Proximal Sequence Research Articles

Cloning of catalase and expression patterns of catalase and selenium-dependent glutathione peroxidase from Exopalaemon carinicauda in response to low salinity stress

Catalase (CAT) and selenium-dependent glutathione peroxidase (Se-GPx) play a vital role in protecting organisms against various oxidative stresses by eliminating H2O2. The objective of this paper is to evaluate the roles of these antioxidant molecules in the ridgetail white prawn Exopalaemon carinicauda in response to low salinity stress. A complementary DNA (cDNA) containing the complete coding sequence of CAT was cloned from the hepatopancreas using reverse-transcription polymerase chain reaction (RT-PCR) and rapid amplification of cDNA ends. The full-length cDNA of CAT (2 649 bp) contains a 5′-untranslated region (UTR) of 78 bp, a 3′-UTR of 1 017 bp, with a poly (A) tail, and an open reading frame of 1 554 bp encoding a 517-amino-acid polypeptide with predicted molecular mass of 58.46 kDa and estimated isoelectric point of 6.64. This CAT sequence contained the proximal active site signature (60FDRERIPERVVHAKGAG76), proximal heme-ligand signature sequence (350RLFSYPDTH358) and three catalytic amino acid residues (His71, Asn144 and Tyr354). Sequence comparison showed that the CAT deduced amino acid sequence of E. carinicauda shared 68%-92% of identities with those of other species. Quantitative real-time PCR analysis revealed that CAT mRNA was widely expressed in the hepatopancreas (highest), hemocyte, eyestalk, heart, gill, muscle, ovary and stomach. Under low salinity stress, CAT and GPx mRNA expression levels both in the gill and hepatopancreas increased significantly at the first 48 h and 6 h respectively, indicating a tissue- and time-dependent antioxidant response in E. carinicauda. All these results indicate that E. carinicauda CAT is a member of the CAT family and might be involved in the acute response against low salinity stress.

Method for trapping affinity chromatography of transcription factors using aldehyde–hydrazide coupling to agarose

The use of a method of coupling DNA was investigated for trapping and purifying transcription factors. Using the GFP–C/EBP (CAAT/enhancer binding protein) fusion protein as a model, trapping gives higher purity and comparable yield to conventional affinity chromatography. The chemistry used is mild and was shown to have no detrimental effect on GFP fluorescence or GFP–C/EBP DNA binding. The method involves introducing a ribose nucleotide to the 3′ end of a DNA sequence. Reaction with mM NaIO4 (sodium metaperiodate) produces a dialdehyde of ribose that couples to hydrazide–agarose. The DNA is combined at nM concentration with a nuclear extract or other protein mixture, and DNA–protein complexes form. The complex is then coupled to hydrazide–agarose for trapping the DNA–protein complex and the protein eluted by increasing NaCl concentration. Using a different oligonucleotide with the proximal E-box sequence from the human telomerase promoter, USF-2 transcription factor was purified by trapping, again with higher purity than results from conventional affinity chromatography and similar yield. Other transcription factors binding E-boxes, including E2A, c-Myc, and Myo-D, were also purified, but myogenin and NFκB were not. Therefore, this approach proved to be valuable for both affinity chromatography and the trapping approach.

Heterogeneous dynamics in DNA site discrimination by the structurally homologous DNA-binding domains of ETS-family transcription factors.

The ETS family of transcription factors exemplifies current uncertainty in how eukaryotic genetic regulators with overlapping DNA sequence preferences achieve target site specificity. PU.1 and Ets-1 represent archetypes for studying site discrimination by ETS proteins because their DNA-binding domains are the most divergent in sequence, yet they share remarkably superimposable DNA-bound structures. To gain insight into the contrasting thermodynamics and kinetics of DNA recognition by these two proteins, we investigated the structure and dynamics of site discrimination by their DNA-binding domains. Electrophoretic mobilities of complexes formed by the two homologs with circularly permuted binding sites showed significant dynamic differences only for DNA complexes of PU.1. Free solution measurements by dynamic light scattering showed PU.1 to be more dynamic than Ets-1; moreover, dynamic changes are strongly coupled to site discrimination by PU.1, but not Ets-1. Interrogation of the protein/DNA interface by DNA footprinting showed similar accessibility to dimethyl sulfate for PU.1/DNA and Ets-1/DNA complexes, indicating that the dynamics of PU.1/DNA complexes reside primarily outside that interface. An information-based analysis of the two homologs’ binding motifs suggests a role for dynamic coupling in PU.1's ability to enforce a more stringent sequence preference than Ets-1 and its proximal sequence homologs.

Tephra from andesitic Shiveluch volcano, Kamchatka, NW Pacific: chronology of explosive eruptions and geochemical fingerprinting of volcanic glass

The ~16-ka-long record of explosive eruptions from Shiveluch volcano (Kamchatka, NW Pacific) is refined using geochemical fingerprinting of tephra and radiocarbon ages. Volcanic glass from 77 prominent Holocene tephras and four Late Glacial tephra packages was analyzed by electron microprobe. Eruption ages were estimated using 113 radiocarbon dates for proximal tephra sequence. These radiocarbon dates were combined with 76 dates for regional Kamchatka marker tephra layers into a single Bayesian framework taking into account the stratigraphic ordering within and between the sites. As a result, we report ~1,700 high-quality glass analyses from Late Glacial–Holocene Shiveluch eruptions of known ages. These define the magmatic evolution of the volcano and provide a reference for correlations with distal fall deposits. Shiveluch tephras represent two major types of magmas, which have been feeding the volcano during the Late Glacial–Holocene time: Baidarny basaltic andesites and Young Shiveluch andesites. Baidarny tephras erupted mostly during the Late Glacial time (~16–12.8 ka BP) but persisted into the Holocene as subordinate admixture to the prevailing Young Shiveluch andesitic tephras (~12.7 ka BP–present). Baidarny basaltic andesite tephras have trachyandesite and trachydacite (SiO2 < 71.5 wt%) glasses. The Young Shiveluch andesite tephras have rhyolitic glasses (SiO2 > 71.5 wt%). Strongly calc-alkaline medium-K characteristics of Shiveluch volcanic glasses along with moderate Cl, CaO and low P2O5 contents permit reliable discrimination of Shiveluch tephras from the majority of other large Holocene tephras of Kamchatka. The Young Shiveluch glasses exhibit wave-like variations in SiO2 contents through time that may reflect alternating periods of high and low frequency/volume of magma supply to deep magma reservoirs beneath the volcano. The compositional variability of Shiveluch glass allows geochemical fingerprinting of individual Shiveluch tephra layers which along with age estimates facilitates their use as a dating tool in paleovolcanological, paleoseismological, paleoenvironmental and archeological studies. Electronic tables accompanying this work offer a tool for statistical correlation of unknown tephras with proximal Shiveluch units taking into account sectors of actual tephra dispersal, eruption size and expected age. Several examples illustrate the effectiveness of the new database. The data are used to assign a few previously enigmatic wide-spread tephras to particular Shiveluch eruptions. Our finding of Shiveluch tephras in sediment cores in the Bering Sea at a distance of ~600 km from the source permits re-assessment of the maximum dispersal distances for Shiveluch tephras and provides links between terrestrial and marine paleoenvironmental records.

A proximal method with logarithmic barrier for nonlinear complementarity problems

We study the proximal method with the regularized logarithmic barrier, originally stated by Attouch and Teboulle for positively constrained optimization problems, in the more general context of nonlinear complementarity problems with monotone operators. We consider two sequences generated by the method. We prove that one of them, called the ergodic sequence, is globally convergent to the solution set of the problem, assuming just monotonicity of the operator and existence of solutions; for convergence of the other one, called the proximal sequence, we demand some stronger property, like paramonotonicity of the operator or the so called "cut property" of the problem.

Sequence and regulation of the porcine FSHR gene promoter

Follicle-stimulating hormone (FSH) plays a crucial role in animal reproduction and exerts its physiological functions by interacting with the FSH receptor (FSHR). The FSHR is exclusively expressed in granulose cells in the ovary and its expression level is closely related to granulose cell differentiation and follicle maturation. In mammal, most of the follicles undergo atresia, while follicle atresia is mainly caused by granulosa cell apoptosis. However, knowledge on the transcriptional regulatory mechanisms of the porcine FSHR gene in granulosa cell is still limited. In this study, approximately 2.1kb of the proximal promoter sequence of the porcine FSHR gene were obtained by genome walking, and the regulatory elements and transcription factors in the porcine FSHR promoter sequence were predicted. Furthermore, the core promoter region (−1195/−598) of the porcine FSHR gene was identified using a luciferase assay. Subsequently, the relationship between expression levels of the porcine FSHR gene and histone H3K9 acetylation levels around the core promoter region (−787/−572) in vivo and invitro were analyzed. Our results showed that an increased FSHR gene expression level was accompanied with an increase in histone H3K9 acetylation levels, suggesting that histone H3K9 acetylation could regulate the expression of the porcine FSHR gene.

The role of palmitoylation and transmembrane domain in sorting of transmembrane adaptor proteins

Plasma membrane proteins synthesised at the endoplasmic reticulum are delivered to the cell surface via sorting pathways. Hydrophobic mismatch theory based on the length of the transmembrane domain (TMD) dominates discussion about determinants required for protein sorting to the plasma membrane. Transmembrane adaptor proteins (TRAP) are involved in signalling events which take place at the plasma membrane. Members of this protein family have TMDs of varying length. We were interested in whether palmitoylation or other motifs contribute to the effective sorting of TRAP proteins. We found that palmitoylation is essential for some, but not all, TRAP proteins independent of their TMD length. We also provide evidence that palmitoylation and proximal sequences can modulate sorting of artificial proteins with TMDs of suboptimal length. Our observations point to a unique character of each TMD defined by its primary amino acid sequence and its impact on membrane protein localisation. We conclude that, in addition to the TMD length, secondary sorting determinants such as palmitoylation or flanking sequences have evolved for the localisation of membrane proteins.

Nucleotide sequence conservation of novel and established cis-regulatory sites within the tyrosine hydroxylase gene promoter.

Tyrosine hydroxylase (TH) is the rate-limiting enzyme in catecholamine biosynthesis and its gene proximal promoter ( < 1 kb upstream from the transcription start site) is essential for regulating transcription in both the developing and adult nervous systems. Several putative regulatory elements within the TH proximal promoter have been reported, but evolutionary conservation of these elements has not been thoroughly investigated. Since many vertebrate species are used to model development, function and disorders of human catecholaminergic neurons, identifying evolutionarily conserved transcription regulatory mechanisms is a high priority. In this study, we align TH proximal promoter nucleotide sequences from several vertebrate species to identify evolutionarily conserved motifs. This analysis identified three elements (a TATA box, cyclic AMP response element (CRE) and a 5'-GGTGG-3' site) that constitute the core of an ancient vertebrate TH promoter. Focusing on only eutherian mammals, two regions of high conservation within the proximal promoter were identified: a ∼250 bp region adjacent to the transcription start site and a ∼85 bp region located approximately 350 bp further upstream. Within both regions, conservation of previously reported cis-regulatory motifs and human single nucleotide variants was evaluated. Transcription reporter assays in a TH -expressing cell line demonstrated the functionality of highly conserved motifs in the proximal promoter regions and electromobility shift assays showed that brain-region specific complexes assemble on these motifs. These studies also identified a non-canonical CRE binding (CREB) protein recognition element in the proximal promoter. Together, these studies provide a detailed analysis of evolutionary conservation within the TH promoter and identify potential cis-regulatory motifs that underlie a core set of regulatory mechanisms in mammals.

Genomic mapping of the MHC transactivator CIITA using an integrated ChIP-seq and genetical genomics approach.

BackgroundThe master transactivator CIITA is essential to the regulation of Major Histocompatibility Complex (MHC) class II genes and an effective immune response. CIITA is known to modulate a small number of non-MHC genes involved in antigen presentation such as CD74 and B2M but its broader genome-wide function and relationship with underlying genetic diversity has not been resolved.ResultsWe report the first genome-wide ChIP-seq map for CIITA and complement this by mapping inter-individual variation in CIITA expression as a quantitative trait. We analyse CIITA recruitment for pathophysiologically relevant primary human B cells and monocytes, resting and treated with interferon-gamma, in the context of the epigenomic regulatory landscape and DNA-binding proteins associated with the CIITA enhanceosome including RFX, CREB1/ATF1 and NFY. We confirm recruitment to proximal promoter sequences in MHC class II genes and more distally involving the canonical CIITA enhanceosome. Overall, we map 843 CIITA binding intervals involving 442 genes and find 95% of intervals are located outside the MHC and 60% not associated with RFX5 binding. Binding intervals are enriched for genes involved in immune function and infectious disease with novel loci including major histone gene clusters. We resolve differentially expressed genes associated in trans with a CIITA intronic sequence variant, integrate with CIITA recruitment and show how this is mediated by allele-specific recruitment of NF-kB.ConclusionsOur results indicate a broader role for CIITA beyond the MHC involving immune-related genes. We provide new insights into allele-specific regulation of CIITA informative for understanding gene function and disease.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0494-z) contains supplementary material, which is available to authorized users.

Targeted sequencing by proximity ligation for comprehensive variant detection and local haplotyping.

Despite developments in targeted gene sequencing and whole-genome analysis techniques, the robust detection of all genetic variation, including structural variants, in and around genes of interest and in an allele-specific manner remains a challenge. Here we present targeted locus amplification (TLA), a strategy to selectively amplify and sequence entire genes on the basis of the crosslinking of physically proximal sequences. We show that, unlike other targeted re-sequencing methods, TLA works without detailed prior locus information, as one or a few primer pairs are sufficient for sequencing tens to hundreds of kilobases of surrounding DNA. This enables robust detection of single nucleotide variants, structural variants and gene fusions in clinically relevant genes, including BRCA1 and BRCA2, and enables haplotyping. We show that TLA can also be used to uncover insertion sites and sequences of integrated transgenes and viruses. TLA therefore promises to be a useful method in genetic research and diagnostics when comprehensive or allele-specific genetic information is needed.

DNA hypermethylation of the forkhead box protein 3 (FOXP3) promoter in CD4+ T cells of patients with systemic sclerosis

Systemic sclerosis (SSc) is a complex autoimmune disease that involves dysregulation of immune homeostasis. The failure of impaired regulatory T cells (Tregs) to maintain immune homeostasis plays a major role in the development of SSc. Transcriptional silencing of the forkhead box protein 3 gene (FOXP3) via hypermethylation of regulatory regions has been identified as a hallmark of committed Tregs and several autoimmune disorders. To investigate whether aberrant expression and methylation of FOXP3 occurs in CD4+ T cells of patients with SSc and their roles in the pathogenesis of SSc. FOXP3 expression in CD4+ T cells was measured by real-time quantitative reverse-itranscriptase polymerase chain reaction and western blot. Bisulfite sequencing was performed to determine the methylation status of the FOXP3 proximal promoter sequence. The percentage of Treg cells was estimated by flow cytometry. Decreased FOXP3 expression was observed in CD4+ T cells from patients with SSc. The methylation levels of the FOXP3 regulatory sequences were elevated and inversely correlated with FOXP3 mRNA expression in patients with SSc. The number of Tregs was significantly reduced in patients with SSc. Treatment of SSc CD4+ T cells with a DNA methylation inhibitor, 5-azacytidine, reduced the mean methylation levels, and enhanced FOXP3 expression and Treg generation. The promoter methylation status and expression level of FOXP3 are significantly associated with disease activity. The contribution of the hypermethylation of the FOXP3 promoter to decreased FOXP3 expression and the subsequent quantitative defects of Tregs may mediate the immune dysfunction in SSc.

Personalized pharmacogenomics profiling using whole-genome sequencing.

Pharmacogenomics holds promise to rationalize drug use by minimizing drug toxicity and at the same time increase drug efficacy. There are currently several assays to screen for known pharmacogenomic biomarkers for the most commonly prescribed drugs. However, these genetic screening assays cannot account for other known or novel pharmacogenomic markers. We analyzed whole-genome sequences of 482 unrelated individuals of various ethnic backgrounds to obtain their personalized pharmacogenomics profiles. Bioinformatics analysis revealed 408,964 variants in 231 pharmacogenes, from which 26,807 were residing on exons and proximal regulatory sequences, whereas 16,487 were novel. In silico analyses indicated that 1012 novel pharmacogene-related variants possibly abolish protein function. We have also performed whole-genome sequencing analysis in a seven-member family of Greek origin in an effort to explain the variable response rate to acenocoumarol treatment in two family members. Overall, our data demonstrate that whole-genome sequencing, unlike conventional genetic screening methods, is necessary to determine an individual's pharmacogenomics profile in a more comprehensive manner, which, combined with the gradually decreasing whole-genome sequencing costs, would expedite bringing personalized medicine closer to reality.

Variability of PSPAL1 (phenylalanine ammonia-lyase gene-1) proximal promoter sequence and expression in pea challenged with Mycosphaerella pinodes

Part of the PSPAL1 gene (corresponding to the proximal promoter, exon 1 and intron) from eight pea varieties was sequenced and compared to the published sequence of PSPAL1 gene from Midoriusui cultivar (GenBank: D10002.1). The sequences showed a very high level of identity (96&amp;ndash;99%), except in five varieties there occurred a motif TTATTACAAAATATTA close to the Goldberg-Hogness (TATA) box, and it was not detected in the other four varieties, including Midoriusui. Plants of eight pea varieties were subjected to controlled infection with Mycosphaerella pinodes and the disease index was determined (it ranged from 5.2 to 42.3%). The PSPAL1 gene of the most resistant cultivar (Walor) contained the above-mentioned motif and that of the most susceptible (Polar) did not. However, the relationship was not clear in varieties with intermediate levels of resistance. In four varieties (Walor, Ezop, Ramrod and Polar) the expression level of PSPAL1 gene in leaves was analysed (1, 3, 6, 9, 12 and 15 h post inoculation) and it showed a weak negative correlation with disease severity (R= &amp;ndash; 0.53). The activation of PSPAL1 gene occurred not only in infected pea leaves but also in stems and &amp;ndash; to a much lower degree &amp;ndash; in roots (with the relative level of PSPAL1 transcripts amounting to 0.15 in roots and 38.75 in leaves), indicating some kind of signal transmission beyond the infected plant tissues.

RNA Polymerase III promoter screen uncovers a novel noncoding RNA family conserved in Caenorhabditis and other clade V nematodes.

RNA Polymerase III is a highly specialized enzyme complex responsible for the transcription of a very distinct set of housekeeping noncoding RNAs including tRNAs, 7SK snRNA, Y RNAs, U6 snRNA, and the RNA components of RNaseP and RNaseMRP. In this work we have utilized the conserved promoter structure of known RNA Polymerase III transcripts consisting of characteristic sequence elements termed proximal sequence elements (PSE) A and B and a TATA-box to uncover a novel RNA Polymerase III-transcribed, noncoding RNA family found to be conserved in Caenorhabditis as well as other clade V nematode species. Homology search in combination with detailed sequence and secondary structure analysis revealed that members of this novel ncRNA family evolve rapidly, and only maintain a potentially functional small stem structure that links the 5' end to the very 3' end of the transcript and a small hairpin structure at the 3' end. This is most likely required for efficient transcription termination. In addition, our study revealed evidence that canonical C/D box snoRNAs are also transcribed from a PSE A-PSE B-TATA-box promoter in Caenorhabditis elegans.

The proximal promoter of a novel interleukin-8-encoding gene in rainbow trout (Oncorhynchus mykiss) is strongly induced by CEBPA, but not NF-κB p65

Interleukin-8 (IL8) is an immediate-early chemokine that has been well characterized in several fish species. Ten IL8 gene variants have already been described in rainbow trout, but none of their promoters has structurally been defined or functionally characterized in teleost fish. To uncover key factors regulating IL8 expression, we intended to functionally characterize an IL8 promoter from rainbow trout. Incidentally, we isolated a novel IL8 gene variant (IL8-G). It is structurally highly similar to the other trout IL8 gene variants and its mRNA concentration increased significantly in secondary lymphoid tissues after infecting healthy fish with Aeromonas salmonicida. The proximal promoter sequence of the IL8-G-encoding gene features in close proximity two consensus elements for CEBP attachment. The proximal site overlaps with a NF-κB-binding site. Cotransfection of an IL8-G promoter-driven reporter gene together with vectors expressing various mammalian CEBP or NF-κB factors revealed in human HEK-293 cells that CEBPA and NF-κB p50, but not NF-κB p65 activate this promoter. The stimulatory effect of NF-κB p50 is likely conveyed by synergizing with CEBPA. Deletion or mutation of either the distal or the proximal CEBP-binding site, respectively, caused a significant decrease in IL8-G promoter activation. We confirmed the significance of the CEBPA factor for IL8-G expression by comparing the stimulatory capacity of the trout CEBPA and -B factors, thereby reducing the evolutionary distance in the inter-species expression assays. Similar promoter induction potential and intracellular localization of the mammalian and teleostean CEBPA and -B factors suggests their functional conservation throughout evolution.

The Rhinovirus Subviral A-Particle Exposes 3′-Terminal Sequences of Its Genomic RNA

Enteroviruses, which represent a large genus within the family Picornaviridae, undergo important conformational modifications during infection of the host cell. Once internalized by receptor-mediated endocytosis, receptor binding and/or the acidic endosomal environment triggers the native virion to expand and convert into the subviral (altered) A-particle. The A-particle is lacking the internal capsid protein VP4 and exposes N-terminal amphipathic sequences of VP1, allowing for its direct interaction with a lipid bilayer. The genomic single-stranded (+)RNA then exits through a hole close to a 2-fold axis of icosahedral symmetry and passes through a pore in the endosomal membrane into the cytosol, leaving behind the empty shell. We demonstrate that in vitro acidification of a prototype of the minor receptor group of common cold viruses, human rhinovirus A2 (HRV-A2), also results in egress of the poly(A) tail of the RNA from the A-particle, along with adjacent nucleotides totaling ∼700 bases. However, even after hours of incubation at pH 5.2, 5'-proximal sequences remain inside the capsid. In contrast, the entire RNA genome is released within minutes of exposure to the acidic endosomal environment in vivo. This finding suggests that the exposed 3'-poly(A) tail facilitates the positioning of the RNA exit site onto the putative channel in the lipid bilayer, thereby preventing the egress of viral RNA into the endosomal lumen, where it may be degraded. For host cell infection, a virus transfers its genome from within the protective capsid into the cytosol; this requires modifications of the viral shell. In common cold viruses, exit of the RNA genome is prepared by the acidic environment in endosomes converting the native virion into the subviral A-particle. We demonstrate that acidification in vitro results in RNA exit starting from the 3'-terminal poly(A). However, the process halts as soon as about 700 bases have left the viral shell. Conversely, inside the cell, RNA egress completes in about 2 min. This suggests the existence of cellular uncoating facilitators.

Down-Regulation of UNC5D in Bladder Cancer: UNC5D as a Possible Mediator of Cisplatin Induced Apoptosis in Bladder Cancer Cells

Identifying potential targets would improve therapeutic planning anddisease management. Therefore, we investigated whether the novel identified dependence receptor UNC5D acts as a tumor suppressor in bladder malignancies. We assessed the UNC5D level in a panel of 15 primary bladder carcinomas and 6 cell lines using real-time reverse transcriptase-polymerase chain reaction and Western blot. MTT assay, TUNEL staining, colony formation assay and Western blot were done in cells untransfected and transfected with UNC5D vector, siUNC5D or siDAPK. UNC5D was dramatically down-regulated in bladder cancer tissue samples and malignant cell lines. Restoration of UNC5D expression in bladder cancer cells lacking endogenous UNC5D expression suppressed cell proliferation and survival. Cisplatin treatment significantly induced UNC5D expression and DAPK dephosphorylation while UNC5D knockdown decreased bladder cancer cell sensitivity to cisplatin. DAPK silencing significantly inhibited the effect of UNC5D on apoptosis induced by cisplatin. Our study suggests that UNC5D may have important roles as a novel suppressor in bladder cancer via the UNC5D/DAPK pathway.

Porcine glucocorticoid receptor (NR3C1) gene: Tissue-specificity of transcriptional strength and glucocorticoid responsiveness of alternative promoters

Glucocorticoid receptor (GR) is transcribed in a tissue- and cell-specific manner with multiple exon 1 mRNA variants driven by selective promoters. We recently cloned and characterized the 5.3kb proximal promoter sequence of porcine GR gene containing 7 untranslated alternative first exons each processed by a distinct promoter. In this study, we showed tissue-specific expression of total GR and its exon 1 mRNA variants in hippocampus, muscle and liver of pigs. Total GR mRNA was most abundant in liver, followed by muscle and hippocampus in descending order. Among all the GR exon 1 mRNA variants detected, GR exon 1–9/10 and 1–4 were the most predominant variants in all the three tissues. The abundance of GR exon 1–4 mRNA was similar to that of 1–10 in muscle, but was significantly lower than 1–10 in liver and hippocampus. The activities of truncated short (S) and long (L) promoters of respective GR exon 1 mRNA variants were analyzed by luciferase reporter assay in 3 representative cell lines, SY5Y, C2C12 and HepG2. S1–10 and S1–4 demonstrated significantly higher activities than other short promoters in all the cell lines examined. Nevertheless, the strongest activity and cell specificity were detected for L1–10 promoter, which was consistent with the predominant exon 1–9/10 expression in porcine tissues. Moreover, with 3 potential nGRE binding sites, L1–10 promoter was more sensitive to dexamethasone (DEX) in HepG2. Our data provide basic knowledge of the transcriptional mechanism underlying the tissue- and cell-specific expression of porcine GR under basal or ligand-stimulated conditions.

Postmitral Valve Replacement Biatrial, Septal Macroreentrant Atrial Tachycardia Developing After Perimitral Flutter Ablation

Atypical macroreentrant atrial tachycardias are frequently encountered in patients after cardiac surgery, correction of congenital heart disease, or atrial fibrillation ablation. Ablation of one circuit can produce an abrupt change in atrial activation sequence giving rise to another macroreentrant circuit. In this report, we present a unique case of a biatrial septal macroreentrant atrial tachycardia, which developed after perimitral flutter ablation in a patient with mitral valve replacement. Editor’s Perspective see p 175 A 59-year-old man with a mitral valve replacement (bioprosthesis) in 1983 for a rheumatic mitral stenosis and a rereplacement in 1999 (right-sided approach with a vertical incision of the interatrial septum) with a mechanical prosthesis attributable to the degeneration of the former prosthesis was referred for the ablation of a sustained atrial tachycardia with a stable cycle length of 260 ms. The echocardiogram demonstrated a left ventricular ejection fraction of 45% to 50%, a moderately dilated right atrium (RA) as well as left atrium (LA) without signs of a prosthetic malfunction. The baseline surface 12-lead ECG showed an atypical atrial flutter with a regular, monomorphic atrial activity and an irregular atrioventricular conduction (Figure 1A). With broad positive flutter waves in unipolar lead V1 and inferior limb leads II, III and avF but negative flutter waves in limb leads I and avL, the ECG morphology suggested LA localization. Any significant 12-lead isoelectric interval was absent. Endocardial activation mapping with a quadripolar catheter in the RA and a steerable octapolar catheter in the coronary sinus (CS; Bard Medical, Covington, GA) demonstrated a distal to proximal activation sequence in the CS with a baseline cycle length of 260 ms, suggestive of a lateral to septal activation of the LA posterior wall (Figure 1B). An irrigated tip ablation catheter (Navistar Thermocool, Biosense Webster, Diamond Bar) was advanced into the LA via …

Genomic mapping of the MHC transactivator CIITA using an integrated ChIP-seq and genetical genomics approach

The master transactivator CIITA is essential to the regulation of Major Histocompatibility Complex (MHC) class II genes and an effective immune response. CIITA is known to modulate a small number of non-MHC genes involved in antigen presentation such as CD74 and B2M but its broader genome-wide function and relationship with underlying genetic diversity has not been resolved. We report the first genome-wide ChIP-seq map for CIITA and complement this by mapping inter-individual variation in CIITA expression as a quantitative trait. We analyse CIITA recruitment for pathophysiologically relevant primary human B cells and monocytes, resting and treated with interferon-gamma, in the context of the epigenomic regulatory landscape and DNA-binding proteins associated with the CIITA enhanceosome including RFX, CREB1/ATF1 and NFY. We confirm recruitment to proximal promoter sequences in MHC class II genes and more distally involving the canonical CIITA enhanceosome. Overall, we map 843 CIITA binding intervals involving 442 genes and find 95% of intervals are located outside the MHC and 60% not associated with RFX5 binding. Binding intervals are enriched for genes involved in immune function and infectious disease with novel loci including major histone gene clusters. We resolve differentially expressed genes associated in trans with a CIITA intronic sequence variant, integrate with CIITA recruitment and show how this is mediated by allele-specific recruitment of NF-kB. Our results indicate a broader role for CIITA beyond the MHC involving immune-related genes. We provide new insights into allele-specific regulation of CIITA informative for understanding gene function and disease.