Exon 1C Research Articles

Corticosterone induces discrete epigenetic signatures in the dorsal and ventral hippocampus that depend upon sex and genotype: focus on methylated Nr3c1 gene

The genomic effects of circulating glucocorticoids are particularly relevant in cortico-limbic structures, which express a high concentration of steroid hormone receptors. To date, no studies have investigated genomic differences in hippocampal subregions, namely the dorsal (dHPC) and ventral (vHPC) hippocampus, in preclinical models treated with exogenous glucocorticoids. Chronic oral corticosterone (CORT) in mouse is a pharmacological approach that disrupts the activity of the hypothalamic-pituitary-adrenal axis, increases affective behavior, and induces genomic changes after stress in the HPC of wildtype (WT) mice and mice heterozygous for the gene coding for brain-derived neurotrophic factor Val66Met (hMet), a variant associated with genetic susceptibility to stress. Using RNA-sequencing, we investigated the genomic signatures of oral CORT in the dHPC and vHPC of WT and hMet male and female mice, and examined sex and genotype differences in response to oral CORT. Males under CORT showed lower glycemia and increased anxiety- and depression-like behavior compared to females that showed instead opposite affective behavior in response to CORT. Rank–rank-hypergeometric overlap (RRHO) was used to identify genes from a continuous gradient of significancy that were concordant across groups. RRHO showed that CORT-induced differentially expressed genes (DEGs) in WT mice and hMet mice converged in the dHPC of males and females, while in the vHPC, DEGs converged in males and diverged in females. The vHPC showed a higher number of DEGs compared to the dHPC and exhibited sex differences related to glucocorticoid receptor (GR)-binding genes and epigenetic modifiers. Methyl-DNA-immunoprecipitation in the vHPC revealed differential methylation of the exons 1C and 1F of the GR gene (Nr3c1) in hMet females. Together, we report behavioral and endocrinological sex differences in response to CORT, as well as epigenetic signatures that i) differ in the dHPC and vHPC,ii) are distinct in males and females, and iii) implicate differential methylation of Nr3c1 selectively in hMet females.

A preferential switch between placental GR exon 1 promoter variants in the presence of maternal asthma or inflammation upregulates GRα D isoforms

IntroductionThe human placenta expresses multiple glucocorticoid receptor (GR) isoforms that may be partially regulated by the untranslated 5’ exon 1 GR gene promoter region which consists of 9 different promoters and 13 splice variants. The objective of this study was to determine which GR exon 1 variants are expressed in the human placenta and relate these findings to GR mRNA and protein expression. MethodsPlacental extracts from pregnancies with or without the complication of maternal asthma and trophoblast cells exposed to an inflammatory challenge in vitro were examined using PCR and Western blot to measure GR exon 1 variants, GR splice variant mRNA and GR protein isoforms, respectively. ResultsAll 9 GR exon 1 variants were detectable in the human placenta and included GR exons 1A, 1B, 1C, 1D, 1E, 1F, 1H, 1I and 1J. In the presence of maternal asthma and a male fetus there was preferential expression of GR exon 1B, 1C, IF and 1J (KW-ANOVA, P < 0.05) which were positively correlated with GRα D3 protein isoform. In female placentae from pregnancies complicated by asthma there was no upregulation of any exon 1 variant (KW-ANOVA, P < 0.05). Exposure of BeWo trophoblast cell line to an inflammatory challenge, lipopolysaccharide, in vitro, resulted in preferential expression of GR exon 1B, 1D, 1E and 1H and associated with GRα-D1 protein upregulation. DiscussionThe preferential expression of different GR exon 1 promoters drive the upregulation of GRα D isoforms and contribute to glucocorticoid resistance observed in male placentae of pregnancies complicated by asthma.

Crosstalk between vrille transcripts, proteins, and regulatory elements controlling circadian rhythms and development in Drosophila.

SummaryThe vrille (vri) gene encodes a transcriptional repressor required for Drosophila development as well as circadian behavior in adults. Alternate first exons produce vri transcripts predicted to produce a short VRI isoform during development and long VRI in adults. A vri mutant (vriΔ679) lacking long VRI transcripts is viable, confirming that short VRI is sufficient for developmental functions, yet behavioral rhythms in vriΔ679 flies persist, showing that short VRI is sufficient for clock output. E-box regulatory elements that drive rhythmic long VRI transcript expression are required for developmental expression of short VRI transcripts. Surprisingly, long VRI transcripts primarily produce short VRI in adults, apparently due to a poor Kozak sequence context, demonstrating that short VRI drives circadian behavior. Thus, E-box-driven long VRI transcripts primarily control circadian rhythms via short VRI, whereas the same E-boxes drive short VRI transcripts that control developmental functions using short VRI.

Hepatocyte nuclear factor-4α regulates expression of the serotonin transporter in intestinal epithelial cells.

The serotonin transporter (SERT) functions to regulate the availability of serotonin (5-HT) in the brain and intestine. An intestine-specific mRNA variant arising from a unique transcription start site and alternative promoter in the SERT gene has been identified (iSERT; spanning exon 1C). A decrease in SERT is implicated in several gut disorders, including inflammatory bowel diseases (IBD). However, little is known about mechanisms regulating the iSERT variant, and a clearer understanding is warranted for targeting SERT for the treatment of gut disorders. The current studies examined the expression of iSERT across different human intestinal regions and investigated its regulation by HNF4α (hepatic nuclear factor-4α), a transcription factor important for diverse cellular functions. iSERT mRNA abundance was highest in the human ileum and Caco-2 cell line. iSERT mRNA expression was downregulated by loss of HNF4α (but not HNF1α, HNF1β, or FOXA1) in Caco-2 cells. Overexpression of HNF4α increased iSERT mRNA concomitant with an increase in SERT protein. Progressive promoter deletion and site-directed mutagenesis revealed that the HNF4α response element spans nucleotides -1,163 to -1150 relative to the translation start site. SERT mRNA levels in the intestine were drastically reduced in the intestine-specific HNF4α-knockout mice relative to HNF4αFL/FL mice. Both HNF4α and SERT mRNA levels were also downregulated in mouse model of ileitis (SAMP) compared with AKR control mice. These results establish the transcriptional regulation of iSERT at the gut-specific internal promoter (hSERTp2) and have identified HNF4α as a critical modulator of basal SERT expression in the intestine.

Vitamin D receptor gene is epigenetically altered and transcriptionally up-regulated in multiple sclerosis.

ObjectiveVitamin D deficiency has been linked to increased risk of multiple sclerosis (MS) and poor outcome. However, the specific role that vitamin D plays in MS still remains unknown. In order to identify potential mechanisms underlying vitamin D effects in MS, we profiled epigenetic changes in vitamin D receptor (VDR) gene to identify genomic regulatory elements relevant to MS pathogenesis.MethodsHuman T cells derived from whole blood by negative selection were isolated in a set of 23 relapsing-remitting MS (RRMS) patients and 12 controls matched by age and gender. DNA methylation levels were assessed by bisulfite cloning sequencing in two regulatory elements of VDR. mRNA levels were measured by RT-qPCR to assess changes in VDR expression between patients and controls.ResultsAn alternative VDR promoter placed at exon 1c showed increased DNA methylation levels in RRMS patients (median 30.08%, interquartile range 19.2%) compared to controls (18.75%, 9.5%), p-value<0.05. Moreover, a 6.5-fold increase in VDR mRNA levels was found in RRMS patients compared to controls (p-value<0.001).ConclusionsAn alternative promoter of the VDR gene shows altered DNA methylation levels in patients with multiple sclerosis, and it is associated with VDR mRNA upregulation. This locus may represent a candidate regulatory element in the genome relevant to MS pathogenesis.

DNA Methylation Analysis of BRD1 Promoter Regions and the Schizophrenia rs138880 Risk Allele.

The bromodomain containing 1 gene, BRD1 is essential for embryogenesis and CNS development. It encodes a protein that participates in histone modifying complexes and thereby regulates the expression of a large number of genes. Genetic variants in the BRD1 locus show association with schizophrenia and bipolar disorder and risk alleles in the promoter region correlate with reduced BRD1 expression. Insights into the transcriptional regulation of BRD1 and the pathogenic mechanisms associated with BRD1 risk variants, however, remain sparse. By studying transcripts in human HeLa and SH-SY5Y cells we provide evidence for differences in relative expression of BRD1 transcripts with three alternative 5’ UTRs (exon 1C, 1B, and 1A). We further show that expression of these transcript variants covaries negatively with DNA methylation proportions in their upstream promoter regions suggesting that promoter usage might be regulated by DNA methylation. In line with findings that the risk allele of the rs138880 SNP in the BRD1 promoter region correlates with reduced BRD1 expression, we find that it is also associated with moderate regional BRD1 promoter hypermethylation in both adipose tissue and blood. Importantly, we demonstrate by inspecting available DNA methylation and expression data that these regions undergo changes in methylation during fetal brain development and that differences in their methylation proportions in fetal compared to postnatal frontal cortex correlate significantly with BRD1 expression. These findings suggest that BRD1 may be dysregulated in both the developing and mature brain of risk allele carriers. Finally, we demonstrate that commonly used mood stabilizers Lithium, Valproate, and Carbamazepine affect the expression of BRD1 in SH-SY5Y cells. Altogether this study indicates a link between genetic risk and epigenetic dysregulation of BRD1 which raises interesting perspectives for targeting the mechanisms pharmacologically.

Molecular Mechanism for Synergistic Anti-Leukemic Activity of Tyrosine Kinase Inhibitors and Glucocorticoids Against Ph+ALL

The tyrosine kinase inhibitors (TKIs) have dramatically altered the management of patients with Philadelphia chromosome-positive ALL (Ph+ALL). In earlier trials of imatinib monotherapy for relapsed or refractory patients with lymphoid blast crisis of CML (CML-LC) and Ph+ALL, complete hematologic remission (CHR) was achieved only in 20%, and relapses occurred in most of the patients within several months. Thereafter, combination induction therapy of imatinib with conventional chemotherapeutic agents profoundly improved the therapeutic outcome in the patients with Ph+ALL. In elderly patients, to reduce the therapy-related toxicities, combination induction therapy of imatinib with glucocorticoids (GCs) followed by imatinib monotherapy was performed. Surprisingly, CHR was obtained in all of the patients with this simple therapy, and median survival from diagnosis was 20 months (Blood 2007). Similarly, induction therapy of dasatinib, a second-generation TKI, combined with GCs achieved CHR in all of the newly diagnosed Ph+ALL patients (Blood 2011). These clinical findings indicate a synergistic anti-leukemic activity of TKIs with GCs in Ph+ALL, but its underlying molecular mechanisms remain totally unknown. Thus, we analyzed synergistic effects of TKIs and dexamethasone (Dex) in a panel of leukemic cell lines derived from Ph+ALL. Indeed, in the presence of 0.5μM of imatinib, IC50 values of Dex were approximately 3-8 times lower than those in the absence of imatinib in the most of Dex-sensitive Ph+ALL cell lines. Since gene expression level of GC receptor (GR; NR3C1) was associated with Dex-sensitivity in Ph+ALL cell lines, we next analyzed the effects of imatinib on gene expression level of NR3C1. Of note, NR3C1 gene expression level was significantly upregulated in the presence of 0.5 μM of imatinib approximately 1.5-7-fold in all of 14 Ph+ALL cell lines except for SK9, which was an imatinib-resistant cell line having a T315I mutation of BCR-ABL, whereas it was unchanged in all of 11 Ph-negative ALL cell lines. Induction of GR was also confirmed by immuno-blotting in representative Ph+ALL cell lines. Moreover, treatment with either dasatinib or nilotinib clearly upregulated the NR3C1 gene expression in the representative Ph+ALL cell lines. Of importance, although gene expression level of NR3C1 was significantly upregulated in the presence of imatinib in the imatinib-sensitive Ph+ALL cell lines, SU-Ph2 and TCCY, it was unchanged in their imatinib-resistant sublines, SU/SR and TCCY/SR, respectively, in which T315I mutation was acquired after the culture with increasing concentrations of imatinib, indicating that upregulation of GR by TKIs in Ph+ALL cell lines was mediated by an inactivation of BCR-ABL. To further verify the downstream pathway of BCR-ABL that is critically involved in the TKI-induced GR upregulation, we treated imatinib-sensitive SU-Ph2 and its imatinib-resistant subline SU/SR with specific inhitors of PI3K (GDC0941, LY294002, and AS606240), JAK2(SD1029), and MAPK(UO126). Among five agents, only UO126 effectively upregulated the NR3C1 gene expression both in SU-Ph2 and in SU/SR, suggesting that TKIs upregulate GR in Ph+ALL cell lines mainly through an inactivation of the MAPK pathway. Previous reports revealed that three promoters, 1A, 1B, and 1C, are mainly involved in the NR3C1 gene expression in ALL cells. We therefore performed real time RT-PCR analysis of the NR3C1 gene using three sets of primers that are specific for exons 1A3, 1B, or 1C. The strongest induction by an imatinib-treatment was observed in the 1A promoter in Ph+ALL cell lines. Finally, since BIM, one of BH3-only pro-apoptotic members of BCL2 family, has been reported to be critically involved in the anti-leukemic activities of both TKIs and GCs, we analyzed BIM expression. Synergistic induction of BIM was confirmed both in mRNA and protein expression levels by a simultaneously treatment of Ph+ALL cell lines with imatinib and Dex. Taken together, these observations in Ph+ALL cell lines indicate that TKIs induce GR expression in Ph+ALL mainly through the MAPK pathway and the 1A promoter of NR3C1 gene by inactivating BCR-ABL and subsequently exert a synergistic anti-leukemic activity with GCs through the induction of BIM. DisclosuresNo relevant conflicts of interest to declare.

Association of genetic polymorphisms of Calpain and Calpastatin genes with growth traits in Bandur sheep

The study was undertaken to determine genetic polymorphisms of Calpain and Calpastatin genes and to study their association with growth traits (birth weight, weaning weight, 6-month body weight and ADG) in Bandur ram lambs (100) distributed over core area of the home tract as well as from Livestock Research and Information Centre (Sheep), KVAFSU, Nagamangala. Miller’s high salt method was used for isolation of genomic DNA from venous blood. A 192 bp of CAPN (exons 5 and 6) and 620 bp of CAST (exons 1C/1D) genes were amplified by PCR using published primers. The PCR-SSCP analysis of CAPN gene revealed genotype frequency for AA (0.672) and AB (0.295) and allele frequency for A (0.820) and B (0.180), respectively. Alignment of A and B allele of Calpain by CLC Main Workbench 6.8.1 showed addition/insertion of one nucleotide ‘A’ in B allele. The PCR-RFLP analysis of CAST gene revealed frequency for genotypes MM (0.24), MN (0.59) and NN (0.17), and for alleles M (0.535) and N (0.465), respectively. The Chi-square test indicated that the studied population was in Hardy-Weinberg equilibrium. Five SNPs were identified in the intron between exon 1C and 1D of CAST. The association of CAPN and CAST genotypes with growth traits was analysed by ANOVA using GLM procedures of SAS 9.3. No significant associations were observed between polymorphisms of CAPN and CAST loci and growth traits.

The role of isoforms in the evolution of cryptic coloration in Peromyscus mice.

A central goal of evolutionary biology is to understand the molecular mechanisms underlying phenotypic adaptation. While the contribution of protein-coding and cis-regulatory mutations to adaptive traits has been well documented, additional sources of variation - such as the production of alternative RNA transcripts from a single gene, or isoforms - have been understudied. Here, we focus on the pigmentation gene Agouti, known to express multiple alternative transcripts, to investigate the role of isoform usage in the evolution of cryptic colour phenotypes in deer mice (genus Peromyscus). We first characterize the Agouti isoforms expressed in the Peromyscus skin and find two novel isoforms not previously identified in Mus. Next, we show that a locally adapted light-coloured population of P.maniculatus living on the Nebraska Sand Hills shows an upregulation of a single Agouti isoform, termed 1C, compared with their ancestral dark-coloured conspecifics. Using invitro assays, we show that this preference for isoform 1C may be driven by isoform-specific differences in translation. In addition, using an admixed population of wild-caught mice, we find that variation in overall Agouti expression maps to a region near exon 1C, which also has patterns of nucleotide variation consistent with strong positive selection. Finally, we show that the independent evolution of cryptic light pigmentation in a different species, P.polionotus, has been driven by a preference for the same Agouti isoform. Together, these findings present an example of the role of alternative transcript processing in adaptation and demonstrate molecular convergence at the level of isoform regulation.

SREBP2 Activation Induces Hepatic Long-chain Acyl-CoA Synthetase 1 (ACSL1) Expression in Vivo and in Vitro through a Sterol Regulatory Element (SRE) Motif of the ACSL1 C-promoter

Long-chain acyl-CoA synthetase 1 (ACSL1) plays a key role in fatty acid metabolism. To identify novel transcriptional modulators of ACSL1, we examined ACSL1 expression in liver tissues of hamsters fed a normal diet, a high fat diet, or a high cholesterol and high fat diet (HCHFD). Feeding hamsters HCHFD markedly reduced hepatic Acsl1 mRNA and protein levels as well as acyl-CoA synthetase activity. Decreases in Acsl1 expression strongly correlated with reductions in hepatic Srebp2 mRNA level and mature Srebp2 protein abundance. Conversely, administration of rosuvastatin (RSV) to hamsters increased hepatic Acsl1 expression. These new findings were reproduced in mice treated with RSV or fed the HCHFD. Furthermore, the RSV induction of acyl-CoA activity in mouse liver resulted in increases in plasma and hepatic cholesterol ester concentrations and reductions in free cholesterol amounts. Investigations on different ACSL1 transcript variants in HepG2 cells revealed that the mRNA expression of C-ACSL1 was specifically regulated by the sterol regulatory element (SRE)-binding protein (SREBP) pathway, and RSV treatment increased the C-ACSL1 abundance from a minor mRNA species to an abundant transcript. We analyzed 5'-flanking sequence of exon 1C of the human ACSL1 gene and identified one putative SRE site. By performing a promoter activity assay and DNA binding assays, we firmly demonstrated the key role of this SRE motif in SREBP2-mediated activation of C-ACSL1 gene transcription. Finally, we demonstrated that knockdown of endogenous SREBP2 in HepG2 cells lowered ACSL1 mRNA and protein levels. Altogether, this work discovered an unprecedented link between ACSL1 and SREBP2 via the specific regulation of the C-ACSL1 transcript.

Alternative transcripts of the SERPINA1 gene in alpha-1 antitrypsin deficiency.

BackgroundSERPINA1 is the gene for alpha-1 antitrypsin (AAT), an acute phase protein with anti-protease and immunoregulatory activities. Mutations in SERPINA1 gene cause AAT deficiency and predispose individuals to early-onset emphysema and liver diseases. Expression of the SERPINA1 gene is regulated by different promoters and alternative splicing events among non-coding exons 1A, 1B and 1C.MethodsWe have developed three quantitative PCR (QT-PCR) assays (1A, 1B and 1C). These assays were applied for the analysis of SERPINA1 alternative transcripts in: (1) 16 human tissues and (2) peripheral blood leukocytes from 33 subjects with AAT mutations and 7 controls.ResultsTissue-specific expression was found for the SERPINA1 transcripts. The 1A transcripts were mainly expressed in leukocytes and lung tissue while those detected with the 1B assay were highly restricted to leukocytes. Only 1B transcripts significantly correlated with serum AAT levels. The 1C transcripts were specifically found in lung, liver, kidney and pancreas. Furthermore, the expression of transcripts was related to AAT genotypes. While deficient variants of AAT had no pronounced effect on the transcript expression, null alleles were associated with significant reduction of different transcripts.ConclusionsThe possibility to discriminate between SERPINA1 alternative splicing products will help us to understand better the regulation of SERPINA1 gene and its association with SERPINA1 mutations-related diseases.Electronic supplementary materialThe online version of this article (doi:10.1186/s12967-015-0585-y) contains supplementary material, which is available to authorized users.

Transcription of human nNOS exon 1c is altered by a single nucleotide promoter polymorphism in patients with infantile hypertrophic pyloric stenosis

Transcription of human nNOS exon 1c is altered by a single nucleotide promoter polymorphism in patients with infantile hypertrophic pyloric stenosis

Identification of Novel Crk-associated Substrate (p130Cas) Variants with Functionally Distinct Focal Adhesion Kinase Binding Activities

Elevated levels of p130(Cas) (Crk-associated substrate)/BCAR1 (breast cancer antiestrogen resistance 1 gene) are associated with aggressiveness of breast tumors. Following phosphorylation of its substrate domain, p130(Cas) promotes the integration of protein complexes involved in multiple signaling pathways and mediates cell proliferation, adhesion, and migration. In addition to the known BCAR1-1A (wild-type) and 1C variants, we identified four novel BCAR1 mRNA variants, generated by alternative first exon usage (1B, 1B1, 1D, and 1E). Exons 1A and 1C encode for four amino acids (aa), whereas 1D and 1E encode for 22 aa and 1B1 encodes for 50 aa. Exon 1B is non-coding, resulting in a truncated p130(Cas) protein (Cas1B). BCAR1-1A, 1B1, and variant 1C mRNAs were ubiquitously expressed in cell lines and a survey of human tissues, whereas 1B, 1D, and 1E expression was more restricted. Reconstitution of all isoforms except for 1B in p130(Cas)-deficient murine fibroblasts induced lamellipodia formation and membrane ruffling, which was unrelated to the substrate domain phosphorylation status. The longer isoforms exhibited increased binding to focal adhesion kinase (FAK), a molecule important for migration and adhesion. The shorter 1B isoform exhibited diminished FAK binding activity and significantly reduced migration and invasion. In contrast, the longest variant 1B1 established the most efficient FAK binding and greatly enhanced migration. Our results indicate that the p130(Cas) exon 1 variants display altered functional properties. The truncated variant 1B and the longer isoform 1B1 may contribute to the diverse effects of p130(Cas) on cell biology and therefore will be the target of future studies.

Expression and Regulation of Intestine Specific Serotonin Transporter (iSERT) Variant

SERT is critical for regulating extracellular availability of serotonin (5-HT) and is a therapeutic target for the treatment for multiple disorders. A novel transcription start site and alternative promoter for SERT gene has recently been demonstrated in human intestine. Decrease in intestinal SERT and consequent high 5-HT levels are implicated in the pathophysiology of inflammatory and diarrheal disorders. Thus, characterization of this novel transcript is warranted for gut-specific SERT-targeted therapies. Therefore, tissue distribution and regulation of intestine specific SERT (iSERT) was investigated. SERT primers spanning exon 1c of human SERT gene were utilized. Human iSERT variant was highly expressed in the small intestine and placenta (Ileum>jejunum>placenta>duodenum) with low expression in colon and brain. Similarly, expression of iSERT mRNA was very low in colonic cell lines HT-29, LS174T, SK015, T84 as compared to Caco-2 (representing ileal phenotype upon differentiation). iSERT mRNA expression was not induced during Caco-2 cells differentiation. iSERT promoter (upstream of exon 2) has potential binding sites for HNF3α. To understand the regulatory mechanisms of this novel transcript, its modulation by hepatocyte nuclear factors (HNFs) was investigated. siRNA mediated knock down of HNF3α suppressed iSERT mRNA levels in Caco-2 cells. Over-expression of HNF4α (but not HNF1α or HNF1β) by Amaxa transfection increased iSERT mRNA levels (~30 fold, p<0.05) as compared to empty vector. These studies demonstrate that HNF3α and HN4α upregulate intestine specific SERT gene expression that may underlie anti-diarrheal or anti-inflammatory effects of HNFs. Supported by NIH & VA.

Isoform-specific imprinting of the MEST gene in porcine parthenogenetic fetuses

Aberrant expression of imprinted genes is the main reason for developmental retardation in mammalian parthenogenetic fetuses. Mesoderm specific transcript (MEST) is a maternally imprinted gene that is linked to cancer and is necessary for normal early embryonic development. Tissue and isoform-specific imprinting of MEST have been identified in humans and mice, but have not yet been identified in pigs. In this study, the three isoforms of porcine MEST were identified using the GenBank and Ensembl sequence databases. Then, we determined MEST isoform-specific mRNA expression and methylation levels by quantitative real-time PCR (qRT-PCR) and bisulfite sequencing PCR analysis (BSP) between porcine parthenogenetic (PA) and control (Con) fetuses, respectively. Altogether, our results demonstrated that the expression of MEST-1A and MEST-1B has no evident differences between PA and Con groups; however, there is no expression of MEST-1C in PA fetuses. In addition, the results of BSP showed that the hypermethylation of exon 1c of MEST-1C was observed in PA samples. Thus, we suggested that MEST-1C was isoform-specific imprinting, which may be contributed to differential methylation status of exon 1c in porcine fetuses.

Severe alpha-1 antitrypsin deficiency in composite heterozygotes inheriting a new splicing mutation QOMadrid.

BackgroundSevere Alpha-1 Antitrypsin (AAT) deficiency is a hereditary condition caused by mutations in the SERPINA1 gene, which predisposes to lung emphysema and liver disease. It is usually related to PI*Z alleles, and less frequent to rare and null (QO) alleles. Null-AAT alleles represent the end of a continuum of variants associated with profound AAT deficiency and extremely increased risk of emphysema.MethodsA family with severe AAT deficiency was analyzed to achieve genetic diagnosis. The complete exons and introns of the SERPINA1 gene were sequenced and transcriptional analysis by RT-PCR was performed to characterize the effect of splicing variants found in the patients. In addition, a minigene MGserpa1_ex1b-1c was cloned into the pSAD vector to in vitro investigate the independent impact of variants on splicing process.ResultsWe report a new identified null allele (PI*QOMadrid) in two adult siblings with practically no detectable serum AAT. The PI*QOMadrid allele consist of a duplication of the thymine (T) in position +2 of the donor splice site of exon 1C (+2dupT). In these two subjects, PI*QOMadrid occurred in compound heterozygote combination with the previously described variant PI*QOPorto. Both QOMadrid and QOPorto variants are located very close together in a regulatory region of the SERPINA1 gene. Analysis of transcripts revealed that QOMadrid variant prevented the expression of transcripts from exon 1C, and then normally spliced RNA products are not expected in the liver of these patients. In addition, aberrant splicing patterns of both variants were clearly distinguished and quantified by functional in vitro assays lending further support to their pathogenicity.ConclusionFinding pathogenic mutations in non-coding regions of the SERPINA1 highlight the importance that regulatory regions might have in the disease. Regulatory regions should be seriously considered in discordant cases with severe AAT deficiency where no coding mutations were found.Electronic supplementary materialThe online version of this article (doi:10.1186/s12931-014-0125-y) contains supplementary material, which is available to authorized users.

Transcription of the Human Sodium Channel SCN1A Gene Is Repressed by a Scaffolding Protein RACK1

Voltage-gated sodium channel α subunit type I (Nav1.1, encoded by SCN1A gene) plays a critical role in the initiation of action potential in the central nervous system. Downregulated expression of SCN1A is believed to be associated with epilepsy. Here, we found that the SCN1A promoter (P1c), located at the 5' untranslated exon 1c, drove the reporter gene expression in human NT2 cells, and a region between nt +53 and +62 downstream of the P1c promoter repressed the promoter activity. Further analyses showed that a scaffolding protein RACK1 (receptor for activated C kinase 1) was involved in binding to this silencer. Knockdown of RACK1 expression in NT2 cells deprived the repressive role of the silencer on the P1c promoter and increased SCN1A transcription, suggesting the potential involvement of RACK1 in negatively regulating SCN1A transcription via interaction with the silencer. Furthermore, we demonstrated that the binding of the protein complex including RACK1 to the SCN1A promoter motif was decreased in neuron-like differentiation of the NT2 cells induced by retinoic acid and resulted in the upregulation of SCN1A transcription. Taken together, this study reports a novel role of RACK1 in regulating SCN1A expression that participates in retinoic acid-induced neuronal differentiation of NT2 cells.

Transcript variants of the porcine glucocorticoid receptor gene (NR3C1)

Glucocorticoid receptor (GR) is a transcription factor activated by circulating glucocorticoids and mediates their effects on various biological functions in the body. The final cellular activity of GR is modulated by alternative splicing and cell-type specific expression of its encoding gene NR3C1. To enhance the current knowledge of alternative processing of NR3C1 in mammalian species and to facilitate future studies of its regulation in the pig we explored here structural diversity, and tissue-specific distribution of transcript variants of the porcine NR3C1, and the correlation between usage of alternative promoters and alternative splicing. We experimentally identified ten alternatively used untranslated first exons (1A, 1B, 1C, 1D, 1E, 1F, 1G, 1H, 1H2 and 1J) and four transcript variants encoding different GR subtypes (GR-alpha, GR-beta, GR-P and GR-gamma). Expression profiling of nine most important target tissues of glucocorticoids revealed that the promoter of exon 1C drives constitutive expression of the predominant GR-alpha subtype. We found compelling evidence that the occurrence of exon 1D influences abundance of the GR-P splice variant, while both seems to play an important role in regulating GR activity in neuroendocrine tissues. Exons 1A and 1B in turn appear to be important for the regulation of the expression of the porcine NR3C1 in liver and spleen. Our results demonstrate that tissue-specific actions of GR depend on the usage of alternative promoter regions that favour the processing of certain GR subtypes.

Methylation of the glucocorticoid receptor gene promoter in bulimic women: Associations with borderline personality disorder, suicidality, and exposure to childhood abuse

To compare levels of methylation of the glucocorticoid receptor (GR) gene (NR3C1) promoter between women with bulimia nervosa (BN) and women with no eating disorder (ED), and also to explore, in women with BN, the extent to which methylation of the GR gene promoter corresponds to childhood abuse, suicidality, or borderline personality disorder (BPD). We measured methylation levels in selected NR3C1 promoter regions using DNA obtained from lymphocytes in 64 women with BN (32 selected as having a history of severe childhood abuse and 32 selected as having no such history) and 32 comparison women with no ED or history of childhood abuse. Compared to noneating disordered women, women with BN and comorbid BPD (or BN with a history of suicidality) showed significantly more methylation of specific exon 1C sites. There was also a (nonsignificant) result indicative of greater methylation in some 1C sites among women with BN, when compared (as a group) to women with no ED. No parallel effects owing to childhood abuse were observed. Our findings associate BN (when accompanied by BPD or suicidality) with hypermethylation of certain GR exon 1C promoter sites. We discuss theoretical and clinical implications of our findings.

Epigenetic Clues to the Biological Embedding of Early Life Adversity

The concept of biological embedding has gained substantial traction as a framework for understanding the roots of complex multifactorial phenomena in health and disease. A body of research over several decades indicates that early life experiences have profound consequences for health in adulthood, including mental health, as a consequence of establishing long-term health gradients ( 1 Hertzman C. Boyce T. How experience gets under the skin to create gradients in developmental health. Annu Rev Public Health. 2010; 31: 329-347 Crossref PubMed Scopus (448) Google Scholar ). Early interventions have been proposed to have an enhanced impact on health trajectories in part because they act at a time of enhanced plasticity ( 2 Hanson M. Godfrey K.M. Lillycrop K.A. Burdge G.C. Gluckman P.D. Developmental plasticity and developmental origins of non-communicable disease: theoretical considerations and epigenetic mechanisms. Prog Biophys Mol Biol. 2010; 106: 272-280 Crossref Scopus (216) Google Scholar ). Early-life adversity in the form of physical and sexual abuse or severe neglect is well recognized to increase the risk of suicide ( 3 Mann J.J. Currier D.M. Stress, genetics and epigenetic effects on the neurobiology of suicidal behavior and depression. Eur Psychiatry. 2010; 25: 268-271 Crossref PubMed Scopus (128) Google Scholar ). It has been challenging, however, to elucidate biologic mechanisms that underlie long-term changes in brain and behavior that are associated with the increased risk. Differential Glucocorticoid Receptor Exon 1B, 1C, and 1H Expression and Methylation in Suicide Completers with a History of Childhood AbuseBiological PsychiatryVol. 72Issue 1PreviewChildhood abuse alters hypothalamic-pituitary-adrenal (HPA) function and increases the risk of suicide. Hippocampal glucocorticoid receptor (GR) activation regulates HPA activity, and human GR expression (hGR) is reduced in the hippocampus of suicide completers with a history of childhood abuse compared with controls. The abuse-related decrease in hGR expression associates with increased DNA methylation of the promoter of the hGR1F variant in the hippocampus. Full-Text PDF