Browsing by Subject "Functional genomics"

Now showing 1 - 7 of 7
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    CASowary: CRISPR-Cas13 guide RNA predictor for transcript depletion
    (BMC, 2022) Krohannon, Alexander; Srivastava, Mansi; Rauch, Simone; Srivastava, Rajneesh; Dickinson, Bryan C.; Janga, Sarath Chandra; BioHealth Informatics, School of Informatics and Computing
    Background: Recent discovery of the gene editing system - CRISPR (Clustered Regularly Interspersed Short Palindromic Repeats) associated proteins (Cas), has resulted in its widespread use for improved understanding of a variety of biological systems. Cas13, a lesser studied Cas protein, has been repurposed to allow for efficient and precise editing of RNA molecules. The Cas13 system utilizes base complementarity between a crRNA/sgRNA (crispr RNA or single guide RNA) and a target RNA transcript, to preferentially bind to only the target transcript. Unlike targeting the upstream regulatory regions of protein coding genes on the genome, the transcriptome is significantly more redundant, leading to many transcripts having wide stretches of identical nucleotide sequences. Transcripts also exhibit complex three-dimensional structures and interact with an array of RBPs (RNA Binding Proteins), both of which may impact the effectiveness of transcript depletion of target sequences. However, our understanding of the features and corresponding methods which can predict whether a specific sgRNA will effectively knockdown a transcript is very limited. Results: Here we present a novel machine learning and computational tool, CASowary, to predict the efficacy of a sgRNA. We used publicly available RNA knockdown data from Cas13 characterization experiments for 555 sgRNAs targeting the transcriptome in HEK293 cells, in conjunction with transcriptome-wide protein occupancy information. Our model utilizes a Decision Tree architecture with a set of 112 sequence and target availability features, to classify sgRNA efficacy into one of four classes, based upon expected level of target transcript knockdown. After accounting for noise in the training data set, the noise-normalized accuracy exceeds 70%. Additionally, highly effective sgRNA predictions have been experimentally validated using an independent RNA targeting Cas system - CIRTS, confirming the robustness and reproducibility of our model's sgRNA predictions. Utilizing transcriptome wide protein occupancy map generated using POP-seq in HeLa cells against publicly available protein-RNA interaction map in Hek293 cells, we show that CASowary can predict high quality guides for numerous transcripts in a cell line specific manner. Conclusions: Application of CASowary to whole transcriptomes should enable rapid deployment of CRISPR/Cas13 systems, facilitating the development of therapeutic interventions linked with aberrations in RNA regulatory processes.
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    Delineation of Molecular Pathways Involved in Cardiomyopathies Caused by Troponin T Mutations
    (American Society for Biochemistry and Molecular Biology, 2016-06) Gilda, Jennifer E.; Lai, Xianyin; Witzmann, Frank A.; Gomes, Aldrin V.; Cellular and Integrative Physiology, School of Medicine
    Familial hypertrophic cardiomyopathy (FHC) is associated with mild to severe cardiac problems and is the leading cause of sudden death in young people and athletes. Although the genetic basis for FHC is well-established, the molecular mechanisms that ultimately lead to cardiac dysfunction are not well understood. To obtain important insights into the molecular mechanism(s) involved in FHC, hearts from two FHC troponin T models (Ile79Asn [I79N] and Arg278Cys [R278C]) were investigated using label-free proteomics and metabolomics. Mutations in troponin T are the third most common cause of FHC, and the I79N mutation is associated with a high risk of sudden cardiac death. Most FHC-causing mutations, including I79N, increase the Ca(2+) sensitivity of the myofilament; however, the R278C mutation does not alter Ca(2+) sensitivity and is associated with a better prognosis than most FHC mutations. Out of more than 1200 identified proteins, 53 and 76 proteins were differentially expressed in I79N and R278C hearts, respectively, when compared with wild-type hearts. Interestingly, more than 400 proteins were differentially expressed when the I79N and R278C hearts were directly compared. The three major pathways affected in I79N hearts relative to R278C and wild-type hearts were the ubiquitin-proteasome system, antioxidant systems, and energy production pathways. Further investigation of the proteasome system using Western blotting and activity assays showed that proteasome dysfunction occurs in I79N hearts. Metabolomic results corroborate the proteomic data and suggest the glycolytic, citric acid, and electron transport chain pathways are important pathways that are altered in I79N hearts relative to R278C or wild-type hearts. Our findings suggest that impaired energy production and protein degradation dysfunction are important mechanisms in FHCs associated with poor prognosis and that cardiac hypertrophy is not likely needed for a switch from fatty acid to glucose metabolism.
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    A functional requirement for sex-determination M/m locus region lncRNA genes in Aedes aegypti female larvae
    (Springer Nature, 2021-05-20) Mysore, Keshava; Hapairai, Limb K.; Li, Ping; Roethele, Joseph B.; Sun, Longhua; Igiede, Jessica; Misenti, Joi K.; Duman‑Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    Although many putative long non-coding RNA (lncRNA) genes have been identified in insect genomes, few of these genes have been functionally validated. A screen for female-specific larvicides that facilitate Aedes aegypti male sex separation uncovered multiple interfering RNAs with target sites in lncRNA genes located in the M/m locus region, including loci within or tightly linked to the sex determination locus. Larval consumption of a Saccharomyces cerevisiae (yeast) strain engineered to express interfering RNA corresponding to lncRNA transcripts resulted in significant female death, yet had no impact on male survival or fitness. Incorporation of the yeast larvicides into mass culturing protocols facilitated scaled production and separation of fit adult males, indicating that yeast larvicides could benefit mosquito population control strategies that rely on mass releases of male mosquitoes. These studies functionally verified a female-specific developmental requirement for M/m locus region lncRNA genes, suggesting that sexually antagonistic lncRNA genes found within this highly repetitive pericentromeric DNA sequence may be contributing to the evolution of A. aegypti sex chromosomes.
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    Integration of Alzheimer’s disease genetics and myeloid genomics identifies disease risk regulatory elements and genes
    (Springer Nature, 2021-03-12) Novikova, Gloriia; Kapoor, Manav; TCW, Julia; Abud, Edsel M.; Efthymiou, Anastasia G.; Chen, Steven X.; Cheng, Haoxiang; Fullard, John F.; Bendl, Jaroslav; Liu, Yiyuan; Roussos, Panos; Björkegren, Johan LM; Liu, Yunlong; Poon, Wayne W.; Hao, Ke; Marcora, Edoardo; Goate, Alison M.; Medical and Molecular Genetics, School of Medicine
    Genome-wide association studies (GWAS) have identified more than 40 loci associated with Alzheimer’s disease (AD), but the causal variants, regulatory elements, genes and pathways remain largely unknown, impeding a mechanistic understanding of AD pathogenesis. Previously, we showed that AD risk alleles are enriched in myeloid-specific epigenomic annotations. Here, we show that they are specifically enriched in active enhancers of monocytes, macrophages and microglia. We integrated AD GWAS with myeloid epigenomic and transcriptomic datasets using analytical approaches to link myeloid enhancer activity to target gene expression regulation and AD risk modification. We identify AD risk enhancers and nominate candidate causal genes among their likely targets (including AP4E1, AP4M1, APBB3, BIN1, MS4A4A, MS4A6A, PILRA, RABEP1, SPI1, TP53INP1, and ZYX) in twenty loci. Fine-mapping of these enhancers nominates candidate functional variants that likely modify AD risk by regulating gene expression in myeloid cells. In the MS4A locus we identified a single candidate functional variant and validated it in human induced pluripotent stem cell (hiPSC)-derived microglia and brain. Taken together, this study integrates AD GWAS with multiple myeloid genomic datasets to investigate the mechanisms of AD risk alleles and nominates candidate functional variants, regulatory elements and genes that likely modulate disease susceptibility.
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    Massively Parallel Reporter Assays for High-Throughput In Vivo Analysis of Cis-Regulatory Elements
    (MDPI, 2023-03-29) Zheng, Yanjiang; VanDusen, Nathan J.; Pediatrics, School of Medicine
    The rapid improvement of descriptive genomic technologies has fueled a dramatic increase in hypothesized connections between cardiovascular gene expression and phenotypes. However, in vivo testing of these hypotheses has predominantly been relegated to slow, expensive, and linear generation of genetically modified mice. In the study of genomic cis-regulatory elements, generation of mice featuring transgenic reporters or cis-regulatory element knockout remains the standard approach. While the data obtained is of high quality, the approach is insufficient to keep pace with candidate identification and therefore results in biases introduced during the selection of candidates for validation. However, recent advances across a range of disciplines are converging to enable functional genomic assays that can be conducted in a high-throughput manner. Here, we review one such method, massively parallel reporter assays (MPRAs), in which the activities of thousands of candidate genomic regulatory elements are simultaneously assessed via the next-generation sequencing of a barcoded reporter transcript. We discuss best practices for MPRA design and use, with a focus on practical considerations, and review how this emerging technology has been successfully deployed in vivo. Finally, we discuss how MPRAs are likely to evolve and be used in future cardiovascular research.
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    PeakMatcher facilitates updated Aedes aegypti embryonic cis-regulatory element map
    (BMC, 2021-01-28) Nowling, Ronald J.; Behura, Susanta; Halfon, Marc S.; Emrich, Scott J.; Duman-Scheel, Molly; Medical and Molecular Genetics, School of Medicine
    Background: The Aedes aegypti mosquito is a threat to human health across the globe. The A. aegypti genome was recently re-sequenced and re-assembled. Due to a combination of long-read PacBio and Hi-C sequencing, the AaegL5 assembly is chromosome complete and significantly improves the assembly in key areas such as the M/m sex-determining locus. Release of the updated genome assembly has precipitated the need to reprocess historical functional genomic data sets, including cis-regulatory element (CRE) maps that had previously been generated for A. aegypti. Results: We re-processed and re-analyzed the A. aegypti whole embryo FAIRE seq data to create an updated embryonic CRE map for the AaegL5 genome. We validated that the new CRE map recapitulates key features of the original AaegL3 CRE map. Further, we built on the improved assembly in the M/m locus to analyze overlaps of open chromatin regions with genes. To support the validation, we created a new method (PeakMatcher) for matching peaks from the same experimental data set across genome assemblies. Conclusion: Use of PeakMatcher software, which is available publicly under an open-source license, facilitated the release of an updated and validated CRE map, which is available through the NIH GEO. These findings demonstrate that PeakMatcher software will be a useful resource for validation and transferring of previous annotations to updated genome assemblies.
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    Systematized reporter assays reveal ZIC protein regulatory abilities are Subclass-specific and dependent upon transcription factor binding site context
    (Nature Publishing group, 2020-08-04) Ahmed, Jehangir N.; Diamand, Koula E. M.; Bellchambers, Helen M.; Arkell, Ruth M.; Pediatrics, School of Medicine
    The ZIC proteins are a family of transcription regulators with a well-defined zinc finger DNA-binding domain and there is evidence that they elicit functional DNA binding at a ZIC DNA binding site. Little is known, however, regarding domains within ZIC proteins that confer trans-activation or -repression. To address this question, a new cell-based trans-activation assay system suitable for ZIC proteins in HEK293T cells was constructed. This identified two previously unannotated evolutionarily conserved regions of ZIC3 that are necessary for trans-activation. These domains are found in all Subclass A ZIC proteins, but not in the Subclass B proteins. Additionally, the Subclass B proteins fail to elicit functional binding at a multimerised ZIC DNA binding site. All ZIC proteins, however, exhibit functional binding when the ZIC DNA binding site is embedded in a multiple transcription factor locus derived from ZIC target genes in the mouse genome. This ability is due to several domains, some of which are found in all ZIC proteins, that exhibit context dependent trans-activation or -repression activity. This knowledge is valuable for assessing the likely pathogenicity of variant ZIC proteins associated with human disorders and for determining factors that influence functional transcription factor binding.