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In Tibetans, noncoding alleles in EPAS1 —whose protein product hypoxia-inducible factor 2α (HIF-2α) drives the response to hypoxia—carry strong signatures of positive selection; however, their functional mechanism has not been systematically examined. Here, we report that high-altitude alleles disrupt the activity of four EPAS1 enhancers in one or more cell types. We further characterize one enhancer (ENH5) whose activity is both allele specific and hypoxia dependent. Deletion of ENH5 results in down-regulation of EPAS1 and HIF-2α targets in acute hypoxia and in a blunting of the transcriptional response to sustained hypoxia. Deletion of ENH5 in mice results in dysregulation of gene expression across multiple tissues. We propose that pleiotropic adaptive effects of the Tibetan alleles in EPAS1 underlie the strong selective signal at this gene.

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as an enhancer-blocking element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that the asthma-associated single nucleotide polymorphism (SNP) rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a mechanism through which a regulatory SNP contributes to genetic risk of asthma., This work was supported by NIH grants R01 HL118758, R01 HL128075, R01 HL119577, R01 HL085197, U19 AI095230, UG3 OD023282 and UM1 AI114271.

Genome-wide association studies (GWAS) have identified many disease-associated variants, yet mechanisms underlying these associations remain unclear. To understand obesity-associated variants, we generate gene regulatory annotations in adipocytes and hypothalamic neurons across cellular differentiation stages. We then test variants in 97 obesity-associated loci using a massively parallel reporter assay and identify putatively causal variants that display cell type specific or cross-tissue enhancer-modulating properties. Integrating these variants with gene regulatory information suggests genes that underlie obesity GWAS associations. We also investigate a complex genomic interval on 16p11.2 where two independent loci exhibit megabase-range, cross-locus chromatin interactions. We demonstrate that variants within these two loci regulate a shared gene set. Together, our data support a model where GWAS loci contain variants that alter enhancer activity across tissues, potentially with temporally restricted effects, to impact the expression of multiple genes. This complex model has broad implications for ongoing efforts to understand GWAS., Many genetic loci have been linked to obesity, but knowledge of their functional mechanisms is limited. Here, the authors perform reporter assays and temporal functional genomics data generation to characterize obesity genetic loci and find that loci often harbor multiple functional variants.

Obesity genes working together The biological causes of obesity are not well understood. Sobreira et al. examined the chromatin interactions between key genes in a locus known for its associations with obesity in human patients. In addition to directly interrogating the connections between these genes and examining the mechanisms that regulate their activity, the authors used mouse models to study the target genes' effects on both adipose tissue and brain cells that play a role in regulating dietary preferences. Science , abf1008, this issue p. 1085

Summary: Various human diseases and pregnancy-related disorders reflect endometrial dysfunction. However, rodent models do not share fundamental biological processes with the human endometrium, such as spontaneous decidualization, and no existing human cell cultures recapitulate the cyclic interactions between endometrial stromal and epithelial compartments necessary for decidualization and implantation. Here we report a protocol differentiating human pluripotent stem cells into endometrial stromal fibroblasts (PSC-ESFs) that are highly pure and able to decidualize. Coculture of PSC-ESFs with placenta-derived endometrial epithelial cells generated organoids used to examine stromal-epithelial interactions. Cocultures exhibited specific endometrial markers in the appropriate compartments, organization with cell polarity, and hormone responsiveness of both cell types. Furthermore, cocultures recapitulate a central feature of the human decidua by cyclically responding to hormone withdrawal followed by hormone retreatment. This advance enables mechanistic studies of the cyclic responses that characterize the human endometrium.

There is a life-long relationship between rhinovirus (RV) infection and the development and clinical manifestations of asthma. In this study we demonstrate that cultured primary bronchial epithelial cells from adults with asthma (n = 9) show different transcriptional and chromatin responses to RV infection compared to those without asthma (n = 9). Both the number and magnitude of transcriptional and chromatin responses to RV were muted in cells from asthma cases compared to controls. Pathway analysis of the transcriptionally responsive genes revealed enrichments of apoptotic pathways in controls but inflammatory pathways in asthma cases. Using promoter capture Hi-C we tethered regions of RV-responsive chromatin to RV-responsive genes and showed enrichment of these regions and genes at asthma GWAS loci. Taken together, our studies indicate a delayed or prolonged inflammatory state in cells from asthma cases and highlight genes that may contribute to genetic risk for asthma., Britney Helling et al. report that cultured bronchial cells from adults with asthma show different gene expression and chromatin accessibility patterns when exposed to rhinovirus than do cells from individuals without asthma. Their data suggest that rhinovirus infection leads to a delayed or elongated activation of inflammatory genes in individuals with asthma compared to those without asthma.

While coding variants often have pleiotropic effects across multiple tissues, non-coding variants are thought to mediate their phenotypic effects by specific tissue and temporal regulation of gene expression. Here, we dissected the genetic and functional architecture of a genomic region within the FTO gene that is strongly associated with obesity risk. We show that multiple variants on a common haplotype modify the regulatory properties of several enhancers targeting IRX3 and IRX5 from megabase distances. We demonstrate that these enhancers impact gene expression in multiple tissues, including adipose and brain, and impart regulatory effects during a restricted temporal window. Our data indicate that the genetic architecture of disease-associated loci may involve extensive pleiotropy, allelic heterogeneity, shared allelic effects across tissues, and temporally-restricted effects.

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. We show that genotype at the asthma-associated SNP rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

Genome-wide association studies (GWAS) have implicated the IL33 locus in asthma, but the underlying mechanisms remain unclear. Here, we identify a 5 kb region within the GWAS-defined segment that acts as a strong regulatory element in vivo and in vitro. Chromatin conformation capture showed that this 5 kb region loops to the IL33 promoter, potentially regulating its expression. Supporting this notion, we show that genotype at an asthma-associated SNP, rs1888909, located within the 5 kb region, is associated with IL33 gene expression in human airway epithelial cells and IL-33 protein expression in human plasma, potentially through differential binding of OCT-1 (POU2F1) to the asthma-risk allele. Our data demonstrate that asthma-associated variants at the IL33 locus mediate allele-specific regulatory activity and IL33 expression, providing a novel mechanism through which a regulatory SNP contributes to genetic risk of asthma.

Human trophoblast stem cells (hTSC) can be isolated from first trimester placenta but not from term placenta. Here we demonstrate that villous cytotrophoblasts (vCTB) from term placenta can be reprogrammed into induced trophoblastic stem-like cells (iTSC) by introducing sets of transcription factors. The iTSCs express TSC markers such as GATA3, TEAD4 and ELF5, and are multipotent, validated by their differentiation into both extravillous trophoblasts (EVT) and syncytiotrophoblasts (STB) in vitro and in vivo. The iTSC can be passaged indefinitely in vitro without slowing of growth. The transcriptome profile of these cells closely resembles the profile of hTSC isolated from first trimester placentae but different from the term placental vCTB from which they originated. The ability to reprogram cells from term placenta into iTSC will allow study of early gestation events which impact placental function later in gestation, including preeclampsia and spontaneous preterm birth.

Mutations in the T-box transcription factor TBX20 are associated with multiple forms of congenital heart defects, including cardiac septal abnormalities, but our understanding of the contributions of endocardial TBX20 to heart development remains incomplete. Here, we investigated how TBX20 interacts with endocardial gene networks to drive the mesenchymal and myocardial movements that are essential for outflow tract and atrioventricular septation. Selective ablation of Tbx20 in murine endocardial lineages reduced the expression of extracellular matrix and cell migration genes that are critical for septation. Using the assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq), we identified accessible chromatin within endocardial lineages and intersected these data with TBX20 ChIP-seq and chromatin loop maps to determine that TBX20 binds a conserved long-range enhancer to regulate versican (Vcan) expression. We also observed reduced Vcan expression in Tbx20-deficient mice, supporting a direct role for TBX20 in Vcan regulation. Further, we show that the Vcan enhancer drove reporter gene expression in endocardial lineages in a TBX20-binding site-dependent manner. This work illuminates gene networks that interact with TBX20 to orchestrate cardiac septation and provides insight into the chromatin landscape of endocardial lineages during septation.

While Mediator plays a key role in eukaryotic transcription, little is known about its mechanism of action. This study combines CRISPR-Cas9 genetic screens, degron assays, Hi-C, and cryoelectron microscopy (cryo-EM) to dissect the function and structure of mammalian Mediator (mMED). Deletion analyses in B, T, and embryonic stem cells (ESC) identified a core of essential subunits required for Pol II recruitment genome-wide. Conversely, loss of non-essential subunits mostly affects promoters linked to multiple enhancers. Contrary to current models, however, mMED and Pol II are dispensable to physically tether regulatory DNA, a topological activity requiring architectural proteins. Cryo-EM analysis revealed a conserved core, with non-essential subunits increasing structural complexity of the tail module, a primary transcription factor target. Changes in tail structure markedly increase Pol II and kinase module interactions. We propose that Mediator's structural pliability enables it to integrate and transmit regulatory signals and act as a functional, rather than an architectural bridge, between promoters and enhancers.

Supplemental Digital Content is available in the text., Rationale: Mutations in the transcription factor TBX20 (T-box 20) are associated with congenital heart disease. Germline ablation of Tbx20 results in abnormal heart development and embryonic lethality by embryonic day 9.5. Because Tbx20 is expressed in multiple cell lineages required for myocardial development, including pharyngeal endoderm, cardiogenic mesoderm, endocardium, and myocardium, the cell type–specific requirement for TBX20 in early myocardial development remains to be explored. Objective: Here, we investigated roles of TBX20 in midgestation cardiomyocytes for heart development. Methods and Results: Ablation of Tbx20 from developing cardiomyocytes using a doxycycline inducible cTnTCre transgene led to embryonic lethality. The circumference of developing ventricular and atrial chambers, and in particular that of prospective left atrium, was significantly reduced in Tbx20 conditional knockout mutants. Cell cycle analysis demonstrated reduced proliferation of Tbx20 mutant cardiomyocytes and their arrest at the G1-S phase transition. Genome-wide transcriptome analysis of mutant cardiomyocytes revealed differential expression of multiple genes critical for cell cycle regulation. Moreover, atrial and ventricular gene programs seemed to be aberrantly regulated. Putative direct TBX20 targets were identified using TBX20 ChIP-Seq (chromatin immunoprecipitation with high throughput sequencing) from embryonic heart and included key cell cycle genes and atrial and ventricular specific genes. Notably, TBX20 bound a conserved enhancer for a gene key to atrial development and identity, COUP-TFII/Nr2f2 (chicken ovalbumin upstream promoter transcription factor 2/nuclear receptor subfamily 2, group F, member 2). This enhancer interacted with the NR2F2 promoter in human cardiomyocytes and conferred atrial specific gene expression in a transgenic mouse in a TBX20-dependent manner. Conclusions: Myocardial TBX20 directly regulates a subset of genes required for fetal cardiomyocyte proliferation, including those required for the G1-S transition. TBX20 also directly downregulates progenitor-specific genes and, in addition to regulating genes that specify chamber versus nonchamber myocardium, directly activates genes required for establishment or maintenance of atrial and ventricular identity. TBX20 plays a previously unappreciated key role in atrial development through direct regulation of an evolutionarily conserved COUPT-FII enhancer.

Transcriptional mediators of cell stress pathways, including HIF1α, ATF4, and p53, are key to normal development and play critical roles in disease, including ischemia and cancer. Despite their importance, mechanisms by which pathways mediated by these transcription factors interact with each other are not fully understood. In addressing the controversial role of HIF1α in cardiomyocytes (CMs) during heart development, we have discovered a mid-gestational requirement for HIF1α for proliferation of hypoxic CMs, involving metabolic switching and a complex interplay between HIF1α, ATF4 and p53. Loss of HIF1α resulted in activation of ATF4 and p53, the latter inhibiting CM proliferation. Bioinformatic and biochemical analyses revealed unexpected mechanisms by which HIF1α intersects with ATF4 and p53 pathways. Our results highlight previously undescribed roles of HIF1α and interactions between major cell stress pathways that could be targeted to enhance proliferation of CMs in ischemia, and may have relevance to other diseases, including cancer.

ATAC-seq is a high-throughput sequencing technique that identifies open chromatin. Depending on the cell type, ATAC-seq samples may contain ~20–80% of mitochondrial sequencing reads. As the regions of open chromatin of interest are usually located in the nuclear genome, mitochondrial reads are typically discarded from the analysis. We tested two approaches to decrease wasted sequencing in ATAC-seq libraries generated from lymphoblastoid cell lines: targeted cleavage of mitochondrial DNA fragments using CRISPR technology and removal of detergent from the cell lysis buffer. We analyzed the effects of these treatments on the number of usable (unique, non-mitochondrial) reads and the number and quality of peaks called, including peaks identified in enhancers and transcription start sites. Both treatments resulted in considerable reduction of mitochondrial reads (1.7 and 3-fold, respectively). The removal of detergent, however, resulted in increased background and fewer peaks. The highest number of peaks and highest quality data was obtained by preparing samples with the original ATAC-seq protocol (using detergent) and treating them with CRISPR. This strategy reduced the amount of sequencing required to call a high number of peaks, which could lead to cost reduction when performing ATAC-seq on large numbers of samples and in cell types that contain a large amount of mitochondria.

Human mutations in or variants of TBX20 are associated with congenital heart disease, cardiomyopathy, and arrhythmias. To investigate whether cardiac disease in patients with these conditions results from an embryonic or ongoing requirement for Tbx20 in myocardium, we ablated Tbx20 specifically in adult cardiomyocytes in mice. This ablation resulted in the onset of severe cardiomyopathy accompanied by arrhythmias, with death ensuing within 1 to 2 weeks of Tbx20 ablation. Accounting for this dramatic phenotype, we identified molecular signatures that posit Tbx20 as a central integrator of a genetic program that maintains cardiomyocyte function in the adult heart. Expression of a number of genes encoding critical transcription factors, ion channels, and cytoskeletal/myofibrillar proteins was downregulated consequent to loss of Tbx20. Genome-wide ChIP analysis of Tbx20-binding regions in the adult heart revealed that many of these genes were direct downstream targets of Tbx20 and uncovered a previously undescribed DNA-binding site for Tbx20. Bioinformatics and in vivo functional analyses revealed a cohort of transcription factors that, working with Tbx20, integrated multiple environmental signals to maintain ion channel gene expression in the adult heart. Our data provide insight into the mechanisms by which mutations in TBX20 cause adult heart disease in humans.

The various organogenic programs deployed during embryonic development rely on the precise expression of a multitude of genes in time and space. Identifying the cis-regulatory elements responsible for this tightly orchestrated regulation of gene expression is an essential step in understanding the genetic pathways involved in development. We describe a strategy to systematically identify tissue-specific cis-regulatory elements that share combinations of sequence motifs. Using heart development as an experimental framework, we employed a combination of Gibbs sampling and linear regression to build a classifier that identifies heart enhancers based on the presence and/or absence of various sequence features, including known and putative transcription factor (TF) binding specificities. In distinguishing heart enhancers from a large pool of random noncoding sequences, the performance of our classifier is vastly superior to four commonly used methods, with an accuracy reaching 92% in cross-validation. Furthermore, most of the binding specificities learned by our method resemble the specificities of TFs widely recognized as key players in heart development and differentiation, such as SRF, MEF2, ETS1, SMAD, and GATA. Using our classifier as a predictor, a genome-wide scan identified over 40,000 novel human heart enhancers. Although the classifier used no gene expression information, these novel enhancers are strongly associated with genes expressed in the heart. Finally, in vivo tests of our predictions in mouse and zebrafish achieved a validation rate of 62%, significantly higher than what is expected by chance. These results support the existence of underlying cis-regulatory codes dictating tissue-specific transcription in mammalian genomes and validate our enhancer classifier strategy as a method to uncover these regulatory codes.

The potency of the immune response has still to be harnessed effectively to combat human cancers. However, the discovery of T-cell targets in melanomas and other tumors has raised the possibility that cancer vaccines can be used to induce a therapeutically effective immune response against cancer. The targets, cancer-testis (CT) antigens, are immunogenic proteins preferentially expressed in normal gametogenic tissues and different histological types of tumors. Therapeutic cancer vaccines directed against CT antigens are currently in late-stage clinical trials testing whether they can delay or prevent recurrence of lung cancer and melanoma following surgical removal of primary tumors. CT antigens constitute a large, but ill-defined, family of proteins that exhibit a remarkably restricted expression. Currently, there is a considerable amount of information about these proteins, but the data are scattered through the literature and in several bioinformatic databases. The database presented here, CTdatabase (http://www.cta.lncc.br), unifies this knowledge to facilitate both the mining of the existing deluge of data, and the identification of proteins alleged to be CT antigens, but that do not have their characteristic restricted expression pattern. CTdatabase is more than a repository of CT antigen data, since all the available information was carefully curated and annotated with most data being specifically processed for CT antigens and stored locally. Starting from a compilation of known CT antigens, CTdatabase provides basic information including gene names and aliases, RefSeq accession numbers, genomic location, known splicing variants, gene duplications and additional family members. Gene expression at the mRNA level in normal and tumor tissues has been collated from publicly available data obtained by several different technologies. Manually curated data related to mRNA and protein expression, and antigen-specific immune responses in cancer patients are also available, together with links to PubMed for relevant CT antigen articles.

Exon-shuffling is an important mechanism accounting for the origin of many new proteins in eukaryotes. However, its role in the creation of proteins in the ancestor of prokaryotes and eukaryotes is still debatable. Excess of symmetric exons is thought to represent evidence for exon-shuffling since the exchange of exons flanked by introns of the same phase does not disrupt the reading frame of the host gene. In this report, we found that there is a significant correlation between symmetric units of shuffling and the age of protein domains. Ancient domains, present in both prokaryotes and eukaryotes, are more frequently bounded by phase 0 introns and their distribution is biased towards the central part of proteins. Modern domains are more frequently bounded by phase 1 introns and are present predominantly at the ends of proteins. We propose a model in which shuffling of ancient domains mainly flanked by phase 0 introns was important in the ancestor of eukaryotes and prokaryotes, during the creation of the central part of proteins. Shuffling of modern domains, predominantly flanked by phase 1 introns, accounted for the origin of the extremities of proteins during eukaryotic evolution.

A significant fraction of the variability found in the human transcriptome is due to alternative splicing, including alternative exon usage (AEU), intron retention and use of cryptic splice sites. We present a comparison of a large-scale analysis of AEU in the human transcriptome through genome mapping of Open Reading Frame ESTs (ORESTES) and conventional ESTs. It is shown here that ORESTES probe low abundant messages more efficiently. In addition, most of the variants detected by ORESTES affect the structure of the corresponding proteins.

PMCID: PMC4113484.-- et al., Genome-wide association studies (GWAS) have reproducibly associated variants within introns of FTO with increased risk for obesity and type 2 diabetes (T2D). Although the molecular mechanisms linking these noncoding variants with obesity are not immediately obvious, subsequent studies in mice demonstrated that FTO expression levels influence body mass and composition phenotypes. However, no direct connection between the obesity-associated variants and FTO expression or function has been made. Here we show that the obesity-associated noncoding sequences within FTO are functionally connected, at megabase distances, with the homeobox gene IRX3. The obesity-associated FTO region directly interacts with the promoters of IRX3 as well as FTO in the human, mouse and zebrafish genomes. Furthermore, long-range enhancers within this region recapitulate aspects of IRX3 expression, suggesting that the obesity-associated interval belongs to the regulatory landscape of IRX3. Consistent with this, obesity-associated single nucleotide polymorphisms are associated with expression of IRX3, but not FTO, in human brains. A direct link between IRX3 expression and regulation of body mass and composition is demonstrated by a reduction in body weight of 25 to 30% in Irx3-deficient mice, primarily through the loss of fat mass and increase in basal metabolic rate with browning of white adipose tissue. Finally, hypothalamic expression of a dominant-negative form of Irx3 reproduces the metabolic phenotypes of Irx3-deficient mice. Our data suggest that IRX3 is a functional long-range target of obesity-associated variants within FTO and represents a novel determinant of body mass and composition., This work was funded by grants from the National Institutes of Health (DK093972, HL119967, HL114010 and DK020595) to M.A.N. and (MH101820, MH090937 and DK20595) to N.J.C. J.L.G.-S. was funded by grants from the Spanish Ministerio de Economía y Competitividad (BFU2010-14839, CSD2007-00008) and the Andalusian Government (CVI-3488). C.-C.H. was supported by a grant from the Canadian Institute of Health Research. K.-H.K. is supported by a fellowship from the Heart and Stroke Foundation of Canada. S.S. is supported by an NIH postdoctoral training grant (T32HL007381)

The ongoing requirement in adult heart for transcription factors with key roles in cardiac development is not well understood. We recently demonstrated that TBX20, a transcriptional regulator required for cardiac development, has key roles in the maintenance of functional and structural phenotypes in adult mouse heart. Conditional ablation of Tbx20 in adult cardiomyocytes leads to a rapid onset and progression of heart failure, with prominent conduction and contractility phenotypes that lead to death. Here we describe a more comprehensive molecular characterization of the functions of TBX20 in adult mouse heart. Coupling genome-wide chromatin immunoprecipitation and transcriptome analyses (RNA-Seq), we identified a subset of genes that change expression in Tbx20 adult cardiomyocyte-specific knockout hearts which are direct downstream targets of TBX20. This analysis revealed a dual role for TBX20 as both a transcriptional activator and a repressor, and that each of these functions regulates genes with very specialized and distinct molecular roles. We also show how TBX20 binds to its targets genome-wide in a context-dependent manner, using various cohorts of co-factors to either promote or repress distinct genetic programs within adult heart. Our integrative approach has uncovered several novel aspects of TBX20 and T-box protein function within adult heart. Sequencing data accession number (http://www.ncbi.nlm.nih.gov/geo): GSE30943.

Expression of eukaryotic genes with complex spatial-temporal regulation during development requires finer regulation than that of genes with simpler expression patterns. Given the high degree of conservation of the developmental gene set across distantly related phylogenetic taxa, it is argued that evolutionary variation has occurred by tweaking regulation of expression of developmental genes, rather than by changes in genes themselves. Complex regulation is often achieved through the coordinated action of transcription regulatory elements spread across the genome up to tens of kilobases from the promoters of their target genes. Disruption of regulatory elements has been implicated in several diseases and studies showing associations between disease traits and nonprotein coding variation hint for a role of regulatory elements as cause of diseases. Therefore, the identification and mapping of regulatory elements in genome scale is crucial to understand how gene expression is regulated, how organisms evolve, and to identify sequence variation causing diseases. Previously developed experimental techniques have been adapted to identify regulatory elements in genome scale and high-throughput, allowing a global view of their biological roles. We review methods as chromatin immunoprecipitation, DNase I hypersensitivity, and computational approaches and how they have been employed to generate maps of histone modifications, open chromatin, nucleosome positioning, and transcription factor binding regions in whole mammalian genomes. Given the importance of non-promoter elements in gene regulation and the recent explosion in the number of studies devoted to them, we focus on these elements and discuss the insights on gene regulation being obtained by these studies.

Exons can be independently gained or created in genes by exon shuffling, duplication and mutation of intronic sequences, especially repetitive elements as Alu. Such new exons are more prone to be alternatively spliced, included in transcripts in low frequencies and evolve faster than ancient exons. Alternative splicing is an important component of the evolution of new exons since it allows new gene parts to be slowly added to existing genes without compromising the original protein function. Eventually, new exons may be integrated to the original gene and evolve new functions. Keywords: exon birth; exon evolution; alternative splicing; exonization of Alu elements

Background One of the least common types of alternative splicing is the complete retention of an intron in a mature transcript. Intron retention (IR) is believed to be the result of intron, rather than exon, definition associated with failure of the recognition of weak splice sites flanking short introns. Although studies on individual retained introns have been published, few systematic surveys of large amounts of data have been conducted on the mechanisms that lead to IR. Results TTo understand how sequence features are associated with or control IR, and to produce a generalized model that could reveal previously unknown signals that regulate this type of alternative splicing, we partitioned intron retention events observed in human cDNAs into two groups based on the relative abundance of both isoforms and compared relevant features. We found that a higher frequency of IR in human is associated with individual introns that have weaker splice sites, genes with shorter intron lengths, higher expression levels and lower density of both a set of exon splicing silencers (ESSs) and the intronic splicing enhancer GGG. Both groups of retained introns presented events conserved in mouse, in which the retained introns were also short and presented weaker splice sites. Conclusion Although our results confirmed that weaker splice sites are associated with IR, they showed that this feature alone cannot explain a non-negligible fraction of events. Our analysis suggests that cis-regulatory elements are likely to play a crucial role in regulating IR and also reveals previously unknown features that seem to influence its occurrence. These results highlight the importance of considering the interplay among these features in the regulation of the relative frequency of IR.

It was recently shown that there is a predominance of phase 1 introns near the cleavage site of signal peptides encoded by human genes [Tordai, H. and Patthy, L. (2004) Insertion of spliceosomal introns in proto-splice sites: the case of secretory signal peptides. FEBS Lett. 575, 109–111]. It was suggested that this biased distribution was due to intron insertion at AG∣G proto-splice sites. However, we found that there is no disproportional excess of AG∣G that would support insertion at proto-splice sites. In fact, all nG∣G sites are enriched in the vicinity of the cleavage site. Additional analyses support an alternative scenario in which exon-shuffling is largely responsible for such excess of phase 1 introns.

Alternative splicing is a very frequent phenomenon in the human transcriptome. There are four major types of alternative splicing: exon skipping, alternative 3′ splice site, alternative 5′ splice site, and intron retention. Here we present a large-scale analysis of intron retention in a set of 21,106 known human genes. We observed that 14.8% of these genes showed evidence of at least one intron retention event. Most of the events are located within the untranslated regions (UTRs) of human transcripts. For those retained introns interrupting the coding region, the GC content, codon usage, and the frequency of stop codons suggest that these sequences are under selection for coding potential. Furthermore, 26% of the introns within the coding region participate in the coding of a protein domain. A comparison with mouse shows that at least 22% of all informative examples of retained introns in human are also present in the mouse transcriptome. We discuss that the data we present suggest that a significant fraction of the observed events is not spurious and might reflect biological significance. The analyses also allowed us to generate a reliable set of intron retention events that can be used for the identification of splicing regulatory elements.

The complex expression patterns observed for many genes are often regulated by distal transcription enhancers. Changes in the nucleotide sequences of enhancers may therefore lead to changes in gene expression, representing a central mechanism by which organisms evolve. With the development of the experimental technique of chromatin immunoprecipitation (ChIP), in which discrete regions of the genome bound by specific proteins can be identified, it is now possible to identify transcription factor binding events (putative cis -regulatory elements) in entire genomes. Comparing protein–DNA binding maps allows us, for the first time, to attempt to identify regulatory differences and infer global patterns of change in gene expression across species. Here, we review studies that used genome-wide ChIP to study the evolution of enhancers. The trend is one of high divergence of cis -regulatory elements between species, possibly compensated by extensive creation and loss of regulatory elements and rewiring of their target genes. We speculate on the meaning of the differences observed and discuss that although ChIP experiments identify the biochemical event of protein–DNA interaction, it cannot determine whether the event results in a biological function, and therefore more studies are required to establish the effect of divergence of binding events on species-specific gene expression.

Distal transcription enhancers are cis-regulatory elements that promote gene expression, enabling spatiotemporal control of genetic programs such as those required in metazoan developmental processes. Because of their importance, their disruption can lead to disease.