Instruct: Given a scientific claim, retrieve documents that support or refute the claim.
Query: Sequence conservation in gene regulatory regions between species is a high-accuracy predictor of conserved functionality.
The mammalian radiation has corresponded with rapid changes in noncoding regions of the genome, but we lack a comprehensive understanding of regulatory evolution in mammals. Here, we track the evolution of promoters and enhancers active in liver across 20 mammalian species from six diverse orders by profiling genomic enrichment of H3K27 acetylation and H3K4 trimethylation. We report that rapid evolution of enhancers is a universal feature of mammalian genomes. Most of the recently evolved enhancers arise from ancestral DNA exaptation, rather than lineage-specific expansions of repeat elements. In contrast, almost all liver promoters are partially or fully conserved across these species. Our data further reveal that recently evolved enhancers can be associated with genes under positive selection, demonstrating the power of this approach for annotating regulatory adaptations in genomic sequences. These results provide important insight into the functional genetics underpinning mammalian regulatory evolution.
Early studies of telomerase suggested that telomeres are maintained by an elegant but relatively simple and highly conserved mechanism of telomerase-mediated replication. As we learn more, it has become clear that the mechanism is elegant but not as simple as first thought. It is also evident that, although many species use similar, sometimes identical, DNA sequences for telomeres, these species express their own individuality in the way they regulate these sequences and, perhaps, in the additional tasks that they have imposed on their telomeric DNA. The striking similarities between telomeres in different species have revealed much about chromosome ends; the differences are proving to be equally informative. In addition to the differences between species that use telomerase, there are also a few exceptional organisms with atypical telomeres for which no telomerase activity has been detected. This review addresses recent studies, the insights they offer, and, perhaps more importantly, the questions they raise.
Increasing numbers of transcripts have been reported to transmit both protein-coding and regulatory information. Apart from challenging our conception of the gene, this observation raises the question as to what extent this phenomenon occurs across the genome and how and why such dual encoding of function has evolved in the eukaryotic genome. To address this question, we consider the evolutionary path of genes in the earliest forms of life on Earth, where it is generally regarded that proteins evolved from a cellular machinery based entirely within RNA. This led to the domination of protein-coding genes in the genomes of microorganisms, although it is likely that RNA never lost its other capacities and functionalities, as evidenced by cis-acting riboswitches and UTRs. On the basis that the subsequent evolution of a more sophisticated regulatory architecture to provide higher levels of epigenetic control and accurate spatiotemporal expression in developmentally complex organisms is a complicated task, we hypothesize: (i) that mRNAs have been and remain subject to secondary selection to provide trans-acting regulatory capability in parallel with protein-coding functions; (ii) that some and perhaps many protein-coding loci, possibly as a consequence of gene duplication, have lost protein-coding functions en route to acquiring more sophisticated trans-regulatory functions; (iii) that many transcripts have become subject to secondary processing to release different products; and (iv) that novel proteins have emerged within loci that previously evolved functionality as regulatory RNAs. In support of the idea that there is a dynamic flux between different types of informational RNAs in both evolutionary and real time, we review recent observations that have arisen from transcriptomic surveys of complex eukaryotes and reconsider how these observations impact on the notion that apparently discrete loci may express transcripts with more than one function. In conclusion, we posit that many eukaryotic loci have evolved the capacity to transact a multitude of overlapping and potentially independent functions as both regulatory and protein-coding RNAs.
We report a database of genes responsive to the Myc oncogenic transcription factor. The database Myc Target Gene prioritizes candidate target genes according to experimental evidence and clusters responsive genes into functional groups. We coupled the prioritization of target genes with phylogenetic sequence comparisons to predict c-Myc target binding sites, which are in turn validated by chromatin immunoprecipitation assays. This database is essential for the understanding of the genetic regulatory networks underlying the genesis of cancers.
UNLABELLED Artificially synthesized short interfering RNAs (siRNAs) are widely used in functional genomics to knock down specific target genes. One ongoing challenge is to guarantee that the siRNA does not elicit off-target effects. Initial reports suggested that siRNAs were highly sequence-specific; however, subsequent data indicates that this is not necessarily the case. It is still uncertain what level of similarity and other rules are required for an off-target effect to be observed, and scoring schemes have not been developed to look beyond simple measures such as the number of mismatches or the number of consecutive matching bases present. We created design rules for predicting the likelihood of a non-specific effect and present a web server that allows the user to check the specificity of a given siRNA in a flexible manner using a combination of methods. The server finds potential off-target matches in the corresponding RefSeq database and ranks them according to a scoring system based on experimental studies of specificity. AVAILABILITY The server is available at http://informatics-eskitis.griffith.edu.au/SpecificityServer.
In this study, we describe the 3D chromosome regulatory landscape of human naive and primed embryonic stem cells. To devise this map, we identified transcriptional enhancers and insulators in these cells and placed them within the context of cohesin-associated CTCF-CTCF loops using cohesin ChIA-PET data. The CTCF-CTCF loops we identified form a chromosomal framework of insulated neighborhoods, which in turn form topologically associating domains (TADs) that are largely preserved during the transition between the naive and primed states. Regulatory changes in enhancer-promoter interactions occur within insulated neighborhoods during cell state transition. The CTCF anchor regions we identified are conserved across species, influence gene expression, and are a frequent site of mutations in cancer cells, underscoring their functional importance in cellular regulation. These 3D regulatory maps of human pluripotent cells therefore provide a foundation for future interrogation of the relationships between chromosome structure and gene control in development and disease.
Studies of replication timing provide a handle into previously impenetrable higher-order levels of chromosome organization and their plasticity during development. Although mechanisms regulating replication timing are not clear, novel genome-wide studies provide a thorough survey of the extent to which replication timing is regulated during most of the early cell fate transitions in mammals, revealing coordinated changes of a defined set of 400–800 kb chromosomal segments that involve at least half the genome. Furthermore, changes in replication time are linked to changes in sub-nuclear organization and domain-wide transcriptional potential, and tissue-specific replication timing profiles are conserved from mouse to human, suggesting that the program has developmental significance. Hence, these studies have provided a solid foundation for linking megabase level chromosome structure to function, and suggest a central role for replication in domain-level genome organization.
Chromatin insulators are DNA elements that regulate the level of gene expression either by preventing gene silencing through the maintenance of heterochromatin boundaries or by preventing gene activation by blocking interactions between enhancers and promoters. CCCTC-binding factor (CTCF), a ubiquitously expressed 11-zinc-finger DNA-binding protein, is the only protein implicated in the establishment of insulators in vertebrates. While CTCF has been implicated in diverse regulatory functions, CTCF has only been studied in a limited number of cell types across human genome. Thus, it is not clear whether the identified cell type-specific differences in CTCF-binding sites are functionally significant. Here, we identify and characterize cell type-specific and ubiquitous CTCF-binding sites in the human genome across 38 cell types designated by the Encyclopedia of DNA Elements (ENCODE) consortium. These cell type-specific and ubiquitous CTCF-binding sites show uniquely versatile transcriptional functions and characteristic chromatin features. In addition, we confirm the insulator barrier function of CTCF-binding and explore the novel function of CTCF in DNA replication. These results represent a critical step toward the comprehensive and systematic understanding of CTCF-dependent insulators and their versatile roles in the human genome.
The similarity in the three-dimensional structures of homologous proteins imposes strong constraints on their sequence variability. It has long been suggested that the resulting correlations among amino acid compositions at different sequence positions can be exploited to infer spatial contacts within the tertiary protein structure. Crucial to this inference is the ability to disentangle direct and indirect correlations, as accomplished by the recently introduced Direct Coupling Analysis (DCA) (Weigt et al. (2009) Proc Natl Acad Sci 106:67). Here we develop a computationally efficient implementation of DCA, which allows us to evaluate the accuracy of contact prediction by DCA for a large number of protein domains, based purely on sequence information. DCA is shown to yield a large number of correctly predicted contacts, recapitulating the global structure of the contact map for the majority of the protein domains examined. Furthermore, our analysis captures clear signals beyond intra- domain residue contacts, arising, e.g., from alternative protein conformations, ligand- mediated residue couplings, and inter-domain interactions in protein oligomers. Our findings suggest that contacts predicted by DCA can be used as a reliable guide to facilitate computational predictions of alternative protein conformations, protein complex formation, and even the de novo prediction of protein domain structures, provided the existence of a large number of homologous sequences which are being rapidly made available due to advances in genome sequencing.
Enhancers control the correct temporal and cell-type-specific activation of gene expression in multicellular eukaryotes. Knowing their properties, regulatory activity and targets is crucial to understand the regulation of differentiation and homeostasis. Here we use the FANTOM5 panel of samples, covering the majority of human tissues and cell types, to produce an atlas of active, in vivo-transcribed enhancers. We show that enhancers share properties with CpG-poor messenger RNA promoters but produce bidirectional, exosome-sensitive, relatively short unspliced RNAs, the generation of which is strongly related to enhancer activity. The atlas is used to compare regulatory programs between different cells at unprecedented depth, to identify disease-associated regulatory single nucleotide polymorphisms, and to classify cell-type-specific and ubiquitous enhancers. We further explore the utility of enhancer redundancy, which explains gene expression strength rather than expression patterns. The online FANTOM5 enhancer atlas represents a unique resource for studies on cell-type-specific enhancers and gene regulation.
Mammalian genomes are extensively transcribed outside the borders of protein-coding genes. Genome-wide studies recently demonstrated that cis-regulatory genomic elements implicated in transcriptional control, such as enhancers and locus-control regions, represent major sites of extragenic noncoding transcription. Enhancer-templated transcripts provide a quantitatively small contribution to the total amount of cellular nonribosomal RNA; nevertheless, the possibility that enhancer transcription and the resulting enhancer RNAs may, in some cases, have functional roles, rather than represent mere transcriptional noise at accessible genomic regions, is supported by an increasing amount of experimental data. In this article we review the current knowledge on enhancer transcription and its functional implications.
Detection of new genomic control elements is critical in understanding transcriptional regulatory networks in their entirety. We studied the genome-wide binding locations of three key regulatory proteins (POU5F1, also known as OCT4; NANOG; and CTCF) in human and mouse embryonic stem cells. In contrast to CTCF, we found that the binding profiles of OCT4 and NANOG are markedly different, with only ∼5% of the regions being homologously occupied. We show that transposable elements contributed up to 25% of the bound sites in humans and mice and have wired new genes into the core regulatory network of embryonic stem cells. These data indicate that species-specific transposable elements have substantially altered the transcriptional circuitry of pluripotent stem cells.
Transcription regulation in higher eukaryotes is controlled by regulatory elements such as enhancers that are recognized by transcription factors. In many cases regulatory elements can be located at distances up to several megabases from their target genes. Recent evidence shows that long-range control of gene expression can be mediated through direct physical interactions between genes and these regulatory elements. Such looping interactions can be detected using the chromosome conformation capture (3C) methodology. Although 3C is experimentally straightforward, to draw meaningful conclusions one must carefully design 3C experiments and implement the conscientious use of controls. The general guidelines presented here should help experimental design and minimize misinterpretation of 3C experiments.
Despite being one of the first eukaryotic transcriptional regulatory elements identified, the sequence of a native TATA box and its significance remain elusive. Applying criteria associated with TATA boxes we queried several Saccharomyces genomes and arrived at the consensus TATA(A/T)A(A/T)(A/G). Approximately 20% of yeast genes contain a TATA box. Strikingly, TATA box-containing genes are associated with responses to stress, are highly regulated, and preferentially utilize SAGA rather than TFIID when compared to TATA-less promoters. Transcriptional regulation in yeast appears to be mechanistically bipolar, possibly reflecting a need to balance inducible stress-related responses with constitutive housekeeping functions.
The YEAst Search for Transcriptional Regulators And Consensus Tracking (YEASTRACT) information system (http://www.yeastract.com) was developed to support the analysis of transcription regulatory associations in Saccharomyces cerevisiae. Last updated in June 2010, this database contains over 48,200 regulatory associations between transcription factors (TFs) and target genes, including 298 specific DNA-binding sites for 110 characterized TFs. All regulatory associations stored in the database were revisited and detailed information on the experimental evidences that sustain those associations was added and classified as direct or indirect evidences. The inclusion of this new data, gathered in response to the requests of YEASTRACT users, allows the user to restrict its queries to subsets of the data based on the existence or not of experimental evidences for the direct action of the TFs in the promoter region of their target genes. Another new feature of this release is the availability of all data through a machine readable web-service interface. Users are no longer restricted to the set of available queries made available through the existing web interface, and can use the web service interface to query, retrieve and exploit the YEASTRACT data using their own implementation of additional functionalities. The YEASTRACT information system is further complemented with several computational tools that facilitate the use of the curated data when answering a number of important biological questions. Since its first release in 2006, YEASTRACT has been extensively used by hundreds of researchers from all over the world. We expect that by making the new data and services available, the system will continue to be instrumental for yeast biologists and systems biology researchers.
Synthetic gene expression is highly sensitive to intragenic compositional context (promoter structure, spacing regions between promoter and coding sequences, and ribosome binding sites). However, much less is known about the effects of intergenic compositional context (spatial arrangement and orientation of entire genes on DNA) on expression levels in synthetic gene networks. We compare expression of induced genes arranged in convergent, divergent, or tandem orientations. Induction of convergent genes yielded up to 400% higher expression, greater ultrasensitivity, and dynamic range than divergent- or tandem-oriented genes. Orientation affects gene expression whether one or both genes are induced. We postulate that transcriptional interference in divergent and tandem genes, mediated by supercoiling, can explain differences in expression and validate this hypothesis through modeling and in vitro supercoiling relaxation experiments. Treatment with gyrase abrogated intergenic context effects, bringing expression levels within 30% of each other. We rebuilt the toggle switch with convergent genes, taking advantage of supercoiling effects to improve threshold detection and switch stability.
In the present study, the relationship between short interfering RNA (siRNA) sequence and RNA interference (RNAi) effect was extensively analyzed using 62 targets of four exogenous and two endogenous genes and three mammalian and Drosophila cells. We present the rules that may govern siRNA sequence preference and in accordance with which highly effective siRNAs essential for systematic mammalian functional genomics can be readily designed. These rules indicate that siRNAs which simultaneously satisfy all four of the following sequence conditions are capable of inducing highly effective gene silencing in mammalian cells: (i) A/U at the 5' end of the antisense strand; (ii) G/C at the 5' end of the sense strand; (iii) at least five A/U residues in the 5' terminal one-third of the antisense strand; and (iv) the absence of any GC stretch of more than 9 nt in length. siRNAs opposite in features with respect to the first three conditions give rise to little or no gene silencing in mammalian cells. Essentially the same rules for siRNA sequence preference were found applicable to DNA-based RNAi in mammalian cells and in ovo RNAi using chick embryos. In contrast to mammalian and chick cells, little siRNA sequence preference could be detected in Drosophila in vivo RNAi.
Evolutionary related multisubunit RNA polymerases (RNAPs) transcribe the genomes of all living organisms. Whereas the core subunits of RNAPs are universally conserved in all three domains of life-indicative of a common evolutionary descent-this only applies to one RNAP-associated transcription factor-Spt5, also known as NusG in bacteria. All other factors that aid RNAP during the transcription cycle are specific for the individual domain or only conserved between archaea and eukaryotes. Spt5 and its bacterial homologue NusG regulate gene expression in several ways by (i) modulating transcription processivity and promoter proximal pausing, (ii) coupling transcription and RNA processing or translation, and (iii) recruiting termination factors and thereby silencing laterally transferred DNA and protecting the genome against double-stranded DNA breaks. This review discusses recent discoveries that identify Spt5-like factors as evolutionary conserved nexus for the regulation and coordination of the machineries responsible for information processing in the cell.
The most highly conserved noncoding elements (HCNEs) in mammalian genomes cluster within regions enriched for genes encoding developmentally important transcription factors (TFs). This suggests that HCNE-rich regions may contain key regulatory controls involved in development. We explored this by examining histone methylation in mouse embryonic stem (ES) cells across 56 large HCNE-rich loci. We identified a specific modification pattern, termed "bivalent domains," consisting of large regions of H3 lysine 27 methylation harboring smaller regions of H3 lysine 4 methylation. Bivalent domains tend to coincide with TF genes expressed at low levels. We propose that bivalent domains silence developmental genes in ES cells while keeping them poised for activation. We also found striking correspondences between genome sequence and histone methylation in ES cells, which become notably weaker in differentiated cells. These results highlight the importance of DNA sequence in defining the initial epigenetic landscape and suggest a novel chromatin-based mechanism for maintaining pluripotency.
Polycomb group (PcG) proteins are required for the epigenetic maintenance of developmental genes in a silent state. Proteins in the Polycomb-repressive complex 1 (PRC1) class of the PcG are conserved from flies to humans and inhibit transcription. One hypothesis for PRC1 mechanism is that it compacts chromatin, based in part on electron microscopy experiments demonstrating that Drosophila PRC1 compacts nucleosomal arrays. We show that this function is conserved between Drosophila and mouse PRC1 complexes and requires a region with an overrepresentation of basic amino acids. While the active region is found in the Posterior Sex Combs (PSC) subunit in Drosophila, it is unexpectedly found in a different PRC1 subunit, a Polycomb homolog called M33, in mice. We provide experimental support for the general importance of a charged region by predicting the compacting capability of PcG proteins from species other than Drosophila and mice and by testing several of these proteins using solution assays and microscopy. We infer that the ability of PcG proteins to compact chromatin in vitro can be predicted by the presence of domains of high positive charge and that PRC1 components from a variety of species conserve this highly charged region. This supports the hypothesis that compaction is a key aspect of PcG function.
Archaea contain a variety of sequence-independent DNA binding proteins consistent with the evolution of several different, sometimes overlapping and exchangeable solutions to the problem of genome compaction. Some of these proteins undergo residue-specific post-translational lysine acetylation or methylation, hinting at analogues of the histone modifications that regulate eukaryotic chromatin structure and transcription. Archaeal transcription initiation most closely resembles the eukaryotic RNA polymerase II (RNAPII) system, but Archaea do not appear to have homologues of the multisubunit complexes that remodel eukaryotic chromatin and activate RNAPII initiation. In contrast, they have sequence-specific regulators that repress and perhaps activate archaeal transcription by mechanisms superficially similar to the bacterial paradigm of regulating promoter binding by RNAP. Repressors compete with archaeal TATA-box binding protein (TBP) and TFB for the TATA-box and TFB-recognition elements (BRE) of the archaeal promoter, or with archaeal RNAP for the site of transcription initiation. Transcript-specific regulation by repressors binding to sites of transcript initiation is consistent with such sites having very little sequence conservation. However, most Archaea have only one TBP and/or TFB that presumably must therefore bind to similar TATA-box and BRE sequences upstream of most genes. Repressors that function by competing with TBP and/or TFB binding must therefore also make additional contacts with transcript-specific regulatory sites adjacent or remote from the TATA-box/BRE region. The fate of the archaeal TBP and TFB following transcription initiation remains to be determined. Based on functional homology with their eukaryotic RNAPII-system counterparts, archaeal TBP and possibly also TFB should remain bound to the TATA-box/BRE region after transcription initiation. However, this seems unlikely as it might limit repressor competition at this site to only the first round of transcription initiation.
The reconstruction of gene regulatory networks underlying cell differentiation from high-throughput gene expression and chromatin data remains a challenge. Here, we derive dynamic gene regulatory networks for human myeloid differentiation using a 5-day time series of RNA-seq and ATAC-seq data. We profile HL-60 promyelocytes differentiating into macrophages, neutrophils, monocytes, and monocyte-derived macrophages. We find a rapid response in the expression of key transcription factors and lineage markers that only regulate a subset of their targets at a given time, which is followed by chromatin accessibility changes that occur later along with further gene expression changes. We observe differences between promyelocyte- and monocyte-derived macrophages at both the transcriptional and chromatin landscape level, despite using the same differentiation stimulus, which suggest that the path taken by cells in the differentiation landscape defines their end cell state. More generally, our approach of combining neighboring time points and replicates to achieve greater sequencing depth can efficiently infer footprint-based regulatory networks from long series data.
RNases P and MRP are ribonucleoprotein complexes involved in tRNA and rRNA processing, respectively. The RNA subunits of these two enzymes are structurally related to each other and play an essential role in the enzymatic reaction. Both of the RNAs have a highly conserved helical region, P4, which is important in the catalytic reaction. We have used a bioinformatics approach based on conserved elements to computationally analyze available genomic sequences of eukaryotic organisms and have identified a large number of novel nuclear RNase P and MRP RNA genes. For MRP RNA for instance, this investigation increases the number of known sequences by a factor of three. We present secondary structure models of many of the predicted RNAs. Although all sequences are able to fold into the consensus secondary structure of P and MRP RNAs, a striking variation in size is observed, ranging from a Nosema locustae MRP RNA of 160 nt to much larger RNAs, e.g. a Plasmodium knowlesi P RNA of 696 nt. The P and MRP RNA genes appear in tandem in some protists, further emphasizing the close evolutionary relationship of these RNAs.
Polycomb Group (PcG) proteins mediate heritable gene silencing by modifying chromatin structure. An essential PcG complex, PRC1, compacts chromatin and inhibits chromatin remodeling. In Drosophila melanogaster, the intrinsically disordered C-terminal region of PSC (PSC-CTR) mediates these noncovalent effects on chromatin, and is essential for viability. Because the PSC-CTR sequence is poorly conserved, the significance of its effects on chromatin outside of Drosophila was unclear. The absence of folded domains also made it difficult to understand how the sequence of PSC-CTR encodes its function. To determine the mechanistic basis and extent of conservation of PSC-CTR activity, we identified 17 metazoan PSC-CTRs spanning chordates to arthropods, and examined their sequence features and biochemical properties. PSC-CTR sequences are poorly conserved, but are all highly charged and structurally disordered. We show that active PSC-CTRs--which bind DNA tightly and inhibit chromatin remodeling efficiently--are distinguished from less active ones by the absence of extended negatively charged stretches. PSC-CTR activity can be increased by dispersing its contiguous negative charge, confirming the importance of this property. Using the sequence properties defined as important for PSC-CTR activity, we predicted the presence of active PSC-CTRs in additional diverse genomes. Our analysis reveals broad conservation of PSC-CTR activity across metazoans. This conclusion could not have been determined from sequence alignments. We further find that plants that lack active PSC-CTRs instead possess a functionally analogous PcG protein, EMF1. Thus, our study suggests that a disordered domain with dispersed negative charges underlies PRC1 activity, and is conserved across metazoans and plants.
To characterize the sequence features surrounding the translation initiation sites on the genome of Synechocystis sp. strain 6803, the total proteins extracted from the cell were resolved by two-dimensional electrophoresis, and the amino-terminal sequences of the relatively abundant protein spots were determined. By comparison of the determined amino-terminal sequences with the nucleotide sequence of the entire genome, the translation initiation sites of a total of 72 proteins were successfully assigned on the genome. The sequence features emerged from the nucleotide sequences at and surrounding the translation initiation sites were as follows: (1) In addition to the three initiation codons, ATG, GTG, and TTG, evidence was obtained that ATT was also used as a rare initiation codon; (2) the core sequences (GAGG, GGAG and AGGA) of the Shine-Dalgarno sequence were identified in the appropriate position preceding the 35 initiation sites (48.6%); and (3) the preferential sequence surrounding the initiation codons was formulated as 5'-YY[...]R-3' where Y and R denote pyrimidine and purine nucleotides, respectively, and three dots represent the initiation codons. The result obtained would provide valuable information for improvement of the gene-finding software, and the approach used in this study should be applicable for comprehensive analysis of the expression profiles of cellular proteins.