmid or pGL3 Enhancer vector into lymphoid NS/0 myeloma cells or 10(1) fibroblasts showed that the L promoter activity was many times higher in the myeloma cells than in fibroblasts. Between the sites of translation and transcription initiation from the L promoter, a nucleotide sequence was identified whose elimination resulted in a significantly higher efficiency of transcription initiation from this promoter. Thus, the oct-1 gene contains at least two alternative promoters: the U promoter apparently provides for the constitutive synthesis of Oct-1 protein, whereas the L promoter manifests tissue specificity, contains octa sites, and appears to be self-regulated.

Four promoters direct expression of the calpastatin gene

Calpastatin is a specific endogenous protein inhibitor of the ubiquitous calcium dependent proteinases μ- and m-calpain. The calpain–calpastatin system is involved in various physiological and pathological processes. In the present study, we determined the bovine calpastatin gene structure and demonstrated that four promoters direct its expression. The gene harbours 35 exons spanning at least 130 kb on genomic DNA. Its structure is similar to that of mouse, pig, and human gene. Transient transfection assays in both C2C12 and COS7 cell lines demonstrated that the putative promoter regions situated 5′ to exon 1xa, 1xb, 1u, and 14t were functional. We also established that the region situated upstream exon 14t is subjected to a tissue specific regulation. The implication of numerous high-scoring cis acting transcriptional motifs which are present in these regions will need to be determined. The existence of four promoters suggests differential expression patterns which must have a physiological significance.

Calpastatin gene promoter activity associated with growth promoter pathways in pigs

β-agonists are known to enhance muscle growth in livestock species, although sometimes at the expense of increased meat toughness. In pigs, administration of porcine somatotropin (pST) produces muscle hypertrophy without detrimental effects on toughness. Calpastatin, a specific inhibitor that regulates the calpain proteinases responsible for cleavage of myofibrillar proteins, has a key role in growth and the rate at which meat tenderises postmortem. In the porcine calpastatin gene there are at least three calpastatin promoters (upstream of exons 1xa, 1xb and 1u respectively), of which 1u is used predominantly in skeletal muscle. All three promoters contain transcription factor binding motifs suggesting sensitivity to β-agonist/cyclic AMP (cAMP)/protein kinase A (PKA) and IGF-1/Ca2+/calcineurin-mediated signalling pathways (Parr et al., 2004). It was previously shown that expression of calpastatin mRNA from all three promoters can be increased in pigs treated with clenbuterol and suppressed in pST-treated animals (Sensky et al., 2004), suggesting differential regulation by the two pathways. In this study, the effect of manipulating these pathways is evaluated in vitro in order to determine if there is a specific element within the porcine 1u promoter that is responsive to both pathways, using rat muscle cells transfected with a series of 5’-deleted porcine 1u promoter constructs and treated with cAMP analogues and the calcium ionophore calcimycin.

Expression of calpastatin isoforms in muscle and functionality of multiple calpastatin promoters

Calpastatin is the specific endogenous inhibitor of calpain proteinase that is encoded by a single gene. Transient transfection assays in both a non-fusing skeletal muscle and non-muscle cell-line demonstrated that the putative porcine calpastatin promoter regions 5 ′ to exons 1xa, 1xb, and 1u were functional. Both real-time quantitative and semi-quantitative RT-PCR on porcine skeletal muscle total RNA indicated that steady-state expression of Type I and III mRNAs containing exons 1xa and 1u, respectively, was at equivalent levels whilst the expression of Type II mRNA containing exon 1xb was significantly less ( p<0.001). Immunoprobing of Western blotted muscle extracts with an antibody raised against a peptide sequence encoded by exon 1xa indicated that Type I protein was expressed and that there was significantly more Type I protein in cardiac than skeletal muscle ( p<0.001). The results suggest that the expression of the single calpastatin gene was differentially controlled at several levels.

The regulation of the Oct-1 gene transcription is mediated by two promoters

The ubiquitous transcription factor Oct-1 is a member of the POU domain family of regulatory proteins. Target genes controlled by Oct-1 include housekeeping genes, e.g. the genes encoding histon H2B or snRNAs, as well as tissue-specific genes, e.g. the genes encoding the light and heavy chains of immunoglobulines, some interleukins, and others. Oct-1 pre-mRNA may be spliced in several ways, resulting in production of several protein isoforms that may differ functionally. The 5′-end of the Oct-1 gene contains two exons—exon 1U and exon 1L that alternatively present in Oct-1 mRNA. We studied regulation of transcription of the Oct-1 gene using reporter gene assays of promoter-luciferase gene-constructs. It was shown that transcription of the Oct-1 gene is regulated by two promoters located upstream of the exon 1U and upstream of the exon 1L. The promoter located upstream of the exon 1U contains G/C-rich sequences and multiple Sp1 sites, while the promoter located upstream of the exon 1L contains A/T-rich motifs and autoregulation-related cis-elements: two octamer sites ATGCAAAT, two octamer related sites and multiple TAAT-core sites. Exons 1U and 1L in the human OTF-1 locus encoding the Oct-1 gene are located at the distance of 108 kbp. In the murine locus otf-1 the distance between exons 1U and 1L is 67 kbp. We suggest that the two promoters can differ functionally.

Spliced oct-1 mRNA isoforms with untranslated exons and a partly deleted region coding for the POU-specific domain

Transcription factor Oct-1 is involved in expression regulation of housekeeping genes, in lymphocyte differentiation, and in the immune response. Tissue-specific oct-1 mRNA isoforms are known to be expressed in lymphoid cells. Four new mouse isoforms were identified. Of these, two were tissue-specific (oct-1Rα and oct-1Rβ) and contained exon 1L. The oct-1Rα was shown to contain an additional fragment, which corresponds to an exon located in the 3"-region of mouse otf-1. No homolog was found in human OTF-1. The oct-1Rβ isoform proved to lack an exon coding for a fragment of the POU domain. This deletion results in a loss of the first helix of the domain, and the mutant protein is devoid of affinity for octamer ATGCAAAT. Two other mRNA isoforms, oct-1d and oct-1e, were shown to contain untranslated regions between exons 1U and 2. The regions correspond to exons 1i and 2i located between exons 1U and 1L in the 5"-region of the mouse oct-1 gene. Human OTF-1 was not found to contain exon 1i. On evidence of these and published data, it was assumed that a set of oct-1 isoforms is present in the cell, reflecting the complexity of expression regulation of оct-l and the multiplicity of its functions.

Human Oct-1L isoform has tissue-specific expression pattern similar to Oct-2

POU homeodomain proteins are important regulators of ubiquitous as well as tissue-specific transcription. Ubiquitously expressed Oct-1 and tissue-specific Oct-2 proteins are members of the POU family and contain very similar DNA-binding POU domains. While Oct-1 is ubiquitous, Oct-2 is predominantly expressed in B cells, in activated T cells and in nervous system. Oct-1 is involved in regulation of some houskeeping genes—histone H2B, snRNAs as well as in tissue-specific regulation of immunoglobuline gene transcription and of some other genes. Here we report that novel alternatively spliced product of the human Oct-1 gene encode Oct-1L isoform with tissue-specific expression pattern, similar to Oct-2. Oct-1L differ from ubiquitously expressed Oct-1A in 5′-terminal exon (exon 1L). Analysis of nucleotide sequences from Human Genome Data Bank has located exon 1L about 108 kbp downstream ubiquitously expressed exon 1U. Amino terminus of Oct-1L show extensive similarity to amino terminus of Oct-2. We suppose, that Oct-1L may has a specific role in gene expression in lymphoid tissues and brain.

Calpastatin Expression in Porcine Cardiac and Skeletal Muscle and Partial Gene Structure

The expression in porcine skeletal and cardiac muscle of calpastatin, the specific endogenous inhibitor of the calpain proteolytic system, was examined 16 h after a single dose of a specific β2-agonist. Immunoblotting of extracts indicated that treatment increased skeletal calpastatin 135-kDa band intensity (P < 0.01), while in cardiac combined 145- and 135-kDa band intensity decreased (P < 0.05). Treatment increased skeletal (P < 0.01) but not cardiac calpastatin mRNA steady-state levels. Three types of cardiac calpastatin mRNA transcripts were identified by 5′-RACE. Types I and II encoded a putative XL region that originated either from exon 1xA or exon 1xB, arranged in tandem. Type III predominated in skeletal muscle and originated from exon 1u, which was located 40–50 kb 3′ to exons 1xA and 1xB. The region 5′ to exon 1u may act as an independent promoter regulated by a cAMP-dependent mechanisms, thereby explaining the differential response of calpastatin to adrenergic stimulation in cardiac and skeletal muscle.

Four Types of Calpastatin Isoforms with Distinct Amino-Terminal Sequences Are Specified by Alternative First Exons and Differentially Expressed in Mouse Tissues

Calpastatin, a specific inhibitor of calpain, consists of a unique N-terminal domain (domain L) and four repetitive protease-inhibitor domains (domains 1-4). The isolated cDNAs from various mammalian species have conspicuous differences in the regions encoding the N-terminal sequences and can be classified into four types. Mouse and bovine calpastatins (Type I and Type II, respectively), which also differ from each other in the uttermost N-terminal regions, possess longer domain L sequences than those of rabbit, pig, and human inhibitors (Type III). A sequence of a shorter isoform, registered in a DNA data bank, starts from a part of domain 2 with a different N-terminal sequence (Type IV). To clarify the source of this molecular diversity, we investigated the entire exon-intron organization of the mouse calpastatin gene. The previously obtained mouse calpastatin cDNA is encoded by as many as 31 exons including the first exon, designated 1xa. Three additional exons specifying the N-terminal sequences of the other types (designated exons 1xb, 1u, and 14t, respectively) were identified in the mouse genomic DNA sequence. While the mRNAs for Types I and III were expressed at high levels in liver, the Type II mRNA was abundant in heart and skeletal muscle and expressed at lower levels in liver, brain and testis. The Type IV mRNA was specifically expressed in testis among the tissues examined. These results suggest that the calpastatin isoforms possessing different N-terminal sequences are generated by alternative transcription initiation from their own promoters and skipping of the mutually exclusive exons.