PhD, CENTRE FOR CELLULAR AND MOLECULAR BIOLOGY
Evolution has been the key player in the generation of complex functions from lower to higher organisms. Lower organism generally contains a large fraction of coding DNA sequences, while in the case of higher organism non-coding DNA constitutes the major part of the genome. In human, protein-coding DNA makes up to the 2% of the genome and rest is non coding DNA. Noncoding DNA part of the genome shows a great level of conservation among different organisms. DNA sequences, which are identical up to 200 bps are known as ultraconserved sequences. Human genome constitutes up to 5% of the ultraconserved sequences. Although the function of ultraconserved sequences is not known but the cell is maintaining these sequences in the genome throughout the evolution. Ultraconserved DNA sequences are known to clustered near developmental genes in high density. We have earlier reported three ultraconserved noncoding DNA sequences, Conserved Regions (CR1, CR2 & CR3), associated with Hox cluster. Hox genes are a set of transcription factors, which regulate early embryonic development. These genes show a collinearity of genomic organization and function along the anterior-posterior body axis. Hox genes and their organization are conserved across phyla. We performed many assays to find out the function of CRs in cell lines as well as in zebrafish. Our results from reporter gene assays in different cell lines using transient transfection assays and FACS suggest minimal or no activity of CRs. Stable cell line transformants in different cell lines suggest that CRs work as repressors. To show the effect of CRs at the organism level, we used zebrafish as an animal model. Transient reporter assays in zebrafish also confirm the time-dependent repressor activity of CRs. To check the regulatory effects of CRs in the regulation of development, we need to generate transgenic and knockout of CR sequences using CRISPR/Cas9 systems. Since CRs are conserved in the different vertebrate system, we want to check the functional conservation of CRs in different organisms. Finally, we will try to decipher the mechanism of CRs during early embryonic development for that first we need to test the transcription potential of CRs. Overall this study for the first time suggests that ultraconserved DNA sequences work as repressors at the cell as well as organism level and opens up a new dimension of research for ultraconserved sequences with respect to their function.
Abstract: Gene-regulatory enhancers have been identified using various approaches, including evolutionary conservation, regulatory protein binding, chromatin modifications, and DNA sequence motifs. To integrate these different approaches, we developed EnhancerFinder, a two-step method for distinguishing developmental enhancers from the genomic background and then predicting their tissue specificity. EnhancerFinder uses a multiple kernel learning approach to integrate DNA sequence motifs, evolutionary patterns, and diverse functional genomics datasets from a variety of cell types. In contrast with prediction approaches that define enhancers based on histone marks or p300 sites from a single cell line, we trained EnhancerFinder on hundreds of experimentally verified human developmental enhancers from the VISTA Enhancer Browser. We comprehensively evaluated EnhancerFinder using cross validation and found that our integrative method improves the identification of enhancers over approaches that consider a single type of data, such as sequence motifs, evolutionary conservation, or the binding of enhancer-associated proteins. We find that VISTA enhancers active in embryonic heart are easier to identify than enhancers active in several other embryonic tissues, likely due to their uniquely high GC content. We applied EnhancerFinder to the entire human genome and predicted 84,301 developmental enhancers and their tissue specificity. These predictions provide specific functional annotations for large amounts of human non-coding DNA, and are significantly enriched near genes with annotated roles in their predicted tissues and lead SNPs from genome-wide association studies. We demonstrate the utility of EnhancerFinder predictions through in vivo validation of novel embryonic gene regulatory enhancers from three developmental transcription factor loci. Our genome-wide developmental enhancer predictions are freely available as a UCSC Genome Browser track, which we hope will enable researchers to further investigate questions in developmental biology.
Pub.: 27 Jun '14, Pinned: 08 Jun '17
Abstract: Cis-regulatory modules are non-protein-coding regions of DNA essential for the control of gene expression. One class of regulatory modules is embryonic enhancers, which drive gene expression during development as a result of transcription factor protein binding at the enhancer sequences. Recent comparative studies have begun to investigate the evolution of the sequence architecture within enhancers. These analyses are illuminating the way that developmental biologists think about enhancers by revealing their molecular mechanism of function.
Pub.: 22 Dec '09, Pinned: 08 Jun '17
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