Browsing by Subject "Gene expression"
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Item 38766 Massively Parallel Reporter Assay Reveals Functional Impact of 3™-UTR SNPs Associated with Neurological and Psychiatric Disorders(Cambridge University Press, 2021) Chen, Andy B.; Thapa, Kriti; Gao, Hongyu; Reiter, Jill L.; Zhang, Junjie; Xuei, Xiaoling; Gu, Hongmei; Wang, Yue; Edenberg, Howard J.; Liu, Yunlong; Medical and Molecular Genetics, School of MedicineABSTRACT IMPACT: Screening the effect of thousands of non-coding genetic variants will help identify variants important in the etiology of diseases OBJECTIVES/GOALS: Massively parallel reporter assays (MPRAs) can experimentally evaluate the impact of genetic variants on gene expression. In this study, our objective was to systematically evaluate the functional activity of 3’-UTR SNPs associated with neurological disorders and use those results to help understand their contributions to disease etiology. METHODS/STUDY POPULATION: To choose variants to evaluate with the MPRA, we first gathered SNPs from the GWAS Catalog that were associated with any neurological disorder trait with p-value < 10-5. For each SNP, we identified the region that was in linkage disequilibrium (r2 > 0.8) and retrieved all the common 3’-UTR SNPs (allele-frequency > 0.05) within that region. We used an MPRA to measure the impact of these 3’-UTR variants in SH-SY5Y neuroblastoma cells and a microglial cell line. These results were then used to train a deep-learning model to predict the impact of variants and identify features that contribute to the predictions. RESULTS/ANTICIPATED RESULTS: Of the 13,515 3’-UTR SNPs tested, 400 and 657 significantly impacted gene expression in SH-SY5Y and microglia, respectively. Of the 84 SNPs significantly impacted in both cells, the direction of impact was the same in 81. The direction of eQTL in GTEx tissues agreed with the assay SNP effect in SH-SY5Y cells but not microglial cells. The deep-learning model predicted sequence activity level correlated with the experimental activity level (Spearman’s corr = 0.45). The deep-learning model identified several predictive motifs similar to motifs of RNA-binding proteins. DISCUSSION/SIGNIFICANCE OF FINDINGS: This study demonstrates that MPRAs can be used to evaluate the effect of non-coding variants, and the results can be used to train a machine learning model and interpret its predictions. Together, these can help identify causal variants and further understand the etiology of diseases.Item 5. Collaborative Study on the Genetics of Alcoholism: Functional genomics(Wiley, 2023) Gameiro-Ros, Isabel; Popova, Dina; Prytkova, Iya; Pang, Zhiping P.; Liu, Yunlong; Dick, Danielle; Bucholz, Kathleen K.; Agrawal, Arpana; Porjesz, Bernice; Goate, Alison M.; Xuei, Xiaoling; Kamarajan, Chella; COGA Collaborators; Tischfield, Jay A.; Edenberg, Howard J.; Slesinger, Paul A.; Hart, Ronald P.; Medical and Molecular Genetics, School of MedicineAlcohol Use Disorder is a complex genetic disorder, involving genetic, neural, and environmental factors, and their interactions. The Collaborative Study on the Genetics of Alcoholism (COGA) has been investigating these factors and identified putative alcohol use disorder risk genes through genome-wide association studies. In this review, we describe advances made by COGA in elucidating the functional changes induced by alcohol use disorder risk genes using multimodal approaches with human cell lines and brain tissue. These studies involve investigating gene regulation in lymphoblastoid cells from COGA participants and in post-mortem brain tissues. High throughput reporter assays are being used to identify single nucleotide polymorphisms in which alternate alleles differ in driving gene expression. Specific single nucleotide polymorphisms (both coding or noncoding) have been modeled using induced pluripotent stem cells derived from COGA participants to evaluate the effects of genetic variants on transcriptomics, neuronal excitability, synaptic physiology, and the response to ethanol in human neurons from individuals with and without alcohol use disorder. We provide a perspective on future studies, such as using polygenic risk scores and populations of induced pluripotent stem cell-derived neurons to identify signaling pathways related with responses to alcohol. Starting with genes or loci associated with alcohol use disorder, COGA has demonstrated that integration of multimodal data within COGA participants and functional studies can reveal mechanisms linking genomic variants with alcohol use disorder, and potential targets for future treatments.Item A CRISPR mis-insertion in the Zic3 5'UTR inhibits in vivo translation and is predicted to result in formation of an mRNA stem-loop hairpin(Company of Biologists, 2025) Bellchambers, Helen M.; Padua, Maria B.; Ware, Stephanie M.; Pediatrics, School of MedicineZic3 loss of function is associated with a range of congenital defects, including heterotaxy and isolated heart defects in humans, as well as neural tube defects, situs anomalies, and tail kinks in model organisms. Here, we describe a novel Zic3ins5V mouse line generated due to a mis-insertion during the CRISPR genome editing process, which altered the Zic3 5'UTR structure. Mice with this insertion developed similar phenotypes to Zic3LacZ null mice, including heterotaxy, isolated heart defects, neural tube defects and tail kinks. Surprisingly, gene expression analysis revealed that the novel Zic3ins5V line displays higher levels of Zic3 mRNA, but western blot analysis confirmed that levels of ZIC3 were greatly reduced in vivo. RNAfold, an RNA secondary structure prediction tool, showed that this mis-insertion may cause the formation of a large stem-loop hairpin incorporating some of the 5'UTR and first exon of Zic3, and the insertion of similar hairpins in a cell-based assay caused the loss of ZIC3 expression. Thus, this mouse line displays a loss of ZIC3 protein consistent with the inhibitory effects of 5'UTR stem-loop hairpin structures.Item A new Down syndrome rat model races forward(Elsevier, 2022) Roper, Randall J.; Goodlett, Charles R.; Biology, School of ScienceAnimal models of Down syndrome (DS) provide an essential resource for understanding genetic, cellular, and molecular contributions to traits associated with trisomy 21 (Ts21). Recent genetic enhancements in the development of DS models, including the new TcHSA21rat model (Kazuki et al.), have potential to transform our understanding of and potential therapies for Ts21.Item A New Method for Stranded Whole Transcriptome RNA-seq(Elsevier, 2013) Miller, David F. B.; Yan, Pearlly S.; Buechlein, Aaron; Rodriguez, Benjamin A.; Yilmaz, Ayse S.; Goel, Shokhi; Lin, Hai; Collins-Burow, Bridgette; Rhodes, Lyndsay V.; Braun, Chris; Pradeep, Sunila; Rupaimoole, Rajesha; Dalkilic, Mehmet; Sood, Anil K.; Burow, Matthew E.; Tang, Haixu; Huang, Tim H.; Liu, Yunlong; Rusch, Douglas B.; Nephew, Kenneth P.; Cellular and Integrative Physiology, School of MedicineThis report describes an improved protocol to generate stranded, barcoded RNA-seq libraries to capture the whole transcriptome. By optimizing the use of duplex specific nuclease (DSN) to remove ribosomal RNA reads from stranded barcoded libraries, we demonstrate improved efficiency of multiplexed next generation sequencing (NGS). This approach detects expression profiles of all RNA types, including miRNA (microRNA), piRNA (Piwi-interacting RNA), snoRNA (small nucleolar RNA), lincRNA (long non-coding RNA), mtRNA (mitochondrial RNA) and mRNA (messenger RNA) without the use of gel electrophoresis. The improved protocol generates high quality data that can be used to identify differential expression in known and novel coding and non-coding transcripts, splice variants, mitochondrial genes and SNPs (single nucleotide polymorphisms).Item Acquisition, processing, and single-cell analysis of normal human breast tissues from a biobank(Cell Press, 2021-12-16) Bhat-Nakshatri, Poornima; Marino, Natascia; Gao, Hongyu; Liu, Yunlong; Storniolo, Anna Maria; Nakshatri, Harikrishna; Surgery, School of MedicineThe Komen Tissue Bank is the only biorepository in the world for normal breast tissues from women. Below we report the acquisition and processing of breast tissue from volunteer donors and describe an experimental and analysis pipeline to generate a single-cell atlas. This atlas is based on single-cell RNA-seq and is useful to derive breast epithelial cell subcluster-specific gene expression signatures, which can be applied to breast cancer gene expression data to identify putative cell-of-origin. For complete details on the use and execution of this protocol, please refer to Bhat-Nakshatri et al. (2021).Item ADARs employ a neural-specific mechanism to regulate PQM-1 expression and survival from hypoxia(bioRxiv, 2023-05-05) Mahapatra, Ananya; Dhakal, Alfa; Noguchi, Aika; Vadlamani, Pranathi; Hundley, Heather A.; Medicine, School of MedicineThe ability to alter gene expression programs in response to changes in environmental conditions is central to the ability of an organism to thrive. For most organisms, the nervous system serves as the master regulator in communicating information about the animal's surroundings to other tissues. The information relay centers on signaling pathways that cue transcription factors in a given cell type to execute a specific gene expression program, but also provide a means to signal between tissues. The transcription factor PQM-1 is an important mediator of the insulin signaling pathway contributing to longevity and the stress response as well as impacting survival from hypoxia. Herein, we reveal a novel mechanism for regulating PQM-1 expression specifically in neural cells of larval animals. Our studies reveal that the RNA binding protein, ADR-1, binds to pqm-1 mRNA in neural cells. This binding is regulated by the presence of a second RNA binding protein, ADR-2, which when absent leads to reduced expression of both pqm-1 and downstream PQM-1 activated genes. Interestingly, we find that neural pqm-1 expression is sufficient to impact gene expression throughout the animal and affect survival from hypoxia; phenotypes that we also observe in adr mutant animals. Together, these studies reveal an important post-transcriptional gene regulatory mechanism that allows the nervous system to sense and respond to environmental conditions to promote organismal survival from hypoxia.Item Adeno-Associated Virus D-Sequence-Mediated Suppression of Expression of a Human Major Histocompatibility Class II Gene: Implications in the Development of Adeno-Associated Virus Vectors for Modulating Humoral Immune Response(Mary Ann Liebert, Inc., 2020-05) Kwon, Hyung-Joo; Qing, Keyun; Ponnazhagan, Selvarangan; Wang, Xu-Shan; Markusic, David M.; Gupte, Siddhant; Boye, Shannon E.; Srivastava, Arun; Pediatrics, School of MedicineA 20-nt long sequence, termed the D-sequence, in the adeno-associated virus (AAV) inverted terminal repeat was observed to share a partial sequence homology with the X-box in the regulatory region of the human leukocyte antigen DRA (HLA-DRA) promoter of the human major histocompatibility complex class II (MHC-II) genes. The D-sequence was also shown to specifically interact with the regulatory factor binding to the X-box (RFX), binding of which to the X-box is a critical step in the MHC-II gene expression, suggesting that D-sequence might compete for RFX transcription factor binding, thereby suppressing expression from the MHC-II promoter. In DNA-mediated transfection experiments, using a reporter gene under the control of the HLA-DRA promoter, D-sequence oligonucleotides were found to inhibit expression of the reporter gene expression in HeLa and 293 cells by ∼93% and 96%, respectively. No inhibition was observed when nonspecific synthetic oligonucleotides were used. D-sequence oligonucleotides had no effect on expression from the cytomegalovirus immediate-early gene promoter. Interferon-γ-mediated activation of MHC-II gene expression was also inhibited by D-sequence oligonucleotides as well as after infection with either the wild-type AAV or transduction with recombinant AAV vectors. These studies suggest that the D-sequence-mediated downregulation of the MHC-II gene expression may be exploited toward the development of novel AAV vectors capable of dampening the host humoral response, which has important implication in the optimal use of these vectors in human gene therapy.Item Aglianico Grape Seed Semi-Polar Extract Exerts Anticancer Effects by Modulating MDM2 Expression and Metabolic Pathways(MDPI, 2023-01-04) Cuciniello, Rossana; Di Meo, Francesco; Sulli, Maria; Demurtas, Olivia Costantina; Tanori, Mirella; Mancuso, Mariateresa; Villano, Clizia; Aversano, Riccardo; Carputo, Domenico; Baldi, Alfonso; Diretto, Gianfranco; Filosa, Stefania; Crispi, Stefania; Medicine, School of MedicineGrapevine (Vitis vinifera L.) seeds are rich in polyphenols including proanthocyanidins, molecules with a variety of biological effects including anticancer action. We have previously reported that the grape seed semi-polar extract of Aglianico cultivar (AGS) was able to induce apoptosis and decrease cancer properties in different mesothelioma cell lines. Concomitantly, this extract resulted in enriched oligomeric proanthocyanidins which might be involved in determining the anticancer activity. Through transcriptomic and metabolomic analyses, we investigated in detail the anticancer pathway induced by AGS. Transcriptomics analysis and functional annotation allowed the identification of the relevant causative genes involved in the apoptotic induction following AGS treatment. Subsequent biological validation strengthened the hypothesis that MDM2 could be the molecular target of AGS and that it could act in both a p53-dependent and independent manner. Finally, AGS significantly inhibited tumor progression in a xenograft mouse model of mesothelioma, confirming also in vivo that MDM2 could act as molecular player responsible for the AGS antitumor effect. Our findings indicated that AGS, exerting a pro-apoptotic effect by hindering MDM2 pathway, could represent a novel source of anticancer molecules.Item AIscEA: unsupervised integration of single-cell gene expression and chromatin accessibility via their biological consistency(Oxford University Press, 2022) Jafari, Elham; Johnson, Travis; Wang, Yue; Liu, Yunlong; Huang, Kun; Wang, Yijie; Biostatistics and Health Data Science, School of MedicineMotivation: The integrative analysis of single-cell gene expression and chromatin accessibility measurements is essential for revealing gene regulation, but it is one of the key challenges in computational biology. Gene expression and chromatin accessibility are measurements from different modalities, and no common features can be directly used to guide integration. Current state-of-the-art methods lack practical solutions for finding heterogeneous clusters. However, previous methods might not generate reliable results when cluster heterogeneity exists. More importantly, current methods lack an effective way to select hyper-parameters under an unsupervised setting. Therefore, applying computational methods to integrate single-cell gene expression and chromatin accessibility measurements remains difficult. Results: We introduce AIscEA-Alignment-based Integration of single-cell gene Expression and chromatin Accessibility-a computational method that integrates single-cell gene expression and chromatin accessibility measurements using their biological consistency. AIscEA first defines a ranked similarity score to quantify the biological consistency between cell clusters across measurements. AIscEA then uses the ranked similarity score and a novel permutation test to identify cluster alignment across measurements. AIscEA further utilizes graph alignment for the aligned cell clusters to align the cells across measurements. We compared AIscEA with the competing methods on several benchmark datasets and demonstrated that AIscEA is highly robust to the choice of hyper-parameters and can better handle the cluster heterogeneity problem. Furthermore, AIscEA significantly outperforms the state-of-the-art methods when integrating real-world SNARE-seq and scMultiome-seq datasets in terms of integration accuracy. Availability and implementation: AIscEA is available at https://figshare.com/articles/software/AIscEA_zip/21291135 on FigShare as well as {https://github.com/elhaam/AIscEA} onGitHub.