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(A) In the upper part of the panel, the three transcripts starting from the P2 promoter, the PCR primers, and the 147-bp and the 255-bp ( ء ) PCR products are represented; black boxes indicate exons common to the three transcripts; grey boxes indicate exons common only to aspartyl-  -hydroxylase and junctate. Oligo(dT) RT-PCR was performed with total RNA isolated from human adult normal tissues or cell lines with the e2F/e3R (lanes a to g) or the e2F1/e5R (lanes h and i) primers and analyzed with agarose gel electrophoresis (lower part of panel). Lanes: a, kidney; b, brain; c, adrenal gland; d, liver; e, heart; f, skeletal muscle; g, RD cell line; h, C2C12-GM cells (C2C12 cells cultured in growth medium in the presence of 10% fetal calf serum); i, C2C12-DM cells (C2C12 cells cultured in differentiation medium in the presence of 2% horse serum and 10 g of insulin/ml); M, pUC Mix Marker 8 (Fermentas). (B) Analysis of 5 Ј RACE products of A  H-J-J exon 2 starting transcripts. cDNAs were synthesized from RD cells (lane m) or human adult skeletal muscle (lane n) total RNA by using the e5R primer. After addition of a poly(G) tail, PCR was performed with the gene-specific e3R primer. One-fifth of the nested PCRs, after a procedure performed with a gene-specific e2R primer, was analyzed by gel electrophoresis. Lanes j to l, control reactions from RD cell RNA performed in the absence of reverse transcriptase, terminal deoxynucleotidyl transferase, and both, respectively. (C) Nucleotide sequence of RACE products, obtained by direct sequencing or cloning. The P2 transcription initiation site ( ϩ 1), the primers used for the last PCR, and the translation start site (ATG) are indicated.
Source publication
Alternative splicing of the locus AbetaH-J-J generates three functionally distinct proteins: an enzyme, AbetaH (aspartyl-beta-hydroxylase), a structural protein of the sarcoplasmic reticulum membrane (junctin), and an integral membrane calcium binding protein (junctate). Junctin and junctate are two important proteins involved in calcium regulation...
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... mouse system and suggest that at least two promoter sequences (tentatively identified as P1 and P2) are present in the 5 region of the AH-J-J locus. In this study, we focused our attention on the P2 promoter, which directs tissue- specific expression of the AH-J-J locus starting from exon 2. The RT-PCR analysis presented in the upper part of Fig. 2A shows that by using an exon 2-specific forward primer we are able to analyze all the transcripts starting from the P2 pro- moter. In the case of human tissues and cells we used primers located in the second and third exons, and in the case of murine C2C12 cell line we used primers located in the second and fifth exons, which exhibit a ...
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... the RD cell line, C2C12-GM cells (C2C12 cells cul- tured in growth medium), and C2C12-DM cells (C2C12 cells cultured in differentiation medium) show that transcription directed from the P2 promoter is tissue specific. Among the analyzed human tissues, high-level transcription was present only in skeletal muscle, cardiac muscle, and brain ( Fig. 2A, lanes f, e, and b). P2 directed transcription was found in RD cells, C2C12-GM, and C2C12-DM cells, with the highest level of expression in differentiated C2C12 cells (Fig. 2A, lane ...
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... directed from the P2 promoter is tissue specific. Among the analyzed human tissues, high-level transcription was present only in skeletal muscle, cardiac muscle, and brain ( Fig. 2A, lanes f, e, and b). P2 directed transcription was found in RD cells, C2C12-GM, and C2C12-DM cells, with the highest level of expression in differentiated C2C12 cells (Fig. 2A, lane ...
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... the transcription initiation site has not been previously described in detail, we also performed 5 RACE to precisely map the origin of transcription. This experiment was per- formed on total RNA isolated from RD cells and human adult skeletal muscle. As shown in Fig. 2B, one band is evident following electrophoresis of 5 RACE products in both cases (lanes m and n). This band was directly sequenced or cloned and then sequenced, giving identical results in both cases. The sequence of the 5 RACE products is shown in Fig. 2C and allows mapping the transcription initiation site, which is indi- cated by a ...
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... per- formed on total RNA isolated from RD cells and human adult skeletal muscle. As shown in Fig. 2B, one band is evident following electrophoresis of 5 RACE products in both cases (lanes m and n). This band was directly sequenced or cloned and then sequenced, giving identical results in both cases. The sequence of the 5 RACE products is shown in Fig. 2C and allows mapping the transcription initiation site, which is indi- cated by a solid arrow. Accordingly, all the nucleotide se- quences located upstream from this site were considered po- tential regulatory regions belonging to the P2 ...
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... binding of pu- rified Sp1 transcription factor to the J-kBmer was found (lane 20). As far as the interactions of J-GRmer with nuclear extracts from C2C12 cells are concerned, the data shown in Fig. 6C demonstrate that the binding is specific, since competition was clearly observed with unlabeled J-GRmer (lane 27) but not with the unrelated oligonucleotides carrying the Ets 1 and MEF-2 binding site (lanes 28 and 26). Surprisingly, the tyrosine-ami- notransferase II (TATII)-GRE double-stranded oligonucleo- tide (8) containing the consensus GRE failed to compete (Fig. 6, lane 25); thus, factors that interact with J-GRmer should be further characterized to establish their relationship, if any, with canonical GRE binding proteins. ...
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... the binding is specific, since competition was clearly observed with unlabeled J-GRmer (lane 27) but not with the unrelated oligonucleotides carrying the Ets 1 and MEF-2 binding site (lanes 28 and 26). Surprisingly, the tyrosine-ami- notransferase II (TATII)-GRE double-stranded oligonucleo- tide (8) containing the consensus GRE failed to compete (Fig. 6, lane 25); thus, factors that interact with J-GRmer should be further characterized to establish their relationship, if any, with canonical GRE binding proteins. This result was reproducibly obtained in several experiments, using high amounts of com- petitor GRE TII mer. Control experiments demonstrated high efficiency of binding of 32 P-labeled ...
Citations
... Human ASPH (NCBI ID: 444 [https://www.ncbi.nlm.nih.gov/gene/? term=444], AβH-J-J locus) has two promoters and undergoes extensive alternative splicing 6,7 resulting in >10 AspH isoforms ( Supplementary Fig. 1). An ASPH mutation resulting in the R735W substitution correlates with Traboulsi syndrome (OMIM 601552/refSNP rs374385878), which manifests as facial dysmorphism and lens dislocation 8 ; another mutation (G434V) affects human kidney function resulting in vesicoureteral reflux 9 . ...
... There is an unmet need for further investigations into the biological functions of human AspH and its role in genetic diseases and cancer. Such work is challenging given the membrane bound nature of AspH 5 , the presence of multiple different isoforms/splice variants (>10 in humans) 6,7 , its location in the ER 5 , and the redox sensitive nature of its disulfide substrates 53 . Our work defining the first substrates for isolated AspH and the roles for the TPR domain in its catalysis provides a structural basis for future work on the cellular and physiological roles of AspH. ...
AspH is an endoplasmic reticulum (ER) membrane-anchored 2-oxoglutarate oxygenase whose C-terminal oxygenase and tetratricopeptide repeat (TPR) domains present in the ER lumen. AspH catalyses hydroxylation of asparaginyl- and aspartyl-residues in epidermal growth factor-like domains (EGFDs). Here we report crystal structures of human AspH, with and without substrate, that reveal substantial conformational changes of the oxygenase and TPR domains during substrate binding. Fe(II)-binding by AspH is unusual, employing only two Fe(II)-binding ligands (His679/His725). Most EGFD structures adopt an established fold with a conserved Cys1–3, 2–4, 5–6 disulfide bonding pattern; an unexpected Cys3–4 disulfide bonding pattern is observed in AspH-EGFD substrate complexes, the catalytic relevance of which is supported by studies involving stable cyclic peptide substrate analogues and by effects of Ca(II) ions on activity. The results have implications for EGFD disulfide pattern processing in the ER and will enable medicinal chemistry efforts targeting human 2OG oxygenases.
... La junctine est une protéine exclusivement localisé dans le muscle et qui appartient à la membrane du RS (Treves et al., 2000) . Elle est issue de l'épissage alternatif du gène ASPH qui code aussi pour la junctate et l'aspartate-β-hydroxylase. Ce gène localisé dans la région 8q12-1 chez l'homme (Feriotto et al., 2005) forme une protéine de 26 kDa (Jones et al., 1995) . Il existe deux isoformes de la junctine, une forme longue composé de six exons et une forme courte composé de cinq exons. ...
La contraction musculaire est provoquée par un relâchement massif de calcium à partir du reticulum sarcoplasmique (RS) des cellules musculaires. Ce relâchement de calcium réalisé par le récepteur de la ryanodine (RyR1), s'effectue dans des structures membranaires spécialisées et très organisées : les triades. Cette architecture spécifique est essentielle à l'activité correcte de RyR1. Cependant, les mécanismes moléculaires mis en jeu dans la formation et le maintien des triades ne sont pas connus. La triadine, qui est une protéine localisée dans la membrane du RS et qui est associée à RyR1, pourrait jouer un rôle dans la structure du reticulum sarcoplasmique pour permettre un relâchement de calcium efficace. L'équipe a montré que l'ablation du gène de la triadine chez la souris induisait une altération des relâchements de calcium et une modification de la forme des triades.Nous avons montré que la triadine pouvait indirectement interagir avec les microtubules et qu'elle pourrait ancrer le RS aux microtubules (Fourest-Lieuvin, J Cell Science, 2012). Par analyse en spectrométrie de masse des protéines co-immunoprécipitées avec la triadine, nous avons identifiéun nouveau partenaire de la triadine, CLIMP-63 qui pourrait être impliqué dans cette fonction. CLIMP-63 est décrite comme une protéine capable d'ancrer le reticulum aux microtubules et de maintenir la forme du reticulum endoplasmique. Nous avons ensuite confirmé son interaction avec la triadine par différentes approches dans différents modèles cellulaires. L'étude et la caractérisation de CLIMP-63 dans le muscle sont tout à fait innovantes et nous avons étudié les conséquences de l'association triadine/CLIMP-63 pour la fonction du muscle et dans la formation ou la maintenance des triades.
... To verify the effects of mithramycin, DNA probes or nuclear extracts, were preincubated for 1 h at 4 • C with different concentration of the compound before the EMSA incubation. Supershift assays were performed as described previously [31,32] by using 2 g of commercially available antibodies specific for Sp1 (cat.07-645) transcription factor and anti-NF-kB (cat. ...
Rapamycin, an inhibitor of mTOR activity, is a potent inducer of erythroid differentiation and fetal hemoglobin production in β-thalassemic patients. Mithramycin (MTH) was studied to see if this inducer of K562 differentiation also operates through inhibition of mTOR. We can conclude from the study that the mTOR pathway is among the major transcript classes affected by mithramycin-treatment in K562 cells and a sharp decrease of raptor protein production and p70S6 kinase is detectable in mithramycin treated K562 cells. The promoter sequence of the raptor gene contains several Sp1 binding sites which may explain its mechanism of action. We hypothesize that the G+C-selective DNA-binding drug mithramycin is able to interact with these sequences and to inhibit the binding of Sp1 to the raptor promoter due to the following results: (a) MTH strongly inhibits the interactions between Sp1 and Sp1-binding sites of the raptor promoter (studied by electrophoretic mobility shift assays, EMSA); (b) MTH strongly reduces the recruitment of Sp1 transcription factor to the raptor promoter in intact K562 cells (studied by chromatin immunoprecipitation experiments, ChIP); (c) Sp1 decoy oligonucleotides are able to specifically inhibit raptor mRNA accumulation in K562 cells. In conclusion, raptor gene expression is involved in mithramycin-mediated induction of erythroid differentiation of K562 cells and one of its mechanism of action is the inhibition of Sp1 binding to the raptor promoter.
Copyright © 2014. Published by Elsevier Ltd.
... Transcripts starting from exon 1 (green box in Fig. 3A), which is under the control of the P1 promoter, are expressed in most tissues and share their NH 2 terminus with aspartyl-β-hydroxylase Dinchuk et al. 2000;Feriotto et al. 2007). Exon 1a (blue box in Fig. 3A) is approximately 8 kb downstream from exon 1 and is under the control of the P2 promoter, whose induction is controlled by the muscle specific transcription factor MEF-2 (Feriotto et al. 2005). Transcripts starting from this exon are expressed in striated muscles and share their NH 2 termini with junctin/junctate Dinchuk et al. 2000). ...
Background Skeletal muscle constitutes approximately 40% of body mass, and age-induced decrease of muscle strength impinge on daily activities and on normal social life in the elderly. Loss of muscle strength has been recognised as a debilitating and life threatening condition also in cachexia in cancer patients and in clinical conditions associated with prolonged bed rest. Skeletal muscle dihydropyridine receptors (Cav1.1) act as Ca2+ channels and voltage sensors to initiate muscle contraction by activating ryanodine receptors, the Ca2+ release channels of the sarcoplasmic reticulum. Cav1.1 activity is enhanced by a retrograde stimulatory signal delivered by the ryanodine receptor. JP45 is a membrane protein interacting with Cav1.1 and the sarcoplasmic reticulum Ca2+ storage protein calsequestrin (CASQ1). We hypothesized that JP45 and CASQ1 form a signalling pathway which modulates Cav1.1 channel activity. Materials and methods We isolated flexor digitorum brevis (FDB) muscle fibres from JP45 and CASQ1 double knockout mice (DKO) and tested whether there were differences in Ca2+ homeostasis between the different mouse lines. Results Our results show that Ca2+ transients evoked by tetanic stimulation in DKO fibres, result from massive Ca2+ influx due to enhanced Cav1.1 channel activity. This enhanced activity causes an increase of muscle strength both in vitro and in vivo. Conclusions We conclude that skeletal muscle contraction is strengthened through the modulation of Cav1.
... Transcripts starting from exon 1 (green box in Fig. 3A), which is under the control of the P1 promoter, are expressed in most tissues and share their NH 2 terminus with aspartyl-β-hydroxylase Dinchuk et al. 2000;Feriotto et al. 2007). Exon 1a (blue box in Fig. 3A) is approximately 8 kb downstream from exon 1 and is under the control of the P2 promoter, whose induction is controlled by the muscle specific transcription factor MEF-2 (Feriotto et al. 2005). Transcripts starting from this exon are expressed in striated muscles and share their NH 2 termini with junctin/junctate Dinchuk et al. 2000). ...
In striated muscle, activation of contraction is initiated by membrane depolarisation caused by an action potential, which triggers the release of Ca(2+) stored in the sarcoplasmic reticulum by a process called excitation-contraction coupling. Excitation-contraction coupling occurs via a highly sophisticated supramolecular signalling complex at the junction between the sarcoplasmic reticulum and the transverse tubules. It is generally accepted that the core components of the excitation-contraction coupling machinery are the dihydropyridine receptors, ryanodine receptors and calsequestrin, which serve as voltage sensor, Ca(2+) release channel, and Ca(2+) storage protein, respectively. Nevertheless, a number of additional proteins have been shown to be essential both for the structural formation of the machinery involved in excitation-contraction coupling and for its fine tuning. In this review we discuss the functional role of minor sarcoplasmic reticulum protein components. The definition of their roles in excitation-contraction coupling is important in order to understand how mutations in genes involved in Ca(2+) signalling cause neuromuscular disorders.
... Junctate is an integral calcium binding protein of sarco(endo)plasmic reticulum membrane, which forms a supramolecular complex with the inositol 1,4,5 trisphosphate receptor and modulates calcium entry through receptor-and store-activated channels [1,12,13]. Our group previously reported the identification of two promoter sequences, present within the human AβH-J-J locus (named P1 and P2), that are expected to regulate the transcription of this locus [1,14,15] . The generated primary transcripts undergo alternative splicing and direct the synthesis of AAH, humbug, junctin, and junctate. ...
... The generated primary transcripts undergo alternative splicing and direct the synthesis of AAH, humbug, junctin, and junctate. We have previously reported the characterization of the P2 promoter, demonstrating that the myocyte enhancer factor 2 (MEF-2) transcription factor binds to this promoter sequence and drives tissue-specific expression, being responsible of inducing transcription during muscle dif- ferentiation [14,15]. In additon, we have recently reported the role of Sp factors in upregulating the P1-directed transcription of the AβH- J-J locus [16]. ...
... HeLa cells transient transfection and luciferase assay were performed in two experiments conducted in triplicate and the data were normalized to Renilla luciferase activity and reported as ratios (means ± SD) to the wild type reporter construct -512 WT; *P < 0.05 and **P < 0.02. and P2), and multiple alternative splicing sites, leading to the synthesis of the functionally distinct proteins, AAH, humbug, junctin, and junctate [1,141516 . The P1 promoter directs AAH and humbug expression in most tissues and is up-regulated by Sp and USF transcription factors. ...
Alternative splicing of the locus A beta H-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) beta-hydroxylase (AAH), truncated homologs of AAH with a role in calcium homeostasis humbug and junctate and a structural protein of the sarcoplasmic reticulum membranes junctin. AAH and humbug are over expressed in a broad range of malignant neoplasms. We have previously reported that this locus contains two promoters, P1 and P2. While AAH and humbug are expressed in most tissues under the regulation of the P1 promoter, AAH, junctin and junctate are predominantly expressed in excitable tissues under the control of the P2 promoter. We previously demonstrated that Sp transcription factors positively regulate the P1 promoter.
In the present study, we extended the functional characterization of the P1 promoter of the A beta H-J-J locus. We demonstrated by quantitative Real-time RT-PCR that mRNAs from the P1 promoter are actively transcribed in all the human cell lines analysed. To investigate the transcription mechanism we transiently transfected HeLa cells with sequentially deleted reporter constructs containing different regions of the -661/+81 P1 nucleotide sequence. Our results showed that (i) this promoter fragment is a powerful activator of the reporter gene in HeLa cell line, (ii) the region spanning 512 bp upstream of the transcription start site exhibits maximal level of transcriptional activity, (iii) progressive deletions from -512 gradually reduce reporter expression. The region responsible for maximal transcription contains an E-box site; we characterized the molecular interactions between USF1/2 with this E-box element by electrophoretic mobility shift assay and supershift analysis. In addition, our USF1 and USF2 chromatin immunoprecipitation results demonstrate that these transcription factors bind the P1 promoter in vivo. A functional role of USF1/USF2 in upregulating P1-directed transcription was demonstrated by analysis of the effects of (i) in vitro mutagenesis of the P1/E-box binding site, (ii) RNA interference targeting USF1 transcripts.
Our results suggest that USF factors positively regulate the core of P1 promoter, and, together with our previously data, we can conclude that both Sp and USF DNA interaction and transcription activity are involved in the P1 promoter dependent expression of AAH and humbug.
... Junctin (JCN), a 210-amino acid (AA) protein (26 kd), was identified as a major calsequestrin (CSQ)-binding protein in junctional sarcoplasmic reticulum (SR) vesicles in canine skeletal and cardiac muscles (Jones et al. 1995, Mitchell et al. 1988). This JCN-encoding gene is shared with two other proteins, junctate and aspartyl β-hydroxylase, through alternative splicing, and exons 2 and 3 are common among the three proteins (Dinchuk et al. 2000, Feriotto et al. 2005, Hong et al. 2001, Treves et al. 2000. Detailed analysis of the protein sequence and topology revealed that JCN is an SR transmembrane protein with a short N-terminus in the cytosol (AAs 1-22), a single transmembrane domain (AAs 23-44), and a bulky C-terminus (AAs 45-210) in the SR lumen (Jones et al. 1995). ...
Junctin (JCN), a 26-kd sarcoplasmic reticulum (SR) transmembrane protein, forms a quaternary protein complex with the ryanodine receptor, calsequestrin, and triadin in the SR lumen of cardiac muscle. Within this complex, calsequestrin, triadin, and JCN appear to be critical for normal regulation of ryanodine receptor-mediated calcium (Ca) release. Junctin and triadin exhibit 60% to 70% amino acid homology in their transmembrane domains, including repeated KEKE motifs important for macromolecular protein-protein interactions within their SR luminal tails. Recent studies have uncovered functional roles of both JCN and triadin in the mouse heart, using transgenic overexpression strategies, which exhibit varying phenotypes including mild SR structural alterations, prolongation of Ca transient decay, impaired relaxation, and cardiac hypertrophy and/or heart failure. More specifically, both in vitro adenoviral gene transfer and in vivo gene-targeting techniques to manipulate JCN expression levels have shown that JCN is an essential factor in maintaining normal cardiac Ca handling and cardiac function. This article reviews the new findings on the regulatory roles of JCN in cardiac SR Ca cycling and contractility, with special emphasis on the effects of JCN ablation on delayed after depolarization-induced arrhythmias and premature mortality in mouse models.
... Our group previously reported the identification of two putative promoter sequences, present within the human AbH-J-J locus (named P1 and P2), that are expected to regulate the transcription of this locus [1,13]. The generated primary transcripts are subjected to alternative splicing and direct the synthesis of AAH, humbug, junctin, and junctate [1,13]. ...
... Our group previously reported the identification of two putative promoter sequences, present within the human AbH-J-J locus (named P1 and P2), that are expected to regulate the transcription of this locus [1,13]. The generated primary transcripts are subjected to alternative splicing and direct the synthesis of AAH, humbug, junctin, and junctate [1,13]. We have recently reported the characterization of the P2 promoter, demonstrating that the myocyte enhancer factor 2 (MEF-2) transcription factor binds this promoter sequence and drives tissue-specific expression, being responsible for inducing transcription during muscle differentiation [1,13]. ...
... The generated primary transcripts are subjected to alternative splicing and direct the synthesis of AAH, humbug, junctin, and junctate [1,13]. We have recently reported the characterization of the P2 promoter, demonstrating that the myocyte enhancer factor 2 (MEF-2) transcription factor binds this promoter sequence and drives tissue-specific expression, being responsible for inducing transcription during muscle differentiation [1,13]. ...
Alternative splicing of the locus AbetaH-J-J generates functionally distinct proteins: the enzyme aspartyl (asparaginyl) beta-hydroxylase, humbug and junctate (truncated homologs of aspartyl (asparaginyl) beta-hydroxylase with a role in calcium regulation), and junctin (a structural protein of the sarcoplasmic reticulum membrane). Aspartyl (asparaginyl) beta-hydroxylase and humbug are overexpressed in a broad range of malignant neoplasms. We have previously reported the gene structure of this locus, showing the presence of two putative promoters, P1 and P2, and characterized the P2 sequences, directing tissue-specific transcription of junctin, aspartyl (asparaginyl) beta-hydroxylase and junctate. In addition, aspartyl (asparaginyl) beta-hydroxylase and humbug are expressed from exon 1 by the P1 promoter. The present study identifies and functionally characterizes the P1 promoter activity of the AbetaH-J-J locus. We demonstrate that mRNAs from the P1 promoter are actively transcribed in all the human tissues and cell lines analyzed, and define the transcription start point in HeLa and RD cells. To investigate the transcription mechanism we cloned 1.7 kb upstream of exon 1 from a human BAC clone, and produced progressively deleted reporter constructs. Our results showed that: (a) the 1.7 kb fragment was a powerful activator of the reporter gene in human hepatoblastoma (HepG2) and human embryonic rhabdomyosarcoma (RD) cell lines; (b) 512 bp upstream of the transcription start site were essential for maximal promoter activity; and (c) progressive deletions from -512 resulted in gradually decreased reporter expression. The region responsible for maximal transcription contains at least 12 GC boxes homologous to binding sequences of specific transcription factor 1 (Sp1); by electrophoretic mobility shift assay and supershift analysis, we identified three GC-rich elements that bind Sp transcription factor family nuclear factors with very high efficiency. A functional role of Sp transcription factors in upregulating P1-directed transcription was demonstrated by analysis of the effects of: (a) in vitro mutagenesis of the Sp1 transcription factor binding sites; (b) transfection with Sp transcription factor 1/3 expression vectors; and (c) treatment with decoy oligonucleotides targeting Sp transcription factors. In addition, Sp1 and Sp3 transcription factor chromatin immunoprecipitation demonstrated in vivo binding of these proteins to P1 promoter. Our results suggest that Sp transcription factors positively regulate the core of the P1 promoter, and the comparison of the two promoters of the AbetaH-J-J locus demonstrates that they are very different with regard to transcriptional efficiency and ability to direct tissue-specific transcription.
... The structural organization of the human AH-J-J locus is shown in FIGURE 1. The scheme presented is based both on results previously reported in detail elsewhere by Treves et al. 1 and Feriotto et al. 4 The combination of data obtained by polymerase chain reaction (PCR) amplification and sequencing allowed us to define the splicing events (FIG. 1) as well as the structure of the 5 region of this locus. The data obtained indicate that the first and second exons are mutually exclusive when mature mRNAs are produced. ...
... On the contrary, the expression directed by the P2 promoter is tissue-specific, with high levels of transcription occurring particularly in skeletal muscle, cardiac muscle, and brain and lower level of transcription in kidney. 4 These results sustain the concept that the roles of the P1 and P2 promoters are sharply different. ...
... 2D), in vivo chromatin immunoprecipitation (ChIP), and co-transfection of C2C12 cells with a plasmid carrying the P2 AH-J-J minimal promoter and an expression plasmid carrying MEF-2 cDNA under the control of CMV promoter. 4 Moreover, the involvement of MEF-2 in transcriptional regulation of the AH-J-J locus is further sustained by experiments employing constructs carrying mutated MEF-2 DNA binding sites. These promoters were found to be less active in sustaining transcription of the luciferase reporter gene both in vitro and in vivo. ...
The human AbetaH-J-J locus is a genomic sequence which generates three functionally distinct proteins, the enzyme aspartyl-beta-hydroxylase (AbetaH), the structural protein of sarcoplasmic reticulum junctin, and the membrane-bound calcium binding protein junctate. The first and second exons are mutually exclusive when mature mRNAs are produced. Moreover, the use of different splice donors has been shown to be involved in the generation of protein diversity by alternative splicing. As to transcriptional regulation, two promoters (P1 and P2) were identified. When the P1 and P2 promoter sequences are compared, important differences are clearly detectable. The most interesting result emerging from studies focused on the P2 promoter is that the calcium-dependent transcriptional factor MEF-2 activates the transcription of junctin, junctate, and AbetaH in excitable tissues and, to a lesser extent, in kidney. No Sp1 binding sites are present in the P2 promoter. In contrast, P1 promoter contains GC-rich sequences, which have homologies with the Sp1 consensus binding site.
Vitamin K is a micronutrient necessary for γ-carboxylation of glutamic acids. This post-translational modification occurs in the endoplasmic reticulum (ER) and affects secreted proteins. Recent clinical studies implicate vitamin K in the pathophysiology of diabetes, but the underlying molecular mechanism remains unknown. Here, we show that mouse β cells lacking γ-carboxylation fail to adapt their insulin secretion in the context of age-related insulin resistance or diet-induced β cell stress. In human islets, γ-carboxylase expression positively correlates with improved insulin secretion in response to glucose. We identify endoplasmic reticulum Gla protein (ERGP) as a γ-carboxylated ER-resident Ca2+-binding protein expressed in β cells. Mechanistically, γ-carboxylation of ERGP protects cells against Ca2+ overfilling by diminishing STIM1 and Orai1 interaction and restraining store-operated Ca2+ entry. These results reveal a critical role of vitamin K-dependent carboxylation in regulation of Ca2+ flux in β cells and in their capacity to adapt to metabolic stress.
