Trans Regulation Research Articles

Cis regulatory effects on A-to-I RNA editing in related Drosophila species.

Adenosine-to-inosine RNA editing modifies maturing mRNAs through the binding of adenosine deaminase acting on RNA (Adar) proteins to double-stranded RNA structures in a process critical for neuronal function. Editing levels at individual editing sites span a broad range and are mediated by both cis-acting elements (surrounding RNA sequence and secondary structure) and trans-acting factors. Here, we aim to determine the roles that cis-acting elements and trans-acting factors play in regulating editing levels. Using two closely related Drosophila species, D. melanogaster and D. sechellia, and their F1 hybrids, we dissect the effects of cis sequences from trans regulators on editing levels by comparing species-specific editing in parents and their hybrids. We report that cis sequence differences are largely responsible for editing level differences between these two Drosophila species. This study presents evidence for cis sequence and structure changes as the dominant evolutionary force that modulates RNA editing levels between these Drosophila species.

A meta-analysis of gene expression signatures of blood pressure and hypertension.

Genome-wide association studies (GWAS) have uncovered numerous genetic variants (SNPs) that are associated with blood pressure (BP). Genetic variants may lead to BP changes by acting on intermediate molecular phenotypes such as coded protein sequence or gene expression, which in turn affect BP variability. Therefore, characterizing genes whose expression is associated with BP may reveal cellular processes involved in BP regulation and uncover how transcripts mediate genetic and environmental effects on BP variability. A meta-analysis of results from six studies of global gene expression profiles of BP and hypertension in whole blood was performed in 7017 individuals who were not receiving antihypertensive drug treatment. We identified 34 genes that were differentially expressed in relation to BP (Bonferroni-corrected p<0.05). Among these genes, FOS and PTGS2 have been previously reported to be involved in BP-related processes; the others are novel. The top BP signature genes in aggregate explain 5%–9% of inter-individual variance in BP. Of note, rs3184504 in SH2B3, which was also reported in GWAS to be associated with BP, was found to be a trans regulator of the expression of 6 of the transcripts we found to be associated with BP (FOS, MYADM, PP1R15A, TAGAP, S100A10, and FGBP2). Gene set enrichment analysis suggested that the BP-related global gene expression changes include genes involved in inflammatory response and apoptosis pathways. Our study provides new insights into molecular mechanisms underlying BP regulation, and suggests novel transcriptomic markers for the treatment and prevention of hypertension.

Messenger RNA processing is altered in autosomal dominant leukodystrophy.

Adult-onset autosomal dominant leukodystrophy (ADLD) is a slowly progressive neurological disorder characterized by autonomic dysfunction, followed by cerebellar and pyramidal features. ADLD is caused by duplication of the lamin B1 gene (LMNB1), which leads to its increased expression. The molecular pathways involved in the disease are still poorly understood. Hence, we analyzed global gene expression in fibroblasts and whole blood of LMNB1 duplication carriers and used Gene Set Enrichment Analysis to explore their gene signatures. We found that LMNB1 duplication is associated with dysregulation of genes involved in the immune system, neuronal and skeletal development. Genes with an altered transcriptional profile clustered in specific genomic regions. Among the dysregulated genes, we further studied the role of RAVER2, which we found to be overexpressed at mRNA and protein level. RAVER2 encodes a putative trans regulator of the splicing repressor polypyrimidine tract binding protein (PTB) and is likely implicated in alternative splicing regulation. Functional studies demonstrated an abnormal splicing pattern of several PTB-target genes and of the myelin protein gene PLP1, previously demonstrated to be involved in ADLD. Mutant mice with different lamin B1 expression levels confirmed that Raver2 expression is dependent on lamin B1 in neural tissue and determines an altered splicing pattern of PTB-target genes and Plp1. Overall our results demonstrate that deregulation of lamin B1 expression induces modified splicing of several genes, likely driven by raver-2 overexpression, and suggest that an alteration of mRNA processing could be a pathogenic mechanism in ADLD.

Junk DNA and the long non-coding RNA twist in cancer genetics.

The central dogma of molecular biology states that the flow of genetic information moves from DNA to RNA to protein. However, in the last decade this dogma has been challenged by new findings on non-coding RNAs (ncRNAs) such as microRNAs (miRNAs). More recently, long non-coding RNAs (lncRNAs) have attracted much attention due to their large number and biological significance. Many lncRNAs have been identified as mapping to regulatory elements including gene promoters and enhancers, ultraconserved regions, and intergenic regions of protein-coding genes. Yet, the biological function and molecular mechanisms of lncRNA in human diseases in general and cancer in particular remain largely unknown. Data from the literature suggest that lncRNA, often via interaction with proteins, functions in specific genomic loci or use their own transcription loci for regulatory activity. In this review, we summarize recent findings supporting the importance of DNA loci in lncRNA function, and the underlying molecular mechanisms via cis or trans regulation, and discuss their implications in cancer. In addition, we use the 8q24 genomic locus, a region containing interactive SNPs, DNA regulatory elements and lncRNAs, as an example to illustrate how single nucleotide polymorphism (SNP) located within lncRNAs may be functionally associated with the individual’s susceptibility to cancer.

Negative feedback buffers effects of regulatory variants

Mechanisms conferring robustness against regulatory variants have been controversial. Previous studies suggested widespread buffering of RNA misexpression on protein levels during translation. We do not find evidence that translational buffering is common. Instead, we find extensive buffering at the level of RNA expression, exerted through negative feedback regulation acting in trans, which reduces the effect of regulatory variants on gene expression. Our approach is based on a novel experimental design in which allelic differential expression in a yeast hybrid strain is compared to allelic differential expression in a pool of its spores. Allelic differential expression in the hybrid is due to cis-regulatory differences only. Instead, in the pool of spores allelic differential expression is not only due to cis-regulatory differences but also due to local trans effects that include negative feedback. We found that buffering through such local trans regulation is widespread, typically compensating for about 15% of cis-regulatory effects on individual genes. Negative feedback is stronger not only for essential genes, indicating its functional relevance, but also for genes with low to middle levels of expression, for which tight regulation matters most. We suggest that negative feedback is one mechanism of Waddington's canalization, facilitating the accumulation of genetic variants that might give selective advantage in different environments.

The SNAI1 3'UTR functions as a sponge for multiple migration-/invasion-related microRNAs.

Accumulating evidence has indicated a large-scale regulatory network generated by 3'untranslated regions (3'UTRs) in cancer. The 3'UTRs act not only in cis but, most likely even more importantly, as trans regulators of gene expression, consequently leading to phenotypic alterations. Here, we found that ectopic expression of SNAI1 3'UTR induced migration and invasion of ovarian cancer cell line RMUG-L without significantly affecting cell viability. Additionally, SNAI1 3'UTR overexpression regulated key epithelial-to-mesenchymal transition (EMT) markers, including SNAI1, Vimentin, and E-cadherin, and functioned as a sponge for multiple migration-/invasion-related microRNAs (miRNAs) in RMUG-L cells. These findings revealed the noncoding function of SNAI1 for the first time.

Overview of Local, State, and National Government Legislation Restricting Trans Fats

Trans fats, also known as partially hydrogenated oils, have long been associated with cardiovascular disease. In 2003, the Food and Drug Administration mandated that trans fat content of ≥0.5 g be listed on food labeling; the next year, the World Health Organization released a recommended daily limit on trans fat intake. During the following decade, legislatures in different municipal and state governments moved trans fat regulation forward, whereas many food companies decided to independently phase out trans fat in their products. The advancement of these parallel processes suggests an emerging best practices phenomenon, integrating public health, law, and the food industry. With both legislation and markets seemingly favoring a limitation on trans fat content in foods, attention has once again shifted to the Food and Drug Administration for a ruling on the safety of trans fats.

Noncoding RNAs in cardiovascular biology and disease.

The genome continues to fascinate the enthusiasts, and the captivation seems unabating because of the continuous stream of new discoveries. What was once considered a junk DNA has now emerged to contain a large number of important regulatory elements.1 As an example of such discoveries is the recent finding that the genome contains ≈6200 enhancer elements that are operational in the human fetal and adult hearts.2 Genes occupy only ≈1.5% and coding exons only ≈1% of the genome, which make up only ≈60 million nucleotides of the 6.4 billion nucleotides (3.2 billion base pairs) in the genome.3 Yet, ≈5% of the human genome has undergone purifying selection and hence are likely functional.4 It is intriguing that about two third of the evolutionary constrained genomic elements are located in introns and the intergenic regions, suggestive of their regulatory roles in the genome.4 These conserved regions are enriched in loci that have been found to be associated with clinical phenotypes in the genome-wide association studies.4 The initial findings of the ENCODE (Encyclopedia of DNA Elements) project, although preliminary, illustrate the presence of numerous regulatory elements in the genome, including the enhancers.1 The discoveries largely made possible by the recent advances in the high-throughput RNA sequencing point to enormous RNA splicing diversity and the plethora of alternative splicing variants. Approximately 95% of the multiexon genes undergo alternative splicing, resulting in ≈100 000 abundant splice variants in various tissues.5 Moreover, it seems that almost all genomic regions in 1 form or shape are transcribed, which seems perplexing as only ≈1% of the genome codes for proteins, as has been understood to date. Only recently we have started to appreciate the diverse biological functions of these nonprotein coding transcripts, which are referred to as noncoding RNAs (ncRNAs). …

Characterizing the genetic basis of transcriptome diversity through RNA-sequencing of 922 individuals

Understanding the consequences of regulatory variation in the human genome remains a major challenge, with important implications for understanding gene regulation and interpreting the many disease-risk variants that fall outside of protein-coding regions. Here, we provide a direct window into the regulatory consequences of genetic variation by sequencing RNA from 922 genotyped individuals. We present a comprehensive description of the distribution of regulatory variation—by the specific expression phenotypes altered, the properties of affected genes, and the genomic characteristics of regulatory variants. We detect variants influencing expression of over ten thousand genes, and through the enhanced resolution offered by RNA-sequencing, for the first time we identify thousands of variants associated with specific phenotypes including splicing and allelic expression. Evaluating the effects of both long-range intra-chromosomal and trans (cross-chromosomal) regulation, we observe modularity in the regulatory network, with three-dimensional chromosomal configuration playing a particular role in regulatory modules within each chromosome. We also observe a significant depletion of regulatory variants affecting central and critical genes, along with a trend of reduced effect sizes as variant frequency increases, providing evidence that purifying selection and buffering have limited the deleterious impact of regulatory variation on the cell. Further, generalizing beyond observed variants, we have analyzed the genomic properties of variants associated with expression and splicing and developed a Bayesian model to predict regulatory consequences of genetic variants, applicable to the interpretation of individual genomes and disease studies. Together, these results represent a critical step toward characterizing the complete landscape of human regulatory variation.

Inheritance of Gene Expression Level and Selective Constraints on Trans- and Cis-Regulatory Changes in Yeast

Gene expression evolution can be caused by changes in cis- or trans-regulatory elements or both. As cis and trans regulation operate through different molecular mechanisms, cis and trans mutations may show different inheritance patterns and may be subjected to different selective constraints. To investigate these issues, we obtained and analyzed gene expression data from two Saccharomyces cerevisiae strains and their hybrid, using high-throughput sequencing. Our data indicate that compared with other types of genes, those with antagonistic cis-trans interactions are more likely to exhibit over- or underdominant inheritance of expression level. Moreover, in accordance with previous studies, genes with trans variants tend to have a dominant inheritance pattern, whereas cis variants are enriched for additive inheritance. In addition, cis regulatory differences contribute more to expression differences between species than within species, whereas trans regulatory differences show a stronger association between divergence and polymorphism. Our data indicate that in the trans component of gene expression differences genes subjected to weaker selective constraints tend to have an excess of polymorphism over divergence compared with those subjected to stronger selective constraints. In contrast, in the cis component, this difference between genes under stronger and weaker selective constraint is mostly absent. To explain these observations, we propose that purifying selection more strongly shapes trans changes than cis changes and that positive selection may have significantly contributed to cis regulatory divergence.

Cis and trans regulatory mechanisms control AP2-mediated B cell receptor endocytosis via select tyrosine-based motifs.

Following antigen recognition, B cell receptor (BCR)-mediated endocytosis is the first step of antigen processing and presentation to CD4+ T cells, a crucial component of the initiation and control of the humoral immune response. Despite this, the molecular mechanism of BCR internalization is poorly understood. Recently, studies of activated B cell-like diffuse large B cell lymphoma (ABC DLBCL) have shown that mutations within the BCR subunit CD79b leads to increased BCR surface expression, suggesting that CD79b may control BCR internalization. Adaptor protein 2 (AP2) is the major mediator of receptor endocytosis via clathrin-coated pits. The BCR contains five putative AP2-binding YxxØ motifs, including four that are present within two immunoreceptor tyrosine-based activation motifs (ITAMs). Using a combination of in vitro and in situ approaches, we establish that the sole mediator of AP2-dependent BCR internalization is the membrane proximal ITAM YxxØ motif in CD79b, which is a major target of mutation in ABC DLBCL. In addition, we establish that BCR internalization can be regulated at a minimum of two different levels: regulation of YxxØ AP2 binding in cis by downstream ITAM-embedded DCSM and QTAT regulatory elements and regulation in trans by the partner cytoplasmic domain of the CD79 heterodimer. Beyond establishing the basic rules governing BCR internalization, these results illustrate an underappreciated role for ITAM residues in controlling clathrin-dependent endocytosis and highlight the complex mechanisms that control the activity of AP2 binding motifs in this receptor system.

Variants in exons and in transcription factors affect gene expression in trans

BackgroundIn recent years many genetic variants (eSNPs) have been reported as associated with expression of transcripts in trans. However, the causal variants and regulatory mechanisms through which they act remain mostly unknown. In this paper we follow two kinds of usual suspects: SNPs that alter coding regions or transcription factors, identifiable by sequencing data with transcriptional profiles in the same cohort. We show these interpretable genomic regions are enriched for eSNP association signals, thereby naturally defining source-target gene pairs. We map these pairs onto a protein-protein interaction (PPI) network and study their topological properties.ResultsFor exonic eSNP sources, we report source-target proximity and high target degree within the PPI network. These pairs are more likely to be co-expressed and the eSNPs tend to have a cis effect, modulating the expression of the source gene. In contrast, transcription factor source-target pairs are not observed to have such properties, but instead a transcription factor source tends to assemble into units of defined functional roles along with its gene targets, and to share with them the same functional cluster of the PPI network.ConclusionsOur results suggest two modes of trans regulation: transcription factor variation frequently acts via a modular regulation mechanism, with multiple targets that share a function with the transcription factor source. Notwithstanding, exon variation often acts by a local cis effect, delineating shorter paths of interacting proteins across functional clusters of the PPI network.

Coevolution within and between Regulatory Loci Can Preserve Promoter Function Despite Evolutionary Rate Acceleration

Phenotypes that appear to be conserved could be maintained not only by strong purifying selection on the underlying genetic systems, but also by stabilizing selection acting via compensatory mutations with balanced effects. Such coevolution has been invoked to explain experimental results, but has rarely been the focus of study. Conserved expression driven by the unc-47 promoters of Caenorhabditis elegans and C. briggsae persists despite divergence within a cis-regulatory element and between this element and the trans-regulatory environment. Compensatory changes in cis and trans are revealed when these promoters are used to drive expression in the other species. Functional changes in the C. briggsae promoter, which has experienced accelerated sequence evolution, did not lead to alteration of gene expression in its endogenous environment. Coevolution among promoter elements suggests that complex epistatic interactions within cis-regulatory elements may facilitate their divergence. Our results offer a detailed picture of regulatory evolution in which subtle, lineage-specific, and compensatory modifications of interacting cis and trans regulators together maintain conserved gene expression patterns.

A 'pivotal' new rule for microRNA-mRNA interactions

Finding biologically relevant targets is a prerequisite for understanding the function of any trans regulator of gene expression, but this can be particularly challenging with microRNAs (miRNAs). A study in this issue addresses the problem by identifying a novel mode of miRNA target recognition.

Cys2His2 zinc finger protein family: Classification, functions, and major members

Cys2His2 (C2H2)-type zinc fingers are widespread DNA binding motifs in eukaryotic transcription factors. Zinc fingers are short protein motifs composed of two or three β-layers and one α-helix. Two cysteine and two histidine residues located in certain positions bind zinc to stabilize the structure. Four other amino acid residues localized in specific positions in the N-terminal region of the α-helix participate in DNA binding by interacting with hydrogen donors and acceptors exposed in the DNA major groove. The number of zinc fingers in a single protein can vary over a wide range, thus enabling variability of target DNA sequences. Besides DNA binding, zinc fingers can also provide protein-protein and RNA-protein interactions. For the most part, proteins containing the C2H2-type zinc fingers are trans regulators of gene expression that play an important role in cellular processes such as development, differentiation, and suppression of malignant cell transformation (oncosuppression).

Joint Modelling of Confounding Factors and Prominent Genetic Regulators Provides Increased Accuracy in Genetical Genomics Studies

Expression quantitative trait loci (eQTL) studies are an integral tool to investigate the genetic component of gene expression variation. A major challenge in the analysis of such studies are hidden confounding factors, such as unobserved covariates or unknown subtle environmental perturbations. These factors can induce a pronounced artifactual correlation structure in the expression profiles, which may create spurious false associations or mask real genetic association signals. Here, we report PANAMA (Probabilistic ANAlysis of genoMic dAta), a novel probabilistic model to account for confounding factors within an eQTL analysis. In contrast to previous methods, PANAMA learns hidden factors jointly with the effect of prominent genetic regulators. As a result, this new model can more accurately distinguish true genetic association signals from confounding variation. We applied our model and compared it to existing methods on different datasets and biological systems. PANAMA consistently performs better than alternative methods, and finds in particular substantially more trans regulators. Importantly, our approach not only identifies a greater number of associations, but also yields hits that are biologically more plausible and can be better reproduced between independent studies. A software implementation of PANAMA is freely available online at http://ml.sheffield.ac.uk/qtl/.

Integrating Genome-Wide Genetic Variations and Monocyte Expression Data Reveals Trans-Regulated Gene Modules in Humans

One major expectation from the transcriptome in humans is to characterize the biological basis of associations identified by genome-wide association studies. So far, few cis expression quantitative trait loci (eQTLs) have been reliably related to disease susceptibility. Trans-regulating mechanisms may play a more prominent role in disease susceptibility. We analyzed 12,808 genes detected in at least 5% of circulating monocyte samples from a population-based sample of 1,490 European unrelated subjects. We applied a method of extraction of expression patterns—independent component analysis—to identify sets of co-regulated genes. These patterns were then related to 675,350 SNPs to identify major trans-acting regulators. We detected three genomic regions significantly associated with co-regulated gene modules. Association of these loci with multiple expression traits was replicated in Cardiogenics, an independent study in which expression profiles of monocytes were available in 758 subjects. The locus 12q13 (lead SNP rs11171739), previously identified as a type 1 diabetes locus, was associated with a pattern including two cis eQTLs, RPS26 and SUOX, and 5 trans eQTLs, one of which (MADCAM1) is a potential candidate for mediating T1D susceptibility. The locus 12q24 (lead SNP rs653178), which has demonstrated extensive disease pleiotropy, including type 1 diabetes, hypertension, and celiac disease, was associated to a pattern strongly correlating to blood pressure level. The strongest trans eQTL in this pattern was CRIP1, a known marker of cellular proliferation in cancer. The locus 12q15 (lead SNP rs11177644) was associated with a pattern driven by two cis eQTLs, LYZ and YEATS4, and including 34 trans eQTLs, several of them tumor-related genes. This study shows that a method exploiting the structure of co-expressions among genes can help identify genomic regions involved in trans regulation of sets of genes and can provide clues for understanding the mechanisms linking genome-wide association loci to disease.

Erratum: Corrigendum: Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes

Nat. Genet. 43, 561–564 (2011); published online 15 May 2011; corrected after print 22 September 2011 In the version of this article initially published, there were several errors in the P values reported in the Adiponectin and HOMA-IR columns of Table 3. These errors have been corrected in the HTMLand PDF versions of the article.

Accurate modeling of confounding variation in eQTL studies leads to a great increase in power to detect trans-regulatory effects

AbstractExpression quantitative trait loci (eQTL) studies are an integral tool to investigate the genetic component of gene expression variation. A major challenge in the analysis of such studies are hidden confounding factors, such as unobserved covariates or unknown environmental influences. These factors can induce a pronounced artifactual correlation structure in the expression profiles, which may create spurious false associations or mask real genetic association signals.Here, we report PANAMA (Probabilistic ANAlysis of genoMic dAta), a novel probabilistic model to account for confounding factors within an eQTL analysis. In contrast to previous methods, PANAMA learns hidden factors jointly with the effect of prominent genetic regulators. As a result, PANAMA can more accurately distinguish between true genetic association signals and confounding variation.We applied our model and compared it to existing methods on a variety of datasets and biological systems. PANAMA consistently performs better than alternative methods, and finds in particular substantially more trans regulators. Importantly, PANAMA not only identified a greater number of associations, but also yields hits that are biologically more plausible and can be better reproduced between independent studies.

Identification of an imprinted master trans regulator at the KLF14 locus related to multiple metabolic phenotypes

Genome-wide association studies have identified many genetic variants associated with complex traits. However, at only a minority of loci have the molecular mechanisms mediating these associations been characterized. In parallel, whilst cis-regulatory patterns of gene expression have been extensively explored, the identification of trans-regulatory effects in humans has attracted less attention. We demonstrate that the Type 2 diabetes and HDL-cholesterol associated cis-acting eQTL of the maternally-expressed transcription factor KLF14 acts as a master trans-regulator of adipose gene expression. Expression levels of genes regulated by this trans-eQTL are highly-correlated with concurrently-measured metabolic traits, and a subset of the trans-genes harbor variants directly-associated with metabolic phenotypes. This trans-eQTL network provides a mechanistic understanding of the effect of the KLF14 locus on metabolic disease risk, providing a potential model for other complex traits.