Biomedical subjects

Claes Wahlestedt

Publications and source records attributed to Claes Wahlestedt.

At least 19 recordsLinked to original sources

PubMed2025

Global Changes in Gene Expression and Splicing in Alcoholic Liver Disease.

Alcohol use disorder is a widespread illness commonly leading to alcoholic liver disease (ALD) and cirrhosis with an increased incidence of hepatocellular carcinoma (HCC), but the mechanisms of alcohol-related oncogenesis in the liver are incompletely understood. We tested the hypothesis that ALD predisposes to HCC via dysregulation of splicing. RNA sequencing was performed on liver biopsies from patients with different stages of ALD: early alcoholic steatohepatitis (eASH), non-severe alcoholic hepatitis (nsAH), and severe alcoholic hepatitis (sAH); furthermore, explants were collected from patients who underwent liver transplantation due to sAH (exAH). We found that alcohol caused widespread changes in transcriptome in all stages of ALD: among ~ 58,000 analyzed genomic features, ~ 4,900 were altered in eASH, ~ 9,100 - in nsAH, 14,100 - in sAH, and ~ 14,300 - in exAH. We observed thousands of missplicing events in all hepatic conditions, with mutually exclusive exons (MEE) being the most common event and exon skipping (ES) - second most common event. Analysis of ~ 600,000 exons revealed that ALD is associated with a genome-wide effect on exon expression, with ~ 50,000 exons being differentially expressed in eASH, ~ 130,000 - in nsAH, ~ 150,000 - in sAH, and ~ 120,000 - in exAH. To determine whether alcohol directly perturbs splicing, we subjected rats to alcohol vapor for 7 weeks and found that the expression of multiple snRNAs was drastically decreased, while expression of splicing factors was not affected. Screening of oncogenes and tumor suppressors, commonly involved in HCC pathogenesis, revealed that ALD affected the hepatic expression and/or splicing of most of these cancer-related genes. In summary, it appears that alcohol causes profound genome-wide changes in gene expression and splicing in the liver, likely via affecting the spliceosome. This results in altered expression and missplicing of key oncogenes and tumor suppressors involved in HCC, suggesting a novel mechanism of oncogenesis in the liver of patients with ALD.

alcohol use disorder↗

PubMed2006

Apoptosis resistance downstream of eIF4E: posttranscriptional activation of an anti-apoptotic transcript carrying a consensus hairpin structure.

Aberrant activation of the translation initiation machinery is a common property of malignant cells, and is essential for breast carcinoma cells to manifest a malignant phenotype. How does sustained activation of the rate limiting step in protein synthesis so fundamentally alter a cell? In this report, we test the post transcriptional operon theory as a possible mechanism, employing a model system in which apoptosis resistance is conferred on NIH 3T3 cells by ectopic expression of eIF4E. We show (i) there is a set of 255 transcripts that manifest an increase in translational efficiency during eIF4E-mediated escape from apoptosis; (ii) there is a novel prototype 55 nt RNA consensus hairpin structure that is overrepresented in the 5'-untranslated region of translationally activated transcripts; (iii) the identified consensus hairpin structure is sufficient to target a reporter mRNA for translational activation under pro-apoptotic stress, but only when eIF4E is deregulated; and (iv) that osteopontin, one of the translationally activated transcripts harboring the identified consensus hairpin structure functions as one mediator of the apoptosis resistance seen in our model. Our findings offer genome-wide insights into the mechanism of eIF4E-mediated apoptosis resistance and provide a paradigm for the systematic study of posttranscriptional control in normal biology and disease.

5' Untranslated Regions↗

PubMed2006

RNA interference is not involved in natural antisense mediated regulation of gene expression in mammals.

BACKGROUND: Antisense transcription, yielding both coding and non-coding RNA, is a widespread phenomenon in mammals. The mechanism by which natural antisense transcripts (NAT) may regulate gene expression are largely unknown. The aim of the present study was to explore the mechanism of reciprocal sense-antisense (S-AS) regulation by studying the effects of a coding and non-coding NAT on corresponding gene expression, and to investigate the possible involvement of endogenous RNA interference (RNAi) in S-AS interactions. RESULTS: We have examined the mechanism of S-AS RNA base pairing, using thymidylate synthase and hypoxia inducible factor-1alpha as primary examples of endogenous genes with coding and non-coding NAT partners, respectively. Here we provide direct evidence against S-AS RNA duplex formation in the cytoplasm of human cells and subsequent activation of RNAi. CONCLUSION: Collectively, our data demonstrate that NAT regulation of gene expression occurs through a pathway independent of Dicer associated RNAi. Moreover, we introduce an experimental strategy with utility for the functional examination of other S-AS pair interactions.

Cell Line↗

PubMed2006

Genome-wide analysis of mammalian promoter architecture and evolution.

Mammalian promoters can be separated into two classes, conserved TATA box-enriched promoters, which initiate at a well-defined site, and more plastic, broad and evolvable CpG-rich promoters. We have sequenced tags corresponding to several hundred thousand transcription start sites (TSSs) in the mouse and human genomes, allowing precise analysis of the sequence architecture and evolution of distinct promoter classes. Different tissues and families of genes differentially use distinct types of promoters. Our tagging methods allow quantitative analysis of promoter usage in different tissues and show that differentially regulated alternative TSSs are a common feature in protein-coding genes and commonly generate alternative N termini. Among the TSSs, we identified new start sites associated with the majority of exons and with 3' UTRs. These data permit genome-scale identification of tissue-specific promoters and analysis of the cis-acting elements associated with them.

3' Untranslated Regions↗

PubMed2006

Pseudo-messenger RNA: phantoms of the transcriptome.

The mammalian transcriptome harbours shadowy entities that resist classification and analysis. In analogy with pseudogenes, we define pseudo-messenger RNA to be RNA molecules that resemble protein-coding mRNA, but cannot encode full-length proteins owing to disruptions of the reading frame. Using a rigorous computational pipeline, which rules out sequencing errors, we identify 10,679 pseudo-messenger RNAs (approximately half of which are transposon-associated) among the 102,801 FANTOM3 mouse cDNAs: just over 10% of the FANTOM3 transcriptome. These comprise not only transcribed pseudogenes, but also disrupted splice variants of otherwise protein-coding genes. Some may encode truncated proteins, only a minority of which appear subject to nonsense-mediated decay. The presence of an excess of transcripts whose only disruptions are opal stop codons suggests that there are more selenoproteins than currently estimated. We also describe compensatory frameshifts, where a segment of the gene has changed frame but remains translatable. In summary, we survey a large class of non-standard but potentially functional transcripts that are likely to encode genetic information and effect biological processes in novel ways. Many of these transcripts do not correspond cleanly to any identifiable object in the genome, implying fundamental limits to the goal of annotating all functional elements at the genome sequence level.

Animals↗

PubMed2006

Complex Loci in human and mouse genomes.

Mammalian genomes harbor a larger than expected number of complex loci, in which multiple genes are coupled by shared transcribed regions in antisense orientation and/or by bidirectional core promoters. To determine the incidence, functional significance, and evolutionary context of mammalian complex loci, we identified and characterized 5,248 cis-antisense pairs, 1,638 bidirectional promoters, and 1,153 chains of multiple cis-antisense and/or bidirectionally promoted pairs from 36,606 mouse transcriptional units (TUs), along with 6,141 cis-antisense pairs, 2,113 bidirectional promoters, and 1,480 chains from 42,887 human TUs. In both human and mouse, 25% of TUs resided in cis-antisense pairs, only 17% of which were conserved between the two organisms, indicating frequent species specificity of antisense gene arrangements. A sampling approach indicated that over 40% of all TUs might actually be in cis-antisense pairs, and that only a minority of these arrangements are likely to be conserved between human and mouse. Bidirectional promoters were characterized by variable transcriptional start sites and an identifiable midpoint at which overall sequence composition changed strand and the direction of transcriptional initiation switched. In microarray data covering a wide range of mouse tissues, genes in cis-antisense and bidirectionally promoted arrangement showed a higher probability of being coordinately expressed than random pairs of genes. In a case study on homeotic loci, we observed extensive transcription of nonconserved sequences on the noncoding strand, implying that the presence rather than the sequence of these transcripts is of functional importance. Complex loci are ubiquitous, host numerous nonconserved gene structures and lineage-specific exonification events, and may have a cis-regulatory impact on the member genes.

Animals↗

PubMed2006

No induction of anti-viral responses in human cell lines HeLa and MCF-7 when transfecting with siRNA or siLNA.

Gene silencing by RNAi and siRNAs has become a well-used tool for researchers. Because of its relatively small size, siRNA was originally thought to avoid activation of anti-viral responses. Recent reports demonstrating so-called "off-target effects" are therefore alarming. One issue raised is that siRNA induces interferon-regulated genes at the transcriptional level. We characterize the anti-viral responses of synthetic siRNA and in vitro-transcribed siRNA by measuring the mRNA levels of IFN-beta and OAS2 in HeLa cells. Transfections with both traditional and LNA-modified synthetic siRNA cause no anti-viral responses, whereas transfection with either long dsRNA or in vitro-transcribed siRNA leads to greater than 1000-fold induction of these genes. The lack of response was also demonstrated at the level of phosphorylated eIF2alpha, and measuring of IFN-beta by ELISA in cell culture media from the human cell line MCF-7. Altogether, transfection with synthetic siRNA does not induce anti-viral responses in these two cell lines. Our results reinforce the role of siRNA as an effective tool for reverse genetics and strengthen siLNA as a tool for future therapeutic applications.

2',5'-Oligoadenylate Synthetase↗

PubMed2006

Nonsynonymous SNPs: validation characteristics, derived allele frequency patterns, and suggestive evidence for natural selection.

We experimentally investigated more than 1,200 entries in dbSNP that would change amino-acids (nsSNPs), using various subsets of DNA samples drawn from 18 global populations (approximately 1,000 subjects in total). First, we mined the data for any SNP features that correlated with a high validation rate. Useful predictors of valid SNPs included multiple submissions to dbSNP, having a dbSNP validation statement, and being present in a low number of ESTs. Together, these features improved validation rates by almost 10-fold. Higher-abundance SNPs (e.g., T/C variants) also validated more frequently. Second, we considered derived alleles and noted a considerably (approximately 10%) increased average derived allele frequency (DAF) in Europeans vs. Africans, plus a further increase in some other populations. This was not primarily due to an SNP ascertainment bias, nor to the effects of natural selection. Instead, it can be explained as a drift-based, progressive increase in DAF that occurs over many generations and becomes exaggerated during population bottlenecks. This observation could be used as the basis for novel DAF-based tests for comparing demographic histories. Finally, we considered individual marker patterns and identified 37 SNPs with allele frequency variance or FST values consistent with the effects of population-specific natural selection. Four particularly striking clusters of these markers were apparent, and three of these coincide with genes/regions from among only several dozen such domains previously suggested by others to carry signatures of selection.

Alleles↗

PubMed2006

Natural antisense and noncoding RNA transcripts as potential drug targets.

Information on the complexity of mammalian RNA transcription has increased greatly in the past few years. Notably, thousands of sense transcripts (conventional protein-coding genes) have antisense transcript partners, most of which are noncoding. Interestingly, a number of antisense transcripts regulate the expression of their sense partners, either in a discordant (antisense knockdown results in sense-transcript elevation) or concordant (antisense knockdown results in concomitant sense-transcript reduction) manner. Two new pharmacological strategies based on the knockdown of antisense RNA transcripts by siRNA (or another RNA targeting principle) are proposed in this review. In the case of discordant regulation, knockdown of antisense transcript elevates the expression of the conventional (sense) gene, thereby conceivably mimicking agonist-activator action. In the case of concordant regulation, knockdown of antisense transcript, or concomitant knockdown of antisense and sense transcripts, results in an additive or even synergistic reduction of the conventional gene expression. Although both strategies have been demonstrated to be valid in cell culture, it remains to be seen whether they provide advantages in other contexts.

Animals↗

PubMed2006

RNA Interference with chemically modified siRNA.

siRNA has become an indispensable tool for functional characterization of genes. It has also demonstrated tremendous potential as a new generation of drug candidates. Although the technology works very well to a great panel of cells in vitro, it is still a challenge to translate the success into in vivo target validation easiness and, even more difficult, into therapeutic applications. With a number of chemically modified compounds under initial clinical trial from several commercial entities, the interests in chemical modification of siRNA have become heightened. In this review we have tried to touch on most of the chemical modifications of RNA that have been tested in the siRNA landscape, but maintained a focus on the backbone modifications, and 2'-modifications on the ribose ring. It is anticipated that more modifications and more systematic comparisons between different modifications will be performed to draw more educated conclusions over some of the modifications.

Hydroxylation↗

PubMed2005

The expression signature of in vitro senescence resembles mouse but not human aging.

BACKGROUND: The biological mechanisms that underlie aging have not yet been fully identified. Senescence, a phenomenon occurring in vitro, limits the number of cell divisions in mammalian cell cultures and has been suggested to contribute to aging. RESULTS: We investigated whether the changes in gene expression that occur during mammalian aging and induction of cellular senescence are similar. We compared changes of gene expression in seven microarray datasets from aging human, mouse and rat, as well as four microarray datasets from senescent cells of man and mouse. The datasets were publicly available or obtained from other laboratories. Correlation measures were used to establish similarities of the expression profiles and gene ontology analyses to identify functional groups of genes that are co-regulated. Robust similarities were established between aging in different species and tissues, indicating that there is an aging transcriptome. Although some cross-species comparisons displayed high correlation, intra-species similarities were more reliable. Similarly, a senescence transcriptome was demonstrated that is conserved across cell types. A similarity between the expression signatures of cellular senescence and aging could be established in mouse, but not in human. CONCLUSION: Our study is the first to use microarray data from several studies and laboratories for dissection of a complex biological phenotype. We demonstrate the presence of a mammalian aging transcriptome, and discuss why similarity between cellular senescence and aging is apparent in aging mice only.

Aging↗

PubMed2005

Expression profiling following local muscle inactivity in humans provides new perspective on diabetes-related genes.

Physical activity enhances muscle mitochondrial gene expression, while inactivity and mitochondrial dysfunction are both risk factors for developing diabetes. Defective activation of the transcriptional coactivator PGC-1alpha may contribute to the gene expression pattern observed in diabetic and insulin-resistant skeletal muscle. We proposed that greater insight into the mitochondrial component of skeletal muscle "diabetes" would be possible if the clinical transcriptome data were contrasted with local muscle inactivity-induced modulation of mitochondrial genes in otherwise healthy subjects. We studied PPARGC1A (PGC-1alpha), PPARGC1B (PGC-1beta), NRF1, and a variety of mitochondrial DNA (mtDNA) and nuclear-encoded mitochondrial genes critical for oxidative phosphorylation in soleus muscle biopsies obtained from six healthy men and women before and after 5 weeks of local muscle inactivity. Muscle inactivity resulted in a coordinated down-regulation of PGC-1alpha and genes involved with mitochondrial metabolism, including muscle substrate delivery genes. Decreased expression of the mtDNA helicase Twinkle was related to the decline in mitochondrial RNA polymerase (r = 0.83, p < 0.04), suggesting that mtDNA transcription and replication are coregulated in human muscle tissue. In contrast to the situation in diabetes, PGC-1beta expression was not significantly altered, while NRF1 expression was actually up-regulated following muscle inactivity. We can conclude that reduced PGC-1alpha expression described in Type 2 diabetes may be partly explained by muscle inactivity. Further, although diabetes patients are typically inactive, our analysis indicates that local muscle inactivity may not be expected to contribute to the decreased NRF1 and PGC-1beta expression noted in insulin-resistant and Type 2 diabetes patients, suggesting these changes may be more disease specific.

Adult↗

PubMed2005

Complex HTR2C linkage disequilibrium and promoter associations with body mass index and serum leptin.

The occurrence of obesity, eating disorders, and related diseases has increased in many parts of the world. Given that few strong genetic factors have been found, it is clear that these are complex multi-factorial diseases. The serotonin receptor 2C, a member of the 5-HTergic system, has been implicated in the control of phagia and obesity. We report a detailed investigation of linkage disequilibrium (LD) within and between the HTR2C promoter and the flanking sequences around a commonly utilized marker in the second coding exon of HTR2C. We suggest that inconsistent associations between HTR2C and several phenotypes, including obesity, may be due to the LD pattern across the gene in which recombination and gene conversion have been influential. The nucleotide and haplotype distribution is consistent with that of the neutral mutation model. The number of haplotypes suggests demographic influences or over dominant selection that may have a function in HTR2C expression. Using the fine LD pattern, we describe a possible association with promoter haplotypes and diplotypes, including a GT microsatellite, and body mass index (BMI) > or =30 kgm(-2) (P<0.0001). SNP -995G>A heterozygotes, as well as promoter diplotypes, were found to marginally influence higher serum leptin corrected for percentage body fat (P=0.01), which might suggest that these subjects are leptin resistant. Our results complement previous studies of HTR2C in both mice and humans, and suggest the importance of genetic variation and elucidating the fine LD structure in uncovering the genetic factors of obesity.

Adult↗

PubMed2005

Considerations when using the significance analysis of microarrays (SAM) algorithm.

BACKGROUND: Users of microarray technology typically strive to use universally acceptable data analysis strategies to determine significant expression changes in their experiments. One of the most frequently utilised methods for gene expression data analysis is SAM (significance analysis of microarrays). The impact of selection thresholds, on the output from SAM, may critically alter the conclusion of a study, yet this consideration has not been systematically evaluated in any publication. RESULTS: We have examined the effect of discrete data selection criteria (qualification criteria for inclusion) and response thresholds (out-put filtering) on the number of significant genes reported by SAM. The use of a reduced data set by applying arbitrary restrictions vis-à-vis abundance calls (e.g. from D-chip) or application of the fold change (FC) option within SAM (named the FC hurdle hereafter), can substantially alter the significant gene list when running SAM in Microsoft Excel. We determined that for a given final FC criteria (e.g. 1.5 fold change) the FC hurdle applied within Microsoft Excel SAM alters the number of reported genes above the final FC criteria. The reason is that the FC hurdle changes the composition of the control data set, such that a different significance level (q-value) is obtained for any given gene. This effect can be so large that it changes subsequent post hoc analysis interpretation, such as ontology overrepresentation analysis. CONCLUSION: Our results argue for caution when using SAM. All data sets analysed with SAM could be reanalysed taking into account the potential impact of the use of arbitrary thresholds to trim data sets before significance testing.

Adult↗

PubMed2005

Human neuropeptide Y signal peptide gain-of-function polymorphism is associated with increased body mass index: possible mode of function.

Neuropeptide Y (NPY) has been implicated in the control of food intake and energy balance based on many observations in animals. We have studied single nucleotide polymorphisms (SNPs) within the regulatory and coding sequences of the human NPY gene. One variant (1128 T>C), which causes an amino acid change from leucine to proline at codon 7 in the signal peptide of NPY, was associated with increased body mass index (BMI) in two separate Swedish populations of normal and overweight individuals. In vitro transcription and translation studies indicated the unlikelihood that this signal peptide variation affects the site of cleavage and targeting or uptake of NPY into the endoplasmic reticulum (ER). However, the mutant, and to a lesser extent the wild-type, signal peptide by themselves markedly potentiated NPY-induced food intake, as well as hypothalamic NPY receptor signaling. Our findings in humans strongly indicate that the NPY signaling system is implicated in body weight regulation and suggest a new and unexpected functional role of a signal peptide.

Animals↗

PubMed2005

A systematic analysis of the silencing effects of an active siRNA at all single-nucleotide mismatched target sites.

The specificity of small interfering RNA (siRNA)-mediated gene silencing is a critical consideration for the application of RNA interference (RNAi). While the discovery of potential off-target effects by siRNAs is of concern, no systematic analysis has been conducted to explore the specificity of RNAi. Here, we present a study where a functionally validated siRNA (siCD46) was examined for silencing specificity on all possible 57 permutated target sites, each carrying a single-nucleotide mutation that would generate a mismatch when paired with siRNA antisense strand. We found that it was not only the position of the mismatched base pair, but also the identity of the nucleotides forming the mismatch that influenced silencing. Surprisingly, mismatches formed between adenine (A) and cytosine (C), in addition to the G:U wobble base pair, were well tolerated and target sites containing such mismatches were silenced almost as efficiently as its fully matched counterpart by siCD46. Northern blots showed that the silencing of fusion genes harboring the mutated target sites involved target mRNA degradation. This study provides direct evidence that the target recognition of siRNA is far more degenerative than previously considered. This finding is instrumental in the understanding of RNAi specificity and may aid the computational prediction of RNA secondary structure.

Base Pair Mismatch↗

PubMed2005

Identification of functional SNPs in the 5-prime flanking sequences of human genes.

BACKGROUND: Over 4 million single nucleotide polymorphisms (SNPs) are currently reported to exist within the human genome. Only a small fraction of these SNPs alter gene function or expression, and therefore might be associated with a cell phenotype. These functional SNPs are consequently important in understanding human health. Information related to functional SNPs in candidate disease genes is critical for cost effective genetic association studies, which attempt to understand the genetics of complex diseases like diabetes, Alzheimer's, etc. Robust methods for the identification of functional SNPs are therefore crucial. We report one such experimental approach. RESULTS: Sequence conserved between mouse and human genomes, within 5 kilobases of the 5-prime end of 176 GPCR genes, were screened for SNPs. Sequences flanking these SNPs were scored for transcription factor binding sites. Allelic pairs resulting in a significant score difference were predicted to influence the binding of transcription factors (TFs). Ten such SNPs were selected for mobility shift assays (EMSA), resulting in 7 of them exhibiting a reproducible shift. The full-length promoter regions with 4 of the 7 SNPs were cloned in a Luciferase based plasmid reporter system. Two out of the 4 SNPs exhibited differential promoter activity in several human cell lines. CONCLUSIONS: We propose a method for effective selection of functional, regulatory SNPs that are located in evolutionary conserved 5-prime flanking regions (5'-FR) regions of human genes and influence the activity of the transcriptional regulatory region. Some SNPs behave differently in different cell types.

Algorithms↗

PubMed2005

A universal plasmid library encoding all permutations of small interfering RNA.

Small interfering RNA (siRNA) is normally designed to silence preselected known genes. Such selections are inevitably prone to bias as a result of limited knowledge about the biological process, transcript identity, and functions. A library that contains all permutations of siRNA could avoid such problems. In this paper, it is shown that 5 x 10(7) siRNA-encoding plasmids can be constructed in a single tube by using vectors with two mutated RNA polymerase III promoters arranged in a convergent manner. Such a library was used to carry out genomewide screening of functional genes in a phenotype-driven manner. Multiple siRNAs that induce a significant increase of cell proliferation speed were identified.

Base Sequence↗