Timing Is Critical for Effective Glucocorticoid Receptor Mediated Repression of the cAMP-Induced CRH Gene

Glucocorticoid negative feedback of the hypothalamus-pituitary-adrenal axis is mediated in part by direct repression of gene transcription in glucocorticoid receptor (GR) expressing cells. We have investigated the cross talk between the two main signaling pathways involved in activation and repression of corticotrophin releasing hormone (CRH) mRNA expression: cyclic AMP (cAMP) and GR. We report that in the At-T20 cell-line the glucocorticoid-mediated repression of the cAMP-induced human CRH proximal promoter activity depends on the relative timing of activation of both signaling pathways. Activation of the GR prior to or in conjunction with cAMP signaling results in an effective repression of the cAMP-induced transcription of the CRH gene. In contrast, activation of the GR 10 minutes after onset of cAMP treatment, results in a significant loss of GR-mediated repression. In addition, translocation of ligand-activated GR to the nucleus was found as early as 10 minutes after glucocorticoid treatment. Interestingly, while both signaling cascades counteract each other on the CRH proximal promoter, they synergize on a synthetic promoter containing ‘positive’ response elements. Since the order of activation of both signaling pathways may vary considerably in vivo, we conclude that a critical time-window exists for effective repression of the CRH gene by glucocorticoids

Caspase-8 binding to cardiolipin in giant unilamellar vesicles provides a functional docking platform for bid

Caspase-8 is involved in death receptor-mediated apoptosis in type II cells, the proapoptotic programme of which is triggered by truncated Bid. Indeed, caspase-8 and Bid are the known intermediates of this signalling pathway. Cardiolipin has been shown to provide an anchor and an essential activating platform for caspase-8 at the mitochondrial membrane surface. Destabilisation of this platform alters receptor-mediated apoptosis in diseases such as Barth Syndrome, which is characterised by the presence of immature cardiolipin which does not allow caspase-8 binding. We used a simplified in vitro system that mimics contact sites and/or cardiolipin-enriched microdomains at the outer mitochondrial surface in which the platform consisting of caspase-8, Bid and cardiolipin was reconstituted in giant unilamellar vesicles. We analysed these vesicles by flow cytometry and confirm previous results that demonstrate the requirement for intact mature cardiolipin for caspase-8 activation and Bid binding and cleavage. We also used confocal microscopy to visualise the rupture of the vesicles and their revesiculation at smaller sizes due to alteration of the curvature following caspase-8 and Bid binding. Biophysical approaches, including Laurdan fluorescence and rupture/tension measurements, were used to determine the ability of these three components (cardiolipin, caspase-8 and Bid) to fulfil the minimal requirements for the formation and function of the platform at the mitochondrial membrane. Our results shed light on the active functional role of cardiolipin, bridging the gap between death receptors and mitochondria

Bias in random forest variable importance measures: Illustrations, sources and a solution

BACKGROUND: Variable importance measures for random forests have been receiving increased attention as a means of variable selection in many classification tasks in bioinformatics and related scientific fields, for instance to select a subset of genetic markers relevant for the prediction of a certain disease. We show that random forest variable importance measures are a sensible means for variable selection in many applications, but are not reliable in situations where potential predictor variables vary in their scale of measurement or their number of categories. This is particularly important in genomics and computational biology, where predictors often include variables of different types, for example when predictors include both sequence data and continuous variables such as folding energy, or when amino acid sequence data show different numbers of categories. RESULTS: Simulation studies are presented illustrating that, when random forest variable importance measures are used with data of varying types, the results are misleading because suboptimal predictor variables may be artificially preferred in variable selection. The two mechanisms underlying this deficiency are biased variable selection in the individual classification trees used to build the random forest on one hand, and effects induced by bootstrap sampling with replacement on the other hand. CONCLUSION: We propose to employ an alternative implementation of random forests, that provides unbiased variable selection in the individual classification trees. When this method is applied using subsampling without replacement, the resulting variable importance measures can be used reliably for variable selection even in situations where the potential predictor variables vary in their scale of measurement or their number of categories. The usage of both random forest algorithms and their variable importance measures in the R system for statistical computing is illustrated and documented thoroughly in an application re-analyzing data from a study on RNA editing. Therefore the suggested method can be applied straightforwardly by scientists in bioinformatics research

Genome-Wide Divergence of DNA Methylation Marks in Cerebral and Cerebellar Cortices

Emerging evidence suggests that DNA methylation plays an expansive role in the central nervous system (CNS). Large-scale whole genome DNA methylation profiling of the normal human brain offers tremendous potential in understanding the role of DNA methylation in brain development and function.Using methylation-sensitive SNP chip analysis (MSNP), we performed whole genome DNA methylation profiling of the prefrontal, occipital, and temporal regions of cerebral cortex, as well as cerebellum. These data provide an unbiased representation of CpG sites comprising 377,509 CpG dinucleotides within both the genic and intergenic euchromatic region of the genome. Our large-scale genome DNA methylation profiling reveals that the prefrontal, occipital, and temporal regions of the cerebral cortex compared to cerebellum have markedly different DNA methylation signatures, with the cerebral cortex being hypermethylated and cerebellum being hypomethylated. Such differences were observed in distinct genomic regions, including genes involved in CNS function. The MSNP data were validated for a subset of these genes, by performing bisulfite cloning and sequencing and confirming that prefrontal, occipital, and temporal cortices are significantly more methylated as compared to the cerebellum.These findings are consistent with known developmental differences in nucleosome repeat lengths in cerebral and cerebellar cortices, with cerebrum exhibiting shorter repeat lengths than cerebellum. Our observed differences in DNA methylation profiles in these regions underscores the potential role of DNA methylation in chromatin structure and organization in CNS, reflecting functional specialization within cortical regions

Hypervariable intronic region in NCX1 is enriched in short insertion-deletion polymorphisms and showed association with cardiovascular traits

<p>Abstract</p> <p>Background</p> <p>Conserved non-coding regions (CNR) have been shown to harbor gene expression regulatory elements. Genetic variations in these regions may potentially contribute to complex disease susceptibility.</p> <p>Methods</p> <p>We targeted CNRs of cardiovascular disease (CVD) candidate gene, <it>Na(+)-Ca(2+) exchanger (NCX1) </it>with polymorphism screening among CVD patients (n = 46) using DHPLC technology. The flanking region (348 bp) of the 14 bp indel in intron 2 was further genotyped by DGGE assay in two Eastern-European CVD samples: essential hypertension (HYPEST; 470 cases, 652 controls) and coronary artery disease, CAD (CADCZ; 257 cases, controls 413). Genotype-phenotype associations were tested by regression analysis implemented in PLINK. Alignments of primate sequences were performed by ClustalW2.</p> <p>Results</p> <p>Nine of the identified <it>NCX1 </it>variants were either singletons or targeted by commercial platforms. The 14 bp intronic indel (rs11274804) was represented with substantial frequency in HYPEST (6.82%) and CADCZ (14.58%). Genotyping in Eastern-Europeans (n = 1792) revealed hypervariable nature of this locus, represented by seven alternative alleles. The alignments of human-chimpanzee-macaque sequences showed that the major human variant (allele frequency 90.45%) was actually a human-specific deletion compared to other primates. In humans, this deletion was surrounded by other short (5-43 bp) deletion variants and a duplication (40 bp) polymorphism possessing overlapping breakpoints. This indicates a potential indel hotspot, triggered by the initial deletion in human lineage. An association was detected between the carrier status of 14 bp indel ancestral allele and CAD (<it>P </it>= 0.0016, OR = 2.02; Bonferroni significance level alpha = 0.0045), but not with hypertension. The risk for the CAD development was even higher among the patients additionally diagnosed with metabolic syndrome (<it>P </it>= 0.0014, OR = 2.34). Consistent with the effect on metabolic processes, suggestive evidence for the association with heart rate, serum triglyceride and LDL levels was detected (<it>P </it>= 0.04).</p> <p>Conclusions</p> <p>Compared to SNPs targeted by large number of locus-specific and genome-wide assays, considerably less attention has been paid to short indel variants in the human genome. The data of genome dynamics, mutation rate and population genetics of short indels, as well as their impact on gene expressional profile and human disease susceptibility is limited. The characterization of <it>NCX1 </it>intronic hypervariable non-coding region enriched in human-specific indel variants contributes to this gap of knowledge.</p

Combined antiviral activity of interferon-α and RNA interference directed against hepatitis C without affecting vector delivery and gene silencing

The current standard interferon-alpha (IFN-α)-based therapy for chronic hepatitis C virus (HCV) infection is only effective in approximately half of the patients, prompting the need for alternative treatments. RNA interference (RNAi) represents novel approach to combat HCV by sequence-specific targeting of viral or host factors involved in infection. Monotherapy of RNAi, however, may lead to therapeutic resistance by mutational escape of the virus. Here, we proposed that combining lentiviral vector-mediated RNAi and IFN-α could be more effective and avoid therapeutic resistance. In this study, we found that IFN-α treatment did not interfere with RNAi-mediated gene silencing. RNAi and IFN-α act independently on HCV replication showing combined antiviral activity when used simultaneously or sequentially. Transduction of mouse hepatocytes in vivo and in vitro was not effected by IFN-α treatment. In conclusion, RNAi and IFN-α can be effectively combined without cross-interference and may represent a promising combinational strategy for the treatment of hepatitis C

Functional Interactions between KCNE1 C-Terminus and the KCNQ1 Channel

The KCNE1 gene product (minK protein) associates with the cardiac KvLQT1 potassium channel (encoded by KCNQ1) to create the cardiac slowly activating delayed rectifier, IKs. Mutations throughout both genes are linked to the hereditary cardiac arrhythmias in the Long QT Syndrome (LQTS). KCNE1 exerts its specific regulation of KCNQ1 activation via interactions between membrane-spanning segments of the two proteins. Less detailed attention has been focused on the role of the KCNE1 C-terminus in regulating channel behavior. We analyzed the effects of an LQT5 point mutation (D76N) and the truncation of the entire C-terminus (Δ70) on channel regulation, assembly and interaction. Both mutations significantly shifted voltage dependence of activation in the depolarizing direction and decreased IKs current density. They also accelerated rates of channel deactivation but notably, did not affect activation kinetics. Truncation of the C-terminus reduced the apparent affinity of KCNE1 for KCNQ1, resulting in impaired channel formation and presentation of KCNQ1/KCNE1 complexes to the surface. Complete saturation of KCNQ1 channels with KCNE1-Δ70 could be achieved by relative over-expression of the KCNE subunit. Rate-dependent facilitation of K+ conductance, a key property of IKs that enables action potential shortening at higher heart rates, was defective for both KCNE1 C-terminal mutations, and may contribute to the clinical phenotype of arrhythmias triggered by heart rate elevations during exercise in LQTS mutations. These results support several roles for KCNE1 C-terminus interaction with KCNQ1: regulation of channel assembly, open-state destabilization, and kinetics of channel deactivation

Identifying Highly Conserved and Highly Differentiated Gene Ontology Categories in Human Populations

Detecting and interpreting certain system-level characteristics associated with human population genetic differences is a challenge for human geneticists. In this study, we conducted a population genetic study using the HapMap genotype data to identify certain special Gene Ontology (GO) categories associated with high/low genetic difference among 11 Hapmap populations. Initially, the genetic differences in each gene region among these populations were measured using allele frequency, linkage disequilibrium (LD) pattern, and transferability of tagSNPs. The associations between each GO term and these genetic differences were then identified. The results showed that cellular process, catalytic activity, binding, and some of their sub-terms were associated with high levels of genetic difference, and genes involved in these functional categories displayed, on average, high genetic diversity among different populations. By contrast, multicellular organismal processes, molecular transducer activity, and some of their sub-terms were associated with low levels of genetic difference. In particular, the neurological system process under the multicellular organismal process category had low levels of genetic difference; the neurological function also showed high evolutionary conservation between species in some previous studies. These results may provide a new insight into the understanding of human evolutionary history at the system-level