Biology subjects

Joshi, T.

Publications and source records attributed to Joshi, T..

30 records · Page 2Linked to original sources

bioRxiv2023

Integrative identification of non-coding regulatory regions driving metastatic prostate cancer

Large-scale sequencing efforts of thousands of tumor samples have been undertaken to understand the mutational landscape of the coding genome. However, the vast majority of germline and somatic variants occur within non-coding portions of the genome. These genomic regions do not directly encode for specific proteins, but can play key roles in cancer progression, for example by driving aberrant gene expression control. Here, we designed an integrative computational and experimental framework to identify recurrently mutated non-coding regulatory regions that drive tumor progression. Application of this approach to whole-genome sequencing (WGS) data from a large cohort of metastatic castration-resistant prostate cancer (mCRPC) revealed a large set of recurrently mutated regions. We used (i) in silico prioritization of functional non-coding mutations, (ii) massively parallel reporter assays, and (iii) in vivo CRISPR-interference (CRISPRi) screens in xenografted mice to systematically identify and validate driver regulatory regions that drive mCRPC. We discovered that one of these enhancer regions, GH22I030351, acts on a bidirectional promoter to simultaneously modulate expression of U2-associated splicing factor SF3A1 and chromosomal protein CCDC157. We found that both SF3A1 and CCDC157 are promoters of tumor growth in xenograft models of prostate cancer. We nominated a number of transcription factors, including SOX6, to be responsible for higher expression of SF3A1 and CCDC157. Collectively, we have established and confirmed an integrative computational and experimental approach that enables the systematic detection of non-coding regulatory regions that drive the progression of human cancers.

cancer biology↗

bioRxiv2023

Adventitial macrophage accumulation impairs perivascular nerve function in mesenteric arteries with inflammatory bowel disease

1.Introduction: Inflammatory bowel disease (IBD) involves aberrant immune responses and is associated with both cardiovascular disease risk and altered intestinal blood flow. However, little is known about how IBD affects regulation of perivascular nerves that mediate blood flow. Previous work found perivascular nerve function is impaired in mesenteric arteries with IBD. The purpose of this study was to determine the mechanism of impaired perivascular nerve function. Methods: RNA sequencing was performed on mesenteric arteries from IL10-/- mice treated with H.hepaticus to induce disease (IBD) or left non-gavaged (Control). For all other studies, Control and IBD mice received either saline or clodronate liposome injections to study the effect of macrophage depletion. Perivascular nerve function was assessed using pressure myography and electrical field stimulation. Leukocyte populations, and perivascular nerves, and adventitial neurotransmitter receptors were labeled using fluorescent immunolabeling. Results: IBD was associated with increased in macrophage-associated gene expression, and immunolabeling showed accumulation of adventitial macrophages. Clodronate liposome injection eliminated adventitial macrophages, which reversed significant attenuation of sensory vasodilation, sympathetic vasoconstriction and sensory inhibition of sympathetic constriction in IBD. Acetylcholine-mediated dilation was impaired in IBD and restored after macrophage depletion, but sensory dilation remained nitric oxide independent regardless of disease and/or macrophage presence. Conclusion: Altered neuro-immune signaling between macrophages and perivascular nerves in the arterial adventitia contributes to impaired vasodilation, particularly via dilatory sensory nerves. Targeting the adventitial macrophage population may help preserve intestinal blood flow in IBD patients.

physiology↗

bioRxiv2023

Inhibition of muscarinic receptor signaling protects human enteric inhibitory neurons against platin chemotherapy toxicity

GI toxicity is a common dose-limiting adverse effect of platin chemotherapy treatment. Up to 50% of cancer survivors continue to experience symptoms of chronic constipation or diarrhea induced by their chemotherapy for many years after their treatment. This drug toxicity is largely attributed to damage to enteric neurons that innervate the GI tract and control GI motility. The mechanisms responsible for platin-induced enteric neurotoxicity and potential preventative strategies have remained unknown. Here, we use human pluripotent stem cell derived enteric neurons to establish a new model system capable of uncovering the mechanism of platin-induced enteric neuropathy. Utilizing this scalable system, we performed a high throughput screen and identified drug candidates and pathways involved in the disease. Our analyses revealed that excitotoxicity through muscarinic cholinergic signaling is a key driver of platin-induced enteric neuropathy. Using single nuclei transcriptomics and functional assays, we discovered that this disease mechanism leads to increased susceptibility of specific neuronal subtypes, including inhibitory nitrergic neurons, to platins. Histological assessment of the enteric nervous system in platin-treated patients confirmed the selective loss of nitrergic neurons. Finally, we demonstrated that pharmacological and genetic inhibition of muscarinic cholinergic signaling is sufficient to rescue enteric neurons from platin excitotoxicity in vitro and can prevent platin-induced constipation and degeneration of nitrergic neurons in mice. These studies define the mechanisms of platin-induced enteric neuropathy and serve as a framework for uncovering cell type-specific manifestations of cellular stress underlying numerous intractable peripheral neuropathies.

pharmacology and toxicology↗

bioRxiv2023

Systematic Identification of Post-Transcriptional Regulatory Modules

In our cells, a limited number of RNA binding proteins (RBPs) are responsible for all aspects of RNA metabolism across the entire transcriptome. To accomplish this, RBPs form regulatory units that act on specific target regulons. However, the landscape of RBP combinatorial interactions remains poorly explored. Here, we performed a systematic annotation of RBP combinatorial interactions via multimodal data integration. We built a large-scale map of RBP protein neighborhoods by generating in vivo proximity-dependent biotinylation datasets of 50 human RBPs. In parallel, we used CRISPR interference with single-cell readout to capture transcriptomic changes upon RBP knockdowns. By combining these physical and functional interaction readouts, along with the atlas of RBP mRNA targets from eCLIP assays, we generated an integrated map of functional RBP interactions. We then used this map to match RBPs to their context-specific functions and validated the predicted functions biochemically for four RBPs. This study highlights the previously underappreciated scale of the inter-RBP interactions, be it genetic or physical, and is a first step towards a more comprehensive understanding of post-transcriptional regulatory processes and their underlying molecular grammar.

systems biology↗

bioRxiv2023

The transcriptome of soybean reproductive tissues subjected to water deficit, heat stress, and a combination of water deficit and heat stress.

Global warming and climate change are driving an alarming increase in the frequency and intensity of extreme climate events, such as droughts, heat waves, and their combination, inflicting heavy losses to agricultural production. Recent studies revealed that the transcriptomic responses of different crops to water deficit (WD) or heat stress (HS) is very different from that to a combination of WD+HS. In addition, it was found that the effects of WD, HS, and WD+HS are significantly more devastating when these stresses occur during the reproductive growth phase of crops, compared to vegetative growth. As the molecular responses of different reproductive and vegetative tissues of plants to WD, HS, or WD+HS could be different from each other, and these differences could impact many current and future breeding and/or engineering attempts to enhance the resilience of crops to climate change, we conducted a transcriptomic analysis of different soybean (Glycine max) tissues to WD, HS, and WD+HS. Here we present a reference transcriptomic dataset that includes the response of soybean leaf, pod, anther, stigma, ovary, and sepal to WD, HS, and WD+HS conditions. Mining this data set for the expression pattern of different stress-response transcripts revealed that each tissue had a unique transcriptomic response to each of the different stress conditions. This finding is important as it suggests that attempting to enhance the overall resilience of crops to climate change could require a coordinated approach that simultaneously alters the expression of different groups of transcripts in different tissues in a stress-specific manner. SIGNIFICANCE STATEMENTA reference transcriptomic dataset of different reproductive tissues of soybean subjected to water deficit, heat stress, and their combination, generated by this study, reveals that different tissues display different responses to these stress conditions. Attempting to enhance the resilience of crops to different stress combinations, associated with climate change, might therefore require simultaneously altering the expression of different sets of transcripts in different tissues in a coordinated and stress-specific manner.

plant biology↗

bioRxiv2022

The effects of multifactorial stress combination on rice and maize

The complexity of environmental factors affecting plants is gradually increasing due to global warming, an increase in the number and intensity of climate change-driven weather events, such as droughts, heat waves, and floods, and the accumulation of different pollutants. The impact of multiple stress conditions on plants was recently termed multifactorial stress combination (MFSC) and defined as the occurrence of three or more stressors that impact plants simultaneously or sequentially. We recently reported that with the increased number and complexity of different stressors, the growth and survival of Arabidopsis thaliana seedlings declines; even if the level of each individual stress is low enough to have no significant effect on plants. This finding is alarming since it reveals that MFSCs of different low-level stressors could impact crops and cause a dramatic reduction in overall growth. However, whether MFSC would impact commercial crop cultivars has not been studied. Here, we reveal that a MFSC of 5 different low level abiotic stresses (salinity, heat, the herbicide paraquat, phosphorus deficiency, and the heavy metal cadmium), applied in an increasing level of complexity, has a significant negative impact on the growth and biomass of a commercial rice (Oryza sativa) cultivar and a maize (Zea mays) hybrid. We further report on the first proteomics analysis of MFSC in plants that identified over 300 proteins common to all 4- and 5-MFSCs. Taken together our findings reveal that the impacts of MFSC on two different crop species are severe, and that MFSC may significantly affect agricultural productivity.

plant biology↗

bioRxiv2022

Differential transpiration between pods and leaves during stress combination in soybean

Climate change is causing an increase in the frequency and intensity of droughts, heat waves, and their combinations, diminishing agricultural productivity and destabilizing societies worldwide. We recently reported that during a combination of water deficit (WD) and heat stress (HS) stomata on leaves of soybean plants are closed, while stomata on flowers are open. This unique stomatal response was accompanied by differential transpiration (higher in flowers, while lower in leaves) that cooled flowers during a combination of WD+HS. Here we reveal that developing pods of soybean plants subjected to a combination of WD+HS use a similar acclimation strategy of differential transpiration to reduce internal pod temperature by about 4{degrees}C. We further show that enhanced expression of transcripts involved in abscisic acid degradation accompanies this response, and that preventing pod transpiration by sealing stomata causes a significant increase in internal pod temperature. Using an RNA-Seq analysis of pods developing on plants subjected to WD+HS, we also show that the response of pods to WD, HS, or WD+HS is distinct from that of leaves or flowers. Interestingly, we report that although flower, pod and seed numbers per plant are decreased under conditions of WD+HS, seed mass of plants subjected to WD+HS is larger than that of plants subjected to HS, and number of seeds with suppressed/aborted development is lower in WD+HS compared to HS. Taken together our findings reveal that differential transpiration occurs in pods of soybean plants subjected to WD+HS and that this process limits heat-induced damage to seed production. One sentence summaryDifferential transpiration between pods and leaves of soybean plants subjected to a combination of water deficit and heat stress buffers internal pod temperature.

plant biology↗

bioRxiv2021

Differential regulation of flower transpiration during abiotic stress in plants

Heat waves, occurring during droughts, can have a devastating impact on yield, especially if they happen during the flowering and seed set stages of the crop cycle. Global warming and climate change are driving an alarming increase in the frequency and intensity of combined drought and heat stress episodes, critically threatening global food security. Previous studies revealed that during a combination of drought and heat stress stomata on leaves of many plants are closed, preventing cooling by transpiration. Because high temperature is detrimental to reproductive processes, essential for plant yield, we measured the inner temperature, transpiration, and sepal stomatal aperture of closed soybean flowers, developing on plants subjected to a combination of drought and heat stress. Here, we report that during a combination of drought and heat stress soybean plants prioritize transpiration through flowers over transpiration through leaves by opening their flower stomata, while keeping their leaf stomata closed. This acclimation strategy, termed differential transpiration, lowers flower inner temperature by about 2-3{degrees}C, protecting reproductive processes at the expense of vegetative tissues. Manipulating stomatal regulation, stomatal size and/or stomatal density of flowers could therefore serve as a viable strategy to enhance the yield of different crops and mitigate some of the current and future impacts of global warming and climate change on agriculture. One sentence summaryDuring stress conditions that result in higher flower inner temperature plants use a differential transpiration strategy to protect reproductive processes at the expense of vegetative tissues.

plant biology↗

bioRxiv2021

An mRNA processing pathway suppresses metastasis by governing translational control from the nucleus

Cancer cells often co-opt post-transcriptional regulatory mechanisms to achieve pathologic expression of gene networks that drive metastasis. Translational control is a major regulatory hub in oncogenesis, however its effects on cancer progression remain poorly understood. To address this, we used ribosome profiling to compare genome-wide translation efficiencies of poorly and highly metastatic breast cancer cells and patient-derived xenografts. We developed novel regression-based methods to analyze ribosome profiling and alternative polyadenylation data, and identified HNRNPC as a translational controller of a specific mRNA regulon. Mechanistically, HNRNPC, in concert with PABPC4, binds near to poly(A) signals, thereby governing the alternative polyadenylation of a set of mRNAs. We found that HNRNPC and PABPC4 are downregulated in highly metastatic cells, which causes HNRNPC-bound mRNAs to undergo 3 UTR lengthening and subsequently, translational repression. We showed that modulating HNRNPC expression impacts the metastatic capacity of breast cancer cells in xenograft mouse models. We also found that a small molecule, previously shown to induce a distal-to-proximal poly(A) site switching, counteracts the HNRNPC-PABPC4 driven deregulation of alternative polyadenylation and decreases the metastatic lung colonization by breast cancer cells in vivo.

cancer biology↗

bioRxiv2021

Allosteric control of Ubp6 and the proteasome via a bidirectional switch

The proteasome is the principal cellular protease, and recognizes target proteins that have been covalently marked by ubiquitin chains. The ubiquitin signal is subject to rapid editing at the proteasome, allowing it to reject substrates based on topological features of their attached ubiquitin chains. Editing is mediated by a key regulator of the proteasome, deubiquitinating enzyme Ubp6. The proteasome activates Ubp6, whereas Ubp6 inhibits the proteasome-both by deubiquitinating proteasome-bound ubiquitin conjugates, and through a noncatalytic effect that does not involve deubiquitination. We report mutants in both Ubp6 and proteasome subunit Rpt1 that abrogate Ubp6 activation. The Ubp6 mutations fall within its ILR element, defined here, which is conserved from yeast to mammals. The ILR is a component of the BL1 blocking loop, other parts of which obstruct ubiquitin access to the catalytic groove in free Ubp6. Rpt1 docking at the ILR opens the catalytic groove by rearranging not only BL1 but also a novel network of three directly interconnected active-site-blocking loops. Ubp6 activation and noncatalytic proteasome inhibition by Ubp6 are linked in that they were eliminated by the same Ubp6 and Rpt1 mutations. Ubp6 and ubiquitin together drive the proteasome into a unique conformational state associated with proteasome inhibition. Our results identify a multicomponent allosteric switch that exerts simultaneous control over the activity of both Ubp6 and the proteasome, and suggest that their active states are in general mutually exclusive. The findings lead to a new paradigm for allosteric control of deubiquitinating enzymes.

biochemistry↗

bioRxiv2020

Inductive Inference of Gene Regulatory Network Using Supervised and Semi-supervised Graph Neural Networks

Discovering gene regulatory relationships and reconstructing gene regulatory networks (GRN) based on gene expression data is a classical, long-standing computational challenge in bioinformatics. Computationally inferring a possible regulatory relationship between two genes can be formulated as a link prediction problem between two nodes in a graph. Graph neural network (GNN) provides an opportunity to construct GRN by integrating topological neighbor propagation through the whole gene network. We propose an end-to-end gene regulatory graph neural network (GRGNN) approach to reconstruct GRNs from scratch utilizing the gene expression data, in both a supervised and a semi-supervised framework. To get better inductive generalization capability, GRN inference is formulated as a graph classification problem, to distinguish whether a subgraph centered at two nodes contains the link between the two nodes. A linked pair between a transcription factor (TF) and a target gene, and their neighbors are labeled as a positive subgraph, while an unlinked TF and target gene pair and their neighbors are labeled as a negative subgraph. A GNN model is constructed with node features from both explicit gene expression and graph embedding. We demonstrate a noisy starting graph structure built from partial information, such as Pearsons correlation coefficient and mutual information can help guide the GRN inference through an appropriate ensemble technique. Furthermore, a semi-supervised scheme is implemented to increase the quality of the classifier. When compared with established methods, GRGNN achieved state-of-the-art performance on the DREAM5 GRN inference benchmarks. GRGNN is publicly available at https://github.com/juexinwang/GRGNN. HighlightsO_LIWe present a novel formulation of graph classification in inferring gene regulatory relationships from gene expression and graph embedding. C_LIO_LIOur method leverages a powerful framework, gene regulatory graph neural network (GRGNN), which is flexible and powerful to ensemble statistical powers from a number of heuristic skeletons. C_LIO_LIOur results show GRGRNN outperforms previous supervised and unsupervised methods inductively on benchmarks. C_LIO_LIGRGNN can be interpreted and explained following the biological network motif hypothesis in gene regulatory networks. C_LI

bioinformatics↗

bioRxiv2020

Traceback of Core Transcription Factors for Soybean Root Growth Maintenance under Water Deficit

Some crops inhibit shoot growth but maintain root growth under water-deficit conditions. Unraveling the molecular mechanisms of root plasticity under water deficit conditions in plants remains a major challenge. We developed an efficient platform for identifying core transcription factors (TFs) that collectively regulate each other and/or themselves in response to water stress, and exploring their interconnected regulatory circuitry involved in root growth maintenance under water deficit in soybean. We performed multi-species phylogenetic footprinting combined with spatial-temporal transcriptome analysis of soybean (Glycine max) roots under water deficit to identify conserved motifs that function in the water-stress response. Using these functional conserved cis-motifs, we applied a new approach to trace back motifs-associated core TFs ingroup as signal mediators, which mediate signaling between abiotic and endogenous stimuli. We integrated a co-functional TF-TF network and conserved motif-centered TF-DNA networks to construct a core TF network defined by mutual cross-regulation among core TFs. We found that core TF ARG (Abscisic acid response element binding factor-like Root Growth regulator) represses BRG (Brassinosteroid enhanced expression-like Root Growth regulator) expression through binding to its promoter at a conserved binding site. ARG and BRG antagonistically regulate Phytochrome-interacting factor-like Root Growth regulator (PRG) and combinatorially regulate some other core TFs. These core TFs form complex regulatory circuits to integrate light and multiple hormone signaling pathways and maintain root growth in response to varying degrees of water stress. Our study provides valuable information to unravel the complicated mechanisms of molecular networks involved in the regulation of root growth under water deficit.

plant biology↗