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Genome-wide identification of conserved regulatory function in diverged sequences.

ABSTRACT: Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and zebrafish syntenic loci, we detected approximately 300 pairs of diverged elements that are likely to share common ancestry and have similar regulatory activity. By analyzing a pool of experimentally validated human enhancers, we demonstrated that 7/8 (88%) of their predicted functional orthologs retained in vivo regulatory control. Moreover, in 5/7 (71%) of assayed enhancer pairs, we observed concordant expression patterns. We argue that TFBS composition is often necessary to retain and sufficient to predict regulatory function in the absence of overt sequence conservation, revealing an entire class of functionally conserved, evolutionarily diverged regulatory elements that we term "covert."

SUBMITTER: Taher L

PROVIDER: S-EPMC3129256 | biostudies-literature | 2011 Jul

REPOSITORIES: biostudies-literature

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Genome-wide identification of conserved regulatory function in diverged sequences.

Taher Leila L McGaughey David M DM Maragh Samantha S Aneas Ivy I Bessling Seneca L SL Miller Webb W Nobrega Marcelo A MA McCallion Andrew S AS Ovcharenko Ivan I

Genome research 20110531 7

Plasticity of gene regulatory encryption can permit DNA sequence divergence without loss of function. Functional information is preserved through conservation of the composition of transcription factor binding sites (TFBS) in a regulatory element. We have developed a method that can accurately identify pairs of functional noncoding orthologs at evolutionarily diverged loci by searching for conserved TFBS arrangements. With an estimated 5% false-positive rate (FPR) in approximately 3000 human and ...[more]

PMID: 21628450

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Project description:BackgroundComputational identification of non-coding RNAs (ncRNAs) is a challenging problem. We describe a genome-wide analysis using Bayesian segmentation to identify intronic elements highly conserved between three evolutionarily distant vertebrate species: human, mouse and zebrafish. We investigate the extent to which these elements include ncRNAs (or conserved domains of ncRNAs) and regulatory sequences.ResultsWe identified 655 deeply conserved intronic sequences in a genome-wide analysis. We also performed a pathway-focussed analysis on genes involved in muscle development, detecting 27 intronic elements, of which 22 were not detected in the genome-wide analysis. At least 87% of the genome-wide and 70% of the pathway-focussed elements have existing annotations indicative of conserved RNA secondary structure. The expression of 26 of the pathway-focused elements was examined using RT-PCR, providing confirmation that they include expressed ncRNAs. Consistent with previous studies, these elements are significantly over-represented in the introns of transcription factors.ConclusionsThis study demonstrates a novel, highly effective, Bayesian approach to identifying conserved non-coding sequences. Our results complement previous findings that these sequences are enriched in transcription factors. However, in contrast to previous studies which suggest the majority of conserved sequences are regulatory factor binding sites, the majority of conserved sequences identified using our approach contain evidence of conserved RNA secondary structures, and our laboratory results suggest most are expressed. Functional roles at DNA and RNA levels are not mutually exclusive, and many of our elements possess evidence of both. Moreover, ncRNAs play roles in transcriptional and post-transcriptional regulation, and this may contribute to the over-representation of these elements in introns of transcription factors. We attribute the higher sensitivity of the pathway-focussed analysis compared to the genome-wide analysis to improved alignment quality, suggesting that enhanced genomic alignments may reveal many more conserved intronic sequences.

Project description:Transcriptional regulatory elements are central to development and interspecific phenotypic variation. Current regulatory element prediction tools rely heavily upon conservation for prediction of putative elements. Recent in vitro observations from the ENCODE project combined with in vivo analyses at the zebrafish phox2b locus suggests that a significant fraction of regulatory elements may fall below commonly applied metrics of conservation. We propose to explore these observations in vivo at the human PHOX2B locus, and also evaluate the potential evidence for genome-wide applicability of these observations through a novel analysis of extant data.Transposon-based transgenic analysis utilizing a tiling path proximal to human PHOX2B in zebrafish recapitulates the observations at the zebrafish phox2b locus of both conserved and non-conserved regulatory elements. Analysis of human sequences conserved with previously identified zebrafish phox2b regulatory elements demonstrates that the orthologous sequences exhibit overlapping regulatory control. Additionally, analysis of non-conserved sequences scattered over 135 kb 5' to PHOX2B, provides evidence of non-conserved regulatory elements positively biased with close proximity to the gene. Furthermore, we provide a novel analysis of data from the ENCODE project, finding a non-uniform distribution of regulatory elements consistent with our in vivo observations at PHOX2B. These observations remain largely unchanged when one accounts for the sequence repeat content of the assayed intervals, when the intervals are sub-classified by biological role (developmental versus non-developmental), or by gene density (gene desert versus non-gene desert).While regulatory elements frequently display evidence of evolutionary conservation, a fraction appears to be undetected by current metrics of conservation. In vivo observations at the PHOX2B locus, supported by our analyses of in vitro data from the ENCODE project, suggest that the risk of excluding non-conserved sequences in a search for regulatory elements may decrease as distance from the gene increases. Our data combined with the ENCODE data suggests that this may represent a genome wide trend.

Project description:BACKGROUND: Gcn5 is a transcriptional coactivator with histone acetyltransferase activity that is conserved with regard to structure as well as its histone substrates throughout the eukaryotes. Gene regulatory networks within cells are thought to be evolutionarily diverged. The use of evolutionarily divergent yeast species, such as S. cerevisiae and S. pombe, which can be studied under similar environmental conditions, provides an opportunity to examine the interface between conserved regulatory components and their cellular applications in different organisms. RESULTS: We show that Gcn5 is important for a common set of stress responses in evolutionarily diverged yeast species and that the activity of the conserved histone acetyltransferase domain is required. We define a group of KCl stress response genes in S. cerevisiae that are specifically dependent on Gcn5. Gcn5 is localised to many Gcn5-dependent genes including Gcn5 repressed targets such as FLO8. Gcn5 regulates divergent sets of KCl responsive genes in S. cerevisiae and S. pombe. Genome-wide localization studies showed a tendency for redistribution of Gcn5 during KCl stress adaptation in S. cerevisiae from short genes to the transcribed regions of long genes. An analogous redistribution was not observed in S. pombe. CONCLUSIONS: Gcn5 is required for the regulation of divergent sets of KCl stress-response genes in S. cerevisiae and S. pombe even though it is required a common group of stress responses, including the response to KCl. Genes that are physically associated with Gcn5 require its activity for their repression or activation during stress adaptation, providing support for a role of Gcn5 as a corepressor as well as a coactivator. The tendency of Gcn5 to re-localise to the transcribed regions of long genes during KCl stress adaptation suggests that Gcn5 plays a specific role in the expression of long genes under adaptive conditions, perhaps by regulating transcriptional elongation as has been seen for Gcn5 in S. pombe. Interestingly an analogous redistribution of Gcn5 is not seen in S. pombe. The study thus provides important new insights in relation to why coregulators like Gcn5 are required for the correct expression of some genes but not others.

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Genome-wide identification of conserved regulatory function in diverged sequences.

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Genome-wide identification of conserved regulatory function in diverged sequences.

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