Revealing posttranscriptional regulatory elements through network-level conservation.
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ABSTRACT:
SUBMITTER: Chan CS
PROVIDER: S-EPMC1309705 | biostudies-literature |
REPOSITORIES: biostudies-literature
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Project description:In higher eukaryotes, introns are usually required for efficient pre-mRNA processing. However, some viruses have alternative approaches involving posttranscriptional regulatory elements (PREs) to enhance intronless heterologous gene expression through enabling stability and 3' end formation, and to facilitate the nucleocytoplasmic export of unspliced mRNAs. In the current study, we compared the human cytomegalovirus (hCMV) immediate/early (IE) intronA, as well as virus-derived PREs-the PRE of Hepatitis B virus (HPRE) and Woodchuck Hepatitis virus (WPRE) on their ability to enhance antigen gene expression in vitro and immune responses induced by DNA vaccination in animal. Among all the constructs, the plasmids carrying the HPRE element showed the highest gene expression level in both in vivo and in vitro models. During immunization of mice with low doses (10 microg) of HIV-1 DNA vaccine, only -intronA/+HPRE and +intronA/+HPRE vaccine constructs induced anti-Gag antibodies, although the -intronA/+WPRE construct also elicited antigen-specific cellular immune responses. In addition, pInHGag (+intronA/+HPRE) at a 10 mug dose could induce higher anti-Gag antibody level than that induced by pGag (-intronA/-HPRE) or pInGag (+intronA/-HPRE) at 40 microg dose (p < 0.05). Our data are useful for the optimization of heterologous expression and immunogenicity of DNA vaccines.
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Project description:BackgroundMetastatic castration-resistant prostate cancer (mCRPC) is an aggressive form of cancer unresponsive to androgen deprivation therapy (ADT) that spreads quickly to other organs. Despite reduced androgen levels after ADT, mCRPC development and lethality continues to be conducted by the androgen receptor (AR) axis. The maintenance of AR signaling in mCRPC is a result of AR alterations, androgen intratumoral production, and the action of regulatory elements, such as noncoding RNAs (ncRNAs). ncRNAs are key elements in cancer signaling, acting in tumor growth, metabolic reprogramming, and tumor progression. In prostate cancer (PCa), the ncRNAs have been reported to be associated with AR expression, PCa proliferation, and castration resistance. In this study, we aimed to reconstruct the lncRNA-centered regulatory network of mCRPC and identify the lncRNAs which act as master regulators (MRs).MethodsWe used publicly available RNA-sequencing to infer the regulatory network of lncRNAs in mCRPC. Five gene signatures were employed to conduct the master regulator analysis. Inferred MRs were then subjected to functional enrichment and symbolic regression modeling. The latter approach was applied to identify the lncRNAs with greater predictive capacity and potential as a biomarker in mCRPC.ResultsWe identified 31 lncRNAs involved in cellular proliferation, tumor metabolism, and invasion-metastasis cascade. SNHG18 and HELLPAR were the highlights of our results. SNHG18 was downregulated in mCRPC and enriched to metastasis signatures. It accurately distinguished both mCRPC and primary CRPC from normal tissue and was associated with epithelial-mesenchymal transition (EMT) and cell-matrix adhesion pathways. HELLPAR consistently distinguished mCRPC from primary CRPC and normal tissue using only its expression.ConclusionOur results contribute to understanding the regulatory behavior of lncRNAs in mCRPC and indicate SNHG18 and HELLPAR as master regulators and potential new diagnostic targets in this tumor.
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Project description:Transcriptional network evolution is central to the development of complex biological systems. Networks can evolve through variation of master regulators and/or by changes in regulation of genes within networks. To gain insight into meaningful evolutionary differences in large networks, it is essential to address the functional consequences of sequence differences in response elements (REs) targeted by transcription factors. Using a combination of custom bioinformatics and multispecies alignment of promoter regions, we investigated the functional evolution of REs in terms of responsiveness to the sequence-specific transcription factor p53, a tumor suppressor and master regulator of stress responses. We identified REs orthologous to known p53 targets in human and rodent cells or alternatively REs related to the established p53 consensus. The orthologous REs were assigned p53 transactivation capabilities based on rules determined from model systems, and a functional heat map was developed to visually summarize conservation of sequence and relative level of responsiveness to p53 for 47 REs in 14 species. Individual REs exhibited marked differences in transactivation potentials and widespread evolutionary turnover. Functional differences were often not predicted from consensus sequence evaluations. Of the established human p53 REs analyzed, 91% had sequence conservation in at least one nonprimate species compared with 67.5% for functional conservation. Surprisingly, there was almost no conservation of functional REs for genes involved in DNA metabolism or repair between humans and rodents, suggesting important differences in p53 stress responses and cancer development.
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Project description:To learn how well ungapped sequence comparisons of multiple species can predict cis-regulatory elements in Caenorhabditis elegans, we made such predictions across the large, complex ceh-13/lin-39 locus and tested them transgenically. We also examined how prediction quality varied with different genomes and parameters in our comparisons. Specifically, we sequenced approximately 0.5% of the C. brenneri and C. sp. 3 PS1010 genomes, and compared five Caenorhabditis genomes (C. elegans, C. briggsae, C. brenneri, C. remanei, and C. sp. 3 PS1010) to find regulatory elements in 22.8 kb of noncoding sequence from the ceh-13/lin-39 Hox subcluster. We developed the MUSSA program to find ungapped DNA sequences with N-way transitive conservation, applied it to the ceh-13/lin-39 locus, and transgenically assayed 21 regions with both high and low degrees of conservation. This identified 10 functional regulatory elements whose activities matched known ceh-13/lin-39 expression, with 100% specificity and a 77% recovery rate. One element was so well conserved that a similar mouse Hox cluster sequence recapitulated the native nematode expression pattern when tested in worms. Our findings suggest that ungapped sequence comparisons can predict regulatory elements genome-wide.
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Project description:The transcription factor p63 is an essential regulator of vertebrate ectoderm development, including epidermis, limbs, and craniofacial tissues. Here, we have investigated the evolutionary conservation of p63 binding sites (BSs) between zebrafish and human. First, we have analyzed sequence conservation of p63 BSs by comparing ChIP-seq data from human keratinocytes and zebrafish embryos, observing a very poor conservation. Next, we compared the gene regulatory network orchestrated by p63 in both species and found a high overlap between them, suggesting a high degree of functional conservation during evolution despite sequence divergence and the large evolutionary distance. Finally, we used transgenic reporter assays in zebrafish embryos to functionally validate a set of equivalent p63 BSs from zebrafish and human located close to genes involved in epidermal development. Reporter expression was driven by human and zebrafish BSs to many common tissues related to p63 expression domains. Therefore, we conclude that the gene regulatory network controlled by p63 is highly conserved across vertebrates despite the fact that p63-bound regulatory elements show high divergence.
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Project description:Identification of transcriptional regulatory elements in the GC-rich Streptomyces genome is essential for the production of novel biochemicals from secondary metabolite biosynthetic gene clusters (smBGCs). Despite many efforts to understand the regulation of transcription initiation in smBGCs, information on the regulation of transcription termination and posttranscriptional processing remains scarce. In this study, we identified the transcriptional regulatory elements in β-lactam antibiotic-producing Streptomyces clavuligerus ATCC 27064 by determining a total of 1,427 transcript 3'-end positions (TEPs) using the term-seq method. Termination of transcription was governed by three classes of TEPs, of which each displayed unique sequence features. The data integration with transcription start sites and transcriptome data generated 1,648 transcription units (TUs) and 610 transcription unit clusters (TUCs). TU architecture showed that the transcript abundance in TU isoforms of a TUC was potentially affected by the sequence context of their TEPs, suggesting that the regulatory elements of TEPs could control the transcription level in additional layers. We also identified TU features of a xenobiotic response element (XRE) family regulator and DUF397 domain-containing protein, particularly showing the abundance of bidirectional TEPs. Finally, we found that 189 noncoding TUs contained potential cis- and trans-regulatory elements that played a major role in regulating the 5' and 3' UTR. These findings highlight the role of transcriptional regulatory elements in transcription termination and posttranscriptional processing in Streptomyces sp.IMPORTANCE Streptomyces sp. is a great source of bioactive secondary metabolites, including antibiotics, antifungal agents, antiparasitic agents, immunosuppressant compounds, and other drugs. Secondary metabolites are synthesized via multistep conversions of the precursor molecules from primary metabolism, governed by multicomplex enzymes from secondary metabolite biosynthetic gene clusters. As their production is closely related with the growth phase and dynamic cellular status in response to various intra- and extracellular signals, complex regulatory systems tightly control the gene expressions related to secondary metabolism. In this study, we determined genome-wide transcript 3'-end positions and transcription units in the β-lactam antibiotic producer Streptomyces clavuligerus ATCC 27064 to elucidate the transcriptional regulatory elements in transcription termination and posttranscriptional processing by integration of multiomics data. These unique features, such as transcript 3'-end sequence, potential riboregulators, and potential 3'-untranslated region (UTR) cis-regulatory elements, can be potentially used to design engineering tools that can regulate the transcript abundance of genes for enhancing secondary metabolite production.
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Project description:BackgroundInfection with hepatitis B virus (HBV) is major public health concern. The limitations of available antiviral drugs require development of novel approaches to inhibit HBV replication. This study was conducted to identify functional elements and new siRNA target sites within the highly conserved regions of the 533 base post-transcriptional regulatory element (PRE) of HBV RNAs.ResultsComputational analysis of the PRE sequence revealed several conserved regulatory elements that are predicted to form local secondary structures some of these within known regulatory regions. A deletion analysis showed that sub-elements of the PRE have different effects on the reporter activity suggesting that the PRE contains multiple regulatory elements. Conserved siRNA targets at nucleotide position 1317-1337 and 1329-1349 were predicted. Although the siRNA at the position 1329-1349 had no effect on the expression of reporter gene, the siRNA target site at the position 1317-1337 was observed to significantly decrease expression of the reporter protein. This siRNA also specifically reduced the level of cccDNA in transiently HBV infected cells.ConclusionThe HBV PRE is likely to contain multiple regulatory elements. A conserved target within this region at 1317-1337 is an effective siRNA target.
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Project description:MotivationRegulatory elements (REs), such as enhancers and promoters, are known as regulatory sequences functional in a heterogeneous regulatory network to control gene expression by recruiting transcription regulators and carrying genetic variants in a context specific way. Annotating those REs relies on costly and labor-intensive next-generation sequencing and RNA-guided editing technologies in many cellular contexts.ResultsWe propose a systematic Gene Ontology Annotation method for Regulatory Elements (RE-GOA) by leveraging the powerful word embedding in natural language processing. We first assemble a heterogeneous network by integrating context specific regulations, protein-protein interactions and gene ontology (GO) terms. Then we perform network embedding and associate regulatory elements with GO terms by assessing their similarity in a low dimensional vector space. With three applications, we show that RE-GOA outperforms existing methods in annotating TFs' binding sites from ChIP-seq data, in functional enrichment analysis of differentially accessible peaks from ATAC-seq data, and in revealing genetic correlation among phenotypes from their GWAS summary statistics data.Availability and implementationThe source code and the systematic RE annotation for human and mouse are available at https://github.com/AMSSwanglab/RE-GOA.Supplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Expression patterns of genes are controlled by short regions of DNA in promoter regions known as cis-regulatory elements. How expression patterns change due to alterations in cis-regulatory elements in the context of gene duplication are not well studied in plants. Over 300 promoter sequences from a small, well-conserved family of plant transcription factors known as Cytokinin Response Factors (CRFs) were examined for conserved motifs across several known clades present in Angiosperms. General CRF and lineage specific motifs were identified. Once identified, significantly enriched motifs were then compared to known transcription factor binding sites to elucidate potential functional roles. Additionally, presence of similar motifs shows that levels of conservation exist between different CRFs across land plants, likely occurring through processes of neo- or sub-functionalization. Furthermore, significant patterns of motif conservation are seen within and between CRF clades suggesting cis-regulatory regions have been conserved throughout CRF evolution.
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