Biology subjects

Krishnan, J.

Publications and source records attributed to Krishnan, J..

24 records · Page 2Linked to original sources

bioRxiv2020

Genome-wide analysis of cis-regulatory changes in the metabolic adaptation of cavefish

Changes in cis-regulatory elements play important roles in adaptation and phenotypic evolution. However, their contribution to metabolic adaptation of organisms is less understood. Here we have utilized a unique vertebrate model, Astyanax mexicanus, different morphotypes of which survive in nutrient-rich surface and nutrient-deprived cave water to uncover gene regulatory networks in metabolic adaptation. We performed genome-wide epigenetic profiling in the liver tissue of one surface and two independently derived cave populations. We find that many cis-regulatory elements differ in their epigenetic status/chromatin accessibility between surface fish and cavefish, while the two independently derived cave populations have evolved remarkably similar regulatory signatures. These differentially accessible regions are associated with genes of key pathways related to lipid metabolism, circadian rhythm and immune system that are known to be altered in cavefish. Using in vitro and in vivo functional testing of the candidate cis-regulatory elements, we find that genetic changes within them cause quantitative expression differences. We characterized one cis-regulatory element in the hpdb gene and found a genomic deletion in cavefish that abolishes binding of the transcriptional repressor IRF2 in vitro and derepresses enhancer activity in reporter assays. Genetic experiments further validated a cis-mediated role of the enhancer and suggest a role of this deletion in the upregulation of hpdb in wild cavefish populations. Selection of this mutation in multiple independent cave populations supports its importance in the adaptation to the cave environment, providing novel molecular insights into the evolutionary trade-off between loss of pigmentation and adaptation to a food-deprived cave environment.

genomics↗

bioRxiv2020

SARS-CoV-2 infects and induces cytotoxic effects in human cardiomyocytes

BackgroundThe coronavirus disease 2019 (COVID-19) is caused by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) and has emerged as global pandemic. SARS-CoV-2 infection can lead to elevated markers of cardiac injury associated with higher risk of mortality in COVID-19 patients. It is unclear whether cardiac injury may have been caused by direct infection of cardiomyocytes or is mainly secondary to lung injury and inflammation. Here we investigate whether human cardiomyocytes are permissive for SARS-CoV-2 infection. MethodsInfection was induced by two strains of SARS-CoV-2 (FFM1 and FFM2) in human induced pluripotent stem cells-derived cardiomyocytes (hiPS-CM) and in two models of human cardiac tissue. ResultsWe show that SARS-CoV-2 infects hiPS-CM as demonstrated by detection of intracellular double strand viral RNA and viral spike glycoprotein protein expression. Increasing concentrations of virus RNA are detected in supernatants of infected cardiomyocytes, which induced infections in CaCo-2 cell lines documenting productive infections. SARS-COV-2 infection induced cytotoxic and pro-apoptotic effects and abolished cardiomyocyte beating. RNA sequencing confirmed a transcriptional response to viral infection as demonstrated by the up-regulation of genes associated with pathways related to viral response and interferon signaling, apoptosis and reactive oxygen stress. SARS-CoV-2 infection and cardiotoxicity was confirmed in a iPS-derived human 3D cardiosphere tissue models. Importantly, viral spike protein and viral particles were detected in living human heart slices after infection with SARS-CoV-2. ConclusionsThe demonstration that cardiomyocytes are permissive for SARS-CoV-2 infection in vitro warrants the further in depth monitoring of cardiotoxic effects in COVID-19 patients. Clinical PerspectiveO_ST_ABSWhat is New?C_ST_ABSO_LIThis study demonstrates that human cardiac myocytes are permissive for SARS-CoV-2 infection. C_LIO_LIThe study documents that SARS-CoV-2 undergoes a full replicatory circle and induces a cytotoxic response in cardiomyocytes. C_LIO_LIInfection was confirmed in two cardiac tissue models, including living human heart slices. C_LI What are the Clinical Implications?O_LIThe study may provide a rational to explain part of the cardiotoxicity observed in COVID-19 patients C_LIO_LIThe demonstration of direct cardiotoxicity induced by SARS-CoV-2 warrants an in depth further analysis of cardiac tissue of COVID-19 patients and a close monitoring for putative direct cardiomyocyte injury. C_LIO_LIThe established models can be used to test novel therapeutic approaches targeting COVID-19. C_LI

cell biology↗

bioRxiv2019

An integrative ENCODE resource for cancer genomics

ENCODE comprises thousands of functional genomics datasets, and the encyclopedia covers hundreds of cell types, providing a universal annotation for genome interpretation. However, for particular applications, it may be advantageous to use a customized annotation. Here, we develop such a custom annotation by leveraging advanced assays, such as eCLIP, Hi-C, and whole-genome STARR-seq on a number of data-rich ENCODE cell types. A key aspect of this annotation is comprehensive and experimentally derived networks of both transcription factors and RNA-binding proteins (TFs and RBPs). Cancer, a disease of system-wide dysregulation, is an ideal application for such a network-based annotation. Specifically, for cancer-associated cell types, we put regulators into hierarchies and measure their network change (rewiring) during oncogenesis. We also extensively survey TF-RBP crosstalk, highlighting how SUB1, a previously uncharacterized RBP, drives aberrant tumor expression and amplifies the effect of MYC, a well-known oncogenic TF. Furthermore, we show how our annotation allows us to place oncogenic transformations in the context of a broad cell space; here, many normal-to-tumor transitions move towards a stem-like state, while oncogene knockdowns show an opposing trend. Finally, we organize the resource into a coherent workflow to prioritize key elements and variants, in addition to regulators. We showcase the application of this prioritization to somatic burdening, cancer differential expression and GWAS. Targeted validations of the prioritized regulators, elements and variants using siRNA knockdowns, CRISPR-based editing, and luciferase assays demonstrate the value of the ENCODE resource.

genomics↗

bioRxiv2019

Single cell analysis reveals modified hematopoietic cell composition affecting inflammatory and immunopathological responses in Astyanax mexicanus

Reduced parasite infection rates in the developed world are suspected to underlie the rising prevalence of autoimmune disorders. However, the long-term evolutionary consequences of decreased parasite exposure on an immune system are not well understood. We used the Mexican tetra Astyanax mexicanus to understand how loss of parasite diversity influences the evolutionary trajectory of the vertebrate immune system by comparing river with cave morphotypes. Here, we present field data that affirms a strong reduction in parasite diversity in the cave ecosystem and show that cavefish immune cells display a more sensitive proinflammatory response towards bacterial endotoxins. Surprisingly, other innate cellular immune responses, such as phagocytosis, are drastically decreased in cavefish. Using two independent single-cell approaches, we identified a shift in the overall immune cell composition in cavefish as the underlying cellular mechanism, indicating strong differences in the immune investment strategy. While surface fish invest evenly into the innate and adaptive immune system, cavefish shifted immune investment to the adaptive immune system, and here, mainly towards specific T-cell populations that promote homeostasis. Additionally, inflammatory responses and immunopathological phenotypes in visceral adipose tissue are drastically reduced in cavefish. Our data indicate that long term adaptation to low parasite diversity coincides with a more sensitive immune system in cavefish, which is accompanied by a reduction of the immune cells that play a role in mediating the proinflammatory response.

immunology↗

bioRxiv2019

Stable transgenesis in Astyanax mexicanus using the Tol2 transposase system.

Astyanax mexicanus is a well-established and widely used fish model system for evolutionary and developmental biology research. These fish exist as surface forms that inhabit rivers and 30 different populations of cavefish. The establishment of A. mexicanus as an emergent model organism for understanding the evolutionary basis of development and behavior has been accelerated by an increasing availability of genomic approaches to identify genotype-phenotype associations. Despite important progress in the deployment of new technologies, deep mechanistic insights into A. mexicanus evolution and development have been limited by a lack of transgenic lines commonly used in genetic model systems. Here, we expand the toolkit of transgenesis by characterizing two novel stable transgenic lines that were generated using the highly efficient Tol2 system, commonly used to generate transgenic zebrafish. A stable transgenic line consisting of the zebrafish ubiquitin promoter fused to eGFP expressed eGFP ubiquitously throughout development in a surface population of Astyanax. To define specific cell-types, we injected fish with a Cntnap2-mCherry construct that labels lateral line mechanosensory neurons in zebrafish. Strikingly, both constructs appear to label the predicted cell types, suggesting many genetic tools and defined promoter regions in zebrafish are directly transferrable to cavefish. The lines provide proof-of-principle for the application of Tol2 transgenic technology in A. mexicanus. Expansion on these initial transgenic lines will provide a platform to address broadly important problems in the quest to bridge the genotype to phenotype gap.

developmental biology↗

bioRxiv2018

Metastable regimes and tipping points of biochemical networks with potential applications in precision medicine

The concept of attractor of dynamic biochemical networks has been used to explain cell types and cell alterations in health and disease. We have recently proposed an extension of the notion of attractor to take into account metastable regimes, de[fi]ned as long lived dynamical states of the network. These regimes correspond to slow dynamics on low dimensional invariant manifolds of the biochemical networks. Methods based on tropical geometry allow to compute the metastable regimes and represent them as polyhedra in the space of logarithms of the species concentrations. We are looking for sensitive parameters and tipping points of the networks by analyzing how these polyhedra depend on the model parameters. Using the coupled MAPK and PI3K/Akt signaling networks as an example, we test the idea that large changes of the metastable states can be associated to cancer disease speci[fi]c alterations of the network. In particular, we show that for model parameters representing protein concentrations, the protein differential level between tumors of different types is reasonably re[fl]ected in the sensitivity scores, with sensitive parameters corresponding to differential proteins.

systems biology↗