IU Indianapolis ScholarWorks :: Browsing by Author "Srivastava, Rajneesh"

Browsing by Author "Srivastava, Rajneesh"

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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.
A Computational Approach for Identification of Potential Regulatory Motifs and Associated TFs Controlling the Expression of UMOD Gene in Kidney
Srivastava, Rajneesh; El-Achkar, Tarek M.; Janga, Sarath Chandra
Uromodujin is known to be involved in various biological processes like regulation of ion homeostasis, cellular defence response, excretion, calcium. ion binding. Alteration in its protein levels leads to several kidney disorders: ADMCKD2, FJHN, HNFJl, MCKD2, UAKD and AKI. Many in vitro, mouse model studies have been conducted to explore regulation of UMOD however its association with various kidney disorders is still unclear. Our study is focused at transcriptional level to uncover transcription factors (TFs) that bind specifically to the regulatory regions of UMOD gene thus contributing to the identity, physiology & development of kidney. UMOD transcripts were found to be specifically expressed in kidney when their transcript levels were compared using Illumina generated RNA-seq data from the Human Body Map 2.0 Project for 16 different human tissues. Application of phylogenetic foot-printing algorithms by employing the MEME-SUITE of tools allowed the identification of 10 high confidence binding motifs and corresponding positions specific weight matrices in the up stream regulatory binding regions of UMOD orthologs from a diverse set of 8 primates and 7 rodents. We further analyzed the predicted binding motifs by Tom-Tom tool from the suite to identify transcription factors, which have a high tendency, and specificity to bind to these discovered motifs. Our study enabled the identification of a reliable list of transcription factors that could potentially bind to these ten discovered motifs of UMOD, which included TFs such as GATA3, HMF- 1, SPl, SOX5, STAT3, FOXA2, KLF4, and SMAD3 etc. Some of the TFs identified have no functional evidence in human but were annotated in other species. Our study allowed the discovery of regulatory motifs as well as the identification of TFs, which can likely bind to these elements to control the expression of the UMOD gene in kidney cells.
A conserved enhancer regulates Il9 expression in multiple lineages
(Nature Research, 2018-11-15) Koh, Byunghee; Qayum, Amina Abdul; Srivastava, Rajneesh; Fu, Yongyao; Ulrich, Benjamin J.; Janga, Sarath Chandra; Kaplan, Mark H.; Pediatrics, School of Medicine
Cytokine genes are regulated by multiple regulatory elements that confer tissue-specific and activation-dependent expression. The cis-regulatory elements of the gene encoding IL-9, a cytokine that promotes allergy, autoimmune inflammation and tumor immunity, have not been defined. Here we identify an enhancer (CNS-25) upstream of the Il9 gene that binds most transcription factors (TFs) that promote Il9 gene expression. Deletion of the enhancer in the mouse germline alters transcription factor binding to the remaining Il9 regulatory elements, and results in diminished IL-9 production in multiple cell types including Th9 cells, and attenuates IL-9-dependent immune responses. Moreover, deletion of the homologous enhancer (CNS-18) in primary human Th9 cultures results in significant decrease of IL-9 production. Thus, Il9 CNS-25/IL9 CNS-18 is a critical and conserved regulatory element for IL-9 production.
Direct RNA-sequencing of human cell lines for transcriptome-wide mapping and annotation of 3' tails at single molecule resolution
Govindaraman, Aniruddhan; Quoseena, Mir; Kadumuri, Raja Shekar Vanna; Srivastava, Mansi; Srivastava, Rajneesh; Janga, Sarath Chandra
The 3' endonucleolytic cleavage of pre-messenger RNA (pre-mRNA) and successive polyadenylation is a fundamental cellular process in eukaryotes. The 3' terminal regions are known to be polyadenylated by canonical poly(A) polymerases during RNA processing of messenger RNA (mRNA) molecules, however, they are also known to harbor additional UnMapped Regions (UMR) composed of uridylation and guanylation[1]. Although short read sequencing technologies are extensively used to study 3' terminal regions, major limitations of these approaches include their inability to detect homopolymeric sequences and sequence full length isoforms [1-2]. Nanopore sequencing enables the long read sequencing and identification of full length transcripts at a single molecule resolution, however currently there are no tools for systematically analyzing 3' terminal UMRs from direct RNA-sequencing datasets. Here, we present RAPTOR (https://github.com/aniram118/RAPTOR), a command line tool for 3' terminal UMR analysis of nanopore direct RNA sequencing data. RAPTOR provides a comprehensive report of UMR sequence information, cognate transcript annotations, nucleotide base composition, conserved hexamer signals and a range of analyses plots at a single molecule resolution. For benchmarking, we sequenced mRNA samples obtained from HepG2 (Liver Hepatocellular Carcinoma) & K562 (Bone Marrow Chronic myelogenous leukemia) cell lines resulting in 243,802 & 598,428 reads respectively. RAPTOR identified high quality UMRs, exhibited median lengths of 201 and 173 nt in HepG2 and K562 transcriptomes respectively. Nucleotide composition analysis of the identified 3' UMRs showed an enrichment for A and U nucleotides in both HepG2 [A: 29%, U: 28%, G:20%, C:23%] and K562 [A : 30%, U: 29%, G:1 9%, C:22%] cells. Several high confidence UMRs were verified by qPCR and sanger sequencing confirming sequence length and identity, respectively. In addition, denovo motif analysis of UMR regions enabled the discovery of several noncanonical motifs beyond Poly A/U patterns. These UMR motifs were identified to be significantly - (p-value <0.01) associated with the established binding motifs of several known RNA Binding Proteins including SART3, HuR (ELAVL1), TIA1 , IGF2BP2/3, PABPCs, PCBPs, SRSFs, HNRNPs and RBM /6, suggesting an unappreciated role of these RBPs in binding to 3' tails of mRNAs.
Embryonic ethanol exposure alters expression of sox2 and other early transcripts in zebrafish, producing gastrulation defects
(Springer Nature, 2020-03-03) Sarmah, Swapnalee; Srivastava, Rajneesh; McClintick, Jeanette N.; Janga, Sarath C.; Edenberg, Howard J.; Marrs, James A.; Biology, School of Science
Ethanol exposure during prenatal development causes fetal alcohol spectrum disorder (FASD), the most frequent preventable birth defect and neurodevelopmental disability syndrome. The molecular targets of ethanol toxicity during development are poorly understood. Developmental stages surrounding gastrulation are very sensitive to ethanol exposure. To understand the effects of ethanol on early transcripts during embryogenesis, we treated zebrafish embryos with ethanol during pre-gastrulation period and examined the transcripts by Affymetrix GeneChip microarray before gastrulation. We identified 521 significantly dysregulated genes, including 61 transcription factors in ethanol-exposed embryos. Sox2, the key regulator of pluripotency and early development was significantly reduced. Functional annotation analysis showed enrichment in transcription regulation, embryonic axes patterning, and signaling pathways, including Wnt, Notch and retinoic acid. We identified all potential genomic targets of 25 dysregulated transcription factors and compared their interactions with the ethanol-dysregulated genes. This analysis predicted that Sox2 targeted a large number of ethanol-dysregulated genes. A gene regulatory network analysis showed that many of the dysregulated genes are targeted by multiple transcription factors. Injection of sox2 mRNA partially rescued ethanol-induced gene expression, epiboly and gastrulation defects. Additional studies of this ethanol dysregulated network may identify therapeutic targets that coordinately regulate early development.
Endothelial Phospholipase Cγ2 Improves Outcomes of Diabetic Ischemic Limb Rescue Following VEGF Therapy
(American Diabetes Association, 2022) Rustagi, Yashika; Abouhashem, Ahmed S.; Verma, Priyanka; Verma, Sumit S.; Hernandez, Edward; Liu, Sheng; Kumar, Manishekhar; Guda, Poornachander R.; Srivastava, Rajneesh; Mohanty, Sujit K.; Kacar, Sedat; Mahajan, Sanskruti; Wanczyk, Kristen E.; Khanna, Savita; Murphy, Michael P.; Gordillo, Gayle M.; Roy, Sashwati; Wan, Jun; Sen, Chandan K.; Singh, Kanhaiya; Medicine, School of Medicine
Therapeutic vascular endothelial growth factor (VEGF) replenishment has met with limited success for the management of critical limb-threatening ischemia. To improve outcomes of VEGF therapy, we applied single-cell RNA sequencing (scRNA-seq) technology to study the endothelial cells of the human diabetic skin. Single-cell suspensions were generated from the human skin followed by cDNA preparation using the Chromium Next GEM Single-cell 3' Kit v3.1. Using appropriate quality control measures, 36,487 cells were chosen for downstream analysis. scRNA-seq studies identified that although VEGF signaling was not significantly altered in diabetic versus nondiabetic skin, phospholipase Cγ2 (PLCγ2) was downregulated. The significance of PLCγ2 in VEGF-mediated increase in endothelial cell metabolism and function was assessed in cultured human microvascular endothelial cells. In these cells, VEGF enhanced mitochondrial function, as indicated by elevation in oxygen consumption rate and extracellular acidification rate. The VEGF-dependent increase in cell metabolism was blunted in response to PLCγ2 inhibition. Follow-up rescue studies therefore focused on understanding the significance of VEGF therapy in presence or absence of endothelial PLCγ2 in type 1 (streptozotocin-injected) and type 2 (db/db) diabetic ischemic tissue. Nonviral topical tissue nanotransfection technology (TNT) delivery of CDH5 promoter-driven PLCγ2 open reading frame promoted the rescue of hindlimb ischemia in diabetic mice. Improvement of blood flow was also associated with higher abundance of VWF+/CD31+ and VWF+/SMA+ immunohistochemical staining. TNT-based gene delivery was not associated with tissue edema, a commonly noted complication associated with proangiogenic gene therapies. Taken together, our study demonstrates that TNT-mediated delivery of endothelial PLCγ2, as part of combination gene therapy, is effective in diabetic ischemic limb rescue.
Express: A database of transcriptome profiles encompassing known and novel transcripts across multiple development stages in eye tissues
(Elsevier, 2018) Budak, Gungor; Dash, Soma; Srivastava, Rajneesh; Lachke, Salil A.; Janga, Sarath Chandra; BioHealth Informatics, School of Informatics and Computing
Advances in sequencing have facilitated nucleotide-resolution genome-wide transcriptomic profiles across multiple mouse eye tissues. However, these RNA sequencing (RNA-seq) based eye developmental transcriptomes are not organized for easy public access, making any further analysis challenging. Here, we present a new database “Express” (http://www.iupui.edu/∼sysbio/express/) that unifies various mouse lens and retina RNA-seq data and provides user-friendly visualization of the transcriptome to facilitate gene discovery in the eye. We obtained RNA-seq data encompassing 7 developmental stages of lens in addition to that on isolated lens epithelial and fibers, as well as on 11 developmental stages of retina/isolated retinal rod photoreceptor cells from publicly available wild-type mouse datasets. These datasets were pre-processed, aligned, quantified and normalized for expression levels of known and novel transcripts using a unified expression quantification framework. Express provides heatmap and browser view allowing easy navigation of the genomic organization of transcripts or gene loci. Further, it allows users to search candidate genes and export both the visualizations and the embedded data to facilitate downstream analysis. We identified total of >81,000 transcripts in the lens and >178,000 transcripts in the retina across all the included developmental stages. This analysis revealed that a significant number of the retina-expressed transcripts are novel. Expression of several transcripts in the lens and retina across multiple developmental stages was independently validated by RT-qPCR for established genes such as Pax6 and Lhx2 as well as for new candidates such as Elavl4, Rbm5, Pabpc1, Tia1 and Tubb2b. Thus, Express serves as an effective portal for analyzing pruned RNA-seq expression datasets presently collected for the lens and retina. It will allow a wild-type context for the detailed analysis of targeted gene-knockout mouse ocular defect models and facilitate the prioritization of candidate genes from Exome-seq data of eye disease patients.
Genome-wide DNA hypermethylation opposes healing in patients with chronic wounds by impairing epithelial-mesenchymal transition
(The American Society for Clinical Investigation, 2022) Singh, Kanhaiya; Rustagi, Yashika; Abouhashem, Ahmed S.; Tabasum, Saba; Verma, Priyanka; Hernandez, Edward; Pal, Durba; Khona, Dolly K.; Mohanty, Sujit K.; Kumar, Manishekhar; Srivastava, Rajneesh; Guda, Poornachander R.; Verma, Sumit S.; Mahajan, Sanskruti; Killian, Jackson A.; Walker, Logan A.; Ghatak, Subhadip; Mathew-Steiner, Shomita S.; Wanczyk, Kristen E.; Liu, Sheng; Wan, Jun; Yan, Pearlly; Bundschuh, Ralf; Khanna, Savita; Gordillo, Gayle M.; Murphy, Michael P.; Roy, Sashwati; Sen, Chandan K.; Surgery, School of Medicine
An extreme chronic wound tissue microenvironment causes epigenetic gene silencing. An unbiased whole-genome methylome was studied in the wound-edge tissue of patients with chronic wounds. A total of 4,689 differentially methylated regions (DMRs) were identified in chronic wound-edge skin compared with unwounded human skin. Hypermethylation was more frequently observed (3,661 DMRs) in the chronic wound-edge tissue compared with hypomethylation (1,028 DMRs). Twenty-six hypermethylated DMRs were involved in epithelial-mesenchymal transition (EMT). Bisulfite sequencing validated hypermethylation of a predicted specific upstream regulator TP53. RNA-Seq analysis was performed to qualify findings from methylome analysis. Analysis of the downregulated genes identified the TP53 signaling pathway as being significantly silenced. Direct comparison of hypermethylation and downregulated genes identified 4 genes, ADAM17, NOTCH, TWIST1, and SMURF1, that functionally represent the EMT pathway. Single-cell RNA-Seq studies revealed that these effects on gene expression were limited to the keratinocyte cell compartment. Experimental murine studies established that tissue ischemia potently induces wound-edge gene methylation and that 5'-azacytidine, inhibitor of methylation, improved wound closure. To specifically address the significance of TP53 methylation, keratinocyte-specific editing of TP53 methylation at the wound edge was achieved by a tissue nanotransfection-based CRISPR/dCas9 approach. This work identified that reversal of methylation-dependent keratinocyte gene silencing represents a productive therapeutic strategy to improve wound closure.
Granzyme A–producing T helper cells are critical for acute graft-versus-host disease
(American Society for Clinical Investigation, 2020-08-18) Park, Sungtae; Griesenauer, Brad; Jiang, Hua; Adom, Djamilatou; Mehrpouya-Bahrami, Pegah; Chakravorty, Srishti; Kazemian, Majid; Imam, Tanbeena; Srivastava, Rajneesh; Hayes, Tristan A.; Pardo, Julian; Janga, Sarath Chandra; Paczesny, Sophie; Kaplan, Mark H.; Olson, Matthew R.; Microbiology and Immunology, School of Medicine
Acute graft-versus-host disease (aGVHD) can occur after hematopoietic cell transplant in patients undergoing treatment for hematological malignancies or inborn errors. Although CD4+ T helper (Th) cells play a major role in aGVHD, the mechanisms by which they contribute, particularly within the intestines, have remained elusive. We have identified a potentially novel subset of Th cells that accumulated in the intestines and produced the serine protease granzyme A (GrA). GrA+ Th cells were distinct from other Th lineages and exhibited a noncytolytic phenotype. In vitro, GrA+ Th cells differentiated in the presence of IL-4, IL-6, and IL-21 and were transcriptionally unique from cells cultured with either IL-4 or the IL-6/IL-21 combination alone. In vivo, both STAT3 and STAT6 were required for GrA+ Th cell differentiation and played roles in maintenance of the lineage identity. Importantly, GrA+ Th cells promoted aGVHD-associated morbidity and mortality and contributed to crypt destruction within intestines but were not required for the beneficial graft-versus-leukemia effect. Our data indicate that GrA+ Th cells represent a distinct Th subset and are critical mediators of aGVHD.
Human fetal dermal fibroblast-myeloid cell diversity is characterized by dominance of pro-healing Annexin1-FPR1 signaling
(Elsevier, 2023-08-02) Srivastava, Rajneesh; Singh, Kanhaiya; Abouhashem, Ahmed S.; Kumar, Manishekhar; Kacar, Sedat; Verma, Sumit S.; Mohanty, Sujit K.; Sinha, Mithun; Ghatak, Subhadip; Xuan, Yi; Sen, Chandan K.; Surgery, School of Medicine
Fetal skin achieves scarless wound repair. Dermal fibroblasts play a central role in extracellular matrix deposition and scarring outcomes. Both fetal and gingival wound repair share minimal scarring outcomes. We tested the hypothesis that compared to adult skin fibroblasts, human fetal skin fibroblast diversity is unique and partly overlaps with gingival skin fibroblasts. Human fetal skin (FS, n = 3), gingiva (HGG, n = 13), and mature skin (MS, n = 13) were compared at single-cell resolution. Dermal fibroblasts, the most abundant cluster, were examined to establish a connectome with other skin cells. Annexin1-FPR1 signaling pathway was dominant in both FS as well as HGG fibroblasts and related myeloid cells while scanty in MS fibroblasts. Myeloid-specific FPR1-ORF delivered in murine wound edge using tissue nanotransfection (TNT) technology significantly enhanced the quality of healing. Pseudotime analyses identified the co-existence of an HGG fibroblast subset with FPR1high myeloid cells of fetal origin indicating common underlying biological processes.

Browsing by Author "Srivastava, Rajneesh"

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