IU Indianapolis ScholarWorks :: Browsing by Author "Reiter, Jill L."

Browsing by Author "Reiter, Jill L."

Now showing 1 - 10 of 26

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 Medicine
ABSTRACT 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.
The Access Technology Program of the Indiana Clinical Translational Sciences Institute (CTSI): A model to facilitate access to cutting-edge technologies across a state
(Cambridge, 2021) Orschell, Christie M.; Skaar, Todd C.; DeFord, Melanie E.; Ybe, Joel; Driscol, Julie; Drury, Christine; Reeves, Lilith; Willis, Monte S.; Reiter, Jill L.; York, Jenna; Orr, Rob; McClintick, Jeanette N.; Sors, Thomas G.; Hunt, Joe; Cornetta, Kenneth; Shekhar, Anantha; Medicine, School of Medicine
Introduction: Access to cutting-edge technologies is essential for investigators to advance translational research. The Indiana Clinical and Translational Sciences Institute (CTSI) spans three major and preeminent universities, four large academic campuses across the state of Indiana, and is mandate to provide best practices to a whole state. Methods: To address the need to facilitate the availability of innovative technologies to its investigators, the Indiana CTSI implemented the Access Technology Program (ATP). The activities of the ATP, or any program of the Indiana CTSI, are challenged to connect technologies and investigators on the multiple Indiana CTSI campuses by the geographical distances between campuses (1–4 hr driving time). Results: Herein, we describe the initiatives developed by the ATP to increase the availability of state-of-the-art technologies to its investigators on all Indiana CTSI campuses, and the methods developed by the ATP to bridge the distance between campuses, technologies, and investigators for the advancement of clinical translational research. Conclusions: The methods and practices described in this publication may inform other approaches to enhance translational research, dissemination, and usage of innovative technologies by translational investigators, especially when distance or multi-campus cultural differences are factors to efficient application.
Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders
(Springer Nature, 2021-04) Rao, Xi; Thapa, Kriti S.; Chen, Andy B.; Lin, Hai; Gao, Hongyu; Reiter, Jill L.; Hargreaves, Katherine A.; Ipe, Joseph; Lai, Dongbing; Xuei, Xiaoling; Wang, Yue; Gu, Hongmei; Kapoor, Manav; Farris, Sean P.; Tischfield, Jay; Foroud, Tatiana; Goate, Alison M.; Skaar, Todd C.; Mayfield, R. Dayne; Edenberg, Howard J.; Liu, Yunlong; Medical and Molecular Genetics, School of Medicine
Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3′ untranslated regions (3′UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3′UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.
Allele-specific expression and high-throughput reporter assay reveal functional genetic variants associated with alcohol use disorders.
(Springer, 2021-04) Rao, Xi; Thapa, Kriti S.; Chen, Andy B.; Lin, Hai; Gao, Hongyu; Reiter, Jill L.; Hargreaves, Katherine A.; Ipe, Joseph; Lai, Dongbing; Xuei, Xiaoling; Wang, Yue; Gu, Hongmei; Kapoor, Manav; Farris, Sean P.; Tischfield, Jay; Foroud, Tatiana; Goate, Alison M.; Skaar, Todd C.; Mayfield, R. Dayne; Edenberg, Howard J.; Liu, Yunlong
Genome-wide association studies (GWAS) of complex traits, such as alcohol use disorders (AUD), usually identify variants in non-coding regions and cannot by themselves distinguish whether the associated variants are functional or in linkage disequilibrium with the functional variants. Transcriptome studies can identify genes whose expression differs between alcoholics and controls. To test which variants associated with AUD may cause expression differences, we integrated data from deep RNA-seq and GWAS of four postmortem brain regions from 30 subjects with AUD and 30 controls to analyze allele-specific expression (ASE). We identified 88 genes with differential ASE in subjects with AUD compared to controls. Next, to test one potential mechanism contributing to the differential ASE, we analyzed single nucleotide polymorphisms (SNPs) in the 3' untranslated regions (3'UTR) of these genes. Of the 88 genes with differential ASE, 61 genes contained 437 SNPs in the 3'UTR with at least one heterozygote among the subjects studied. Using a modified PASSPORT-seq (parallel assessment of polymorphisms in miRNA target-sites by sequencing) assay, we identified 25 SNPs that affected RNA levels in a consistent manner in two neuroblastoma cell lines, SH-SY5Y and SK-N-BE(2). Many of these SNPs are in binding sites of miRNAs and RNA-binding proteins, indicating that these SNPs are likely causal variants of AUD-associated differential ASE. In sum, we demonstrate that a combination of computational and experimental approaches provides a powerful strategy to uncover functionally relevant variants associated with the risk for AUD.
ASSESSMENT OF PROCEDURAL ASPECTS AND QUALITY CONTROL IN HUMAN PLACENTAL RNA ISOLATION PROTOCOLS
(Office of the Vice Chancellor for Research, 2012-04-13) Maasa, Robinah K.; Sanders, Kerry L.; Creekmore, Amy L.; Lee, Men-Jean; Reiter, Jill L.
High quality RNA is of paramount importance in accurately interpreting gene expression changes in the placenta throughout pregnancy, as well as in common placental pathologies. The purpose of this study was to develop a standard operating procedure for the collection of human placental tissue and isolation of high quality RNA for pregnancy-related molecular studies. To accomplish this task, we compared several different parameters to minimize RNA degradation, including preservation (liquid nitrogen vs. RNAlater), dis-ruption (mortar/pestle vs. homogenization), and isolation (Trizol vs. RNeasy). We performed 150 RNA isolations from 30 term placentas. The overall yield was 365 ± 197 ng RNA per mg of tissue. The A260/280 ratio for all samples was 2.11 ± 0.1 (mean ± s.d.) and the RQI was 7.1 ± 1.4. No significant differences in RNA purity, yield, or quality were observed between different placental collections or RNA isolation techniques. However, poor RQI values of 2.7 to 3.3 were obtained after brief thawing of frozen placental samples. We also compared storage of RNAlater stabilized tissue at 4 de-grees or room temperature for 1 day, 7 days, and 30 days. The integrity of RNA stored at room temperature for 1 day was significantly better (P‹0.05 RQI 7.3 ± 0.58, mean ± s.d) than RNA stored at room temperature for 30 days (RQI 5.0 ±1.2, mean ± s.d). The results of these studies will be useful for establishing standard procedures for placenta collection for pregnancy biobanks.
Bioinformatics detection of modulators controlling splicing factor‐dependent intron retention in the human brain
(Wiley, 2022) Chen, Steven X.; Simpson, Ed; Reiter, Jill L.; Liu, Yunlong; Medical and Molecular Genetics, School of Medicine
Alternative RNA splicing is an important means of genetic control and transcriptome diversity. However, when alternative splicing events are studied independently, coordinated splicing modulated by common factors is often not recognized. As a result, the molecular mechanisms of how splicing regulators promote or repress splice site recognition in a context‐dependent manner are not well understood. The functional coupling between multiple gene regulatory layers suggests that splicing is modulated by additional genetic or epigenetic components. Here, we developed a bioinformatics approach to identify causal modulators of splicing activity based on the variation of gene expression in large RNA sequencing datasets. We applied this approach in a neurological context with hundreds of dorsolateral prefrontal cortex samples. Our model is strengthened with the incorporation of genetic variants to impute gene expression in a Mendelian randomization‐based approach. We identified novel modulators of the splicing factor SRSF1, including UIMC1 and the long noncoding RNA CBR3‐AS1, that function over dozens of SRSF1 intron retention splicing targets. This strategy can be widely used to identify modulators of RNA‐binding proteins involved in tissue‐specific alternative splicing.
Cryopreservation Preserves Cell-Type Composition and Gene Expression Profiles in Bone Marrow Aspirates From Multiple Myeloma Patients
(Frontiers Media, 2021-04-21) Chen, Duojiao; Abu Zaid, Mohammad I.; Reiter, Jill L.; Czader, Magdalena; Wang, Lin; McGuire, Patrick; Xuei, Xiaoling; Gao, Hongyu; Huang, Kun; Abonour, Rafat; Walker, Brian A.; Liu, Yunlong; Medical and Molecular Genetics, School of Medicine
Single-cell RNA sequencing reveals gene expression differences between individual cells and also identifies different cell populations that are present in the bulk starting material. To obtain an accurate assessment of patient samples, single-cell suspensions need to be generated as soon as possible once the tissue or sample has been collected. However, this requirement poses logistical challenges for experimental designs involving multiple samples from the same subject since these samples would ideally be processed at the same time to minimize technical variation in data analysis. Although cryopreservation has been shown to largely preserve the transcriptome, it is unclear whether the freeze-thaw process might alter gene expression profiles in a cell-type specific manner or whether changes in cell-type proportions might also occur. To address these questions in the context of multiple myeloma clinical studies, we performed single-cell RNA sequencing (scRNA-seq) to compare fresh and frozen cells isolated from bone marrow aspirates of six multiple myeloma patients, analyzing both myeloma cells (CD138+) and cells constituting the microenvironment (CD138-). We found that cryopreservation using 90% fetal calf serum and 10% dimethyl sulfoxide resulted in highly consistent gene expression profiles when comparing fresh and frozen samples from the same patient for both CD138+ myeloma cells (R ≥ 0.96) and for CD138- cells (R ≥ 0.9). We also demonstrate that CD138- cell-type proportions showed minimal alterations, which were mainly related to small differences in immune cell subtype sensitivity to the freeze-thaw procedures. Therefore, when processing fresh multiple myeloma samples is not feasible, cryopreservation is a useful option in single-cell profiling studies.
Cytogenetic features of human trophoblast cell lines SWAN-71 and 3A-subE
(Elsevier, 2017-04) Reiter, Jill L.; Drendel, Holli M.; Chakraborty, Sujata; Schellinger, Megan M.; Lee, Men-Jean; Mor, Gil; Department of Obstetrics and Gynecology, IU School of Medicine
Immortalization of primary cells with telomerase is thought to maintain normal phenotypic properties and avoid chromosomal abnormalities and other cancer-associated changes that occur following simian virus 40 tumor antigen (SV40 Tag) induced immortalization. However, we report that the human telomerase reverse transcriptase (hTERT)-immortalized SWAN-71 trophoblast cell line has a near pentaploid 103∼119,XXXX[cp20] karyotype. Additionally, DNA typing analysis indicated that SWAN-71 cells have acquired microsatellite instability. In comparison, the post-crisis SV40-transformed trophoblast cell line 3A-subE was hypertriploid 69∼81,XX[cp20]. Both cell lines contained multiple specific clonal rearrangements. These findings emphasize the need to monitor for genetic instability in hTERT-immortalized cells.
Differential Splicing of Skipped Exons Predicts Drug Response in Cancer Cell Lines
(Elsevier, 2021-12) Simpson, Edward; Chen, Steven; Reiter, Jill L.; Liu, Yunlong; BioHealth Informatics, School of Informatics and Computing
Alternative splicing of pre-mRNA transcripts is an important regulatory mechanism that increases the diversity of gene products in eukaryotes. Various studies have linked specific transcript isoforms to altered drug response in cancer; however, few algorithms have incorporated splicing information into drug response prediction. In this study, we evaluated whether basal-level splicing information could be used to predict drug sensitivity by constructing doxorubicin-sensitivity classification models with splicing and expression data. We detailed splicing differences between sensitive and resistant cell lines by implementing quasi-binomial generalized linear modeling (QBGLM) and found altered inclusion of 277 skipped exons. We additionally conducted RNA-binding protein (RBP) binding motif enrichment and differential expression analysis to characterize cis- and trans-acting elements that potentially influence doxorubicin response-mediating splicing alterations. Our results showed that a classification model built with skipped exon data exhibited strong predictive power. We discovered an association between differentially spliced events and epithelial-mesenchymal transition (EMT) and observed motif enrichment, as well as differential expression of RBFOX and ELAVL RBP family members. Our work demonstrates the potential of incorporating splicing data into drug response algorithms and the utility of a QBGLM approach for fast, scalable identification of relevant splicing differences between large groups of samples.
Dynamic chromatin accessibility and transcriptome changes following PDGF-BB treatment of bone-marrow derived mesenchymal stem cells
(Springer Nature, 2024-10-15) Liu, Sheng; Chu, Xiaona; Reiter, Jill L.; Yu, Xuhong; Fang, Fang; McGuire, Patrick; Gao, Hongyu; Liu, Yunlong; Wan, Jun; Wang, Yue; Medical and Molecular Genetics, School of Medicine
Background: Mesenchymal stem cells (MSCs) are multipotent stem cells that are under investigation for use in clinical trials because they are capable of self-renewal and differentiating into different cell types under defined conditions. Nonetheless, the therapeutic effects of MSCs have been constrained by low engraftment rates, cell fusion, and cell survival. Various strategies have been explored to improve the therapeutic efficacy of MSCs, with platelet-derived growth factor (PDGF)-BB emerging as a promising candidate. To enhance our comprehension of the impact of PDGF-BB on the gene expression profile and chromosomal accessibility of MSCs, RNA-sequencing and analysis of chromatin accessibility profiles were conducted on three human primary MSCs in culture, both with and without stimulation by PDGF-BB. Results: Integrative analysis of gene expression and chromatin accessibility demonstrated that PDGF-BB treatment modified the chromatin accessibility landscape, marking regions for activation or repression through the AP-1 family transcription factors TEAD, CEBP, and RUNX2. These changes in AP-1 transcription factor expression, in turn, led to cell proliferation and differentiation potential towards osteoblasts, adipocytes, or chondrocytes. The degree of MSC differentiation varies among cells isolated from different donors. The presence of an enrichment of exosome-related genes is also noted among all the differentially expressed genes. Conclusions: In conclusion, the observed changes in AP-1 transcription factor expression not only induced cellular proliferation and differentiation, but also revealed variations in the degree of MSC differentiation based on donor-specific differences. Moreover, the enrichment of exosome-related genes among differentially expressed genes suggests a potential significant role for PDGF-BB in facilitating intercellular communication.

Browsing by Author "Reiter, Jill L."

Results Per Page

Sort Options