Biology subjects

Lee, H. J.

Publications and source records attributed to Lee, H. J..

11 recordsLinked to original sources

bioRxiv2018

Meteorin-like (Metrnl) adipomyokine improves glucose tolerance in type 2 diabetes via AMPK pathway

Meteorin-like (metrnl) is a recently identified adipomyokine that has beneficial effects on glucose metabolism. However, its underlying mechanism of action is not completely understood. In this study, we have shown that a level of metrnl increase in vitro under electrical-pulse-stimulation (EPS) and in vivo in exercise mice, suggesting that metrnl is an exercise-induced myokine. In addition, metrnl increases glucose uptake through the calcium-dependent AMPK pathway. Metrnl also increases the phosphorylation of HDAC5, a transcriptional repressor of GLUT4, in an AMPK-dependent manner. Phosphorylated HDAC5 interacts with 14-3-3 proteins and sequesters them in the cytoplasm, resulting in the activation of GLUT4 transcription. The intraperitoneal injection of recombinant metrnl improves glucose tolerance in mice with high fat-induced obesity or type 2 diabetes (db/db), but this is not seen in AMPK {beta}1{beta}2 muscle-specific null mice (AMPK {beta}1{beta}2 MKO). In conclusion, we have demonstrated that metrnl induces beneficial effects on glucose metabolism via AMPK and is a promising therapeutic candidate for glucose-related diseases such as type 2 diabetes.

biochemistry↗

bioRxiv2018

Genome-scale oscillations in DNA methylation during exit from pluripotency

Pluripotency is accompanied by the erasure of parental epigenetic memory with naive pluripotent cells exhibiting global DNA hypomethylation both in vitro and in vivo. Exit from pluripotency and priming for differentiation into somatic lineages is associated with genome-wide de novo DNA methylation. We show that during this phase, coexpression of enzymes required for DNA methylation turnover, DNMT3s and TETs, promotes cell-to-cell variability in this epigenetic mark. Using a combination of single-cell sequencing and quantitative biophysical modelling, we show that this variability is associated with coherent, genome-scale, oscillations in DNA methylation with an amplitude dependent on CpG density. Analysis of parallel single-cell transcriptional and epigenetic profiling provides evidence for oscillatory dynamics both in vitro and in vivo. These observations provide fresh insights into the emergence of epigenetic heterogeneity during early embryo development, indicating that dynamic changes in DNA methylation might influence early cell fate decisions.\n\nHighlightsO_LICo-expression of DNMT3s and TETs drive genome-scale oscillations of DNA methylation\nC_LIO_LIOscillation amplitude is greatest at a CpG density characteristic of enhancers\nC_LIO_LICell synchronisation reveals oscillation period and link with primary transcripts\nC_LIO_LIMultiomic single-cell profiling provides evidence for oscillatory dynamics in vivo\nC_LI

genomics↗

bioRxiv2018

Connecting the dots between mechanosensitive channel abundance, osmotic shock, and survival at single-cell resolution

Rapid changes in extracellular osmolarity are one of many insults microbial cells face on a daily basis. To protect against such shocks, Escherichia coli and other microbes express several types of transmembrane channels which open and close in response to changes in membrane tension. In E. coli, one of the most abundant channels is the mechanosensitive channel of large conductance (MscL). While this channel has been heavily characterized through structural methods, electrophysiology, and theoretical modeling, our understanding of its physiological role in preventing cell death by alleviating high membrane tension remains tenuous. In this work, we examine the contribution of MscL alone to cell survival after osmotic shock at single cell resolution using quantitative fluorescence microscopy. We conduct these experiments in an E. coli strain which is lacking all mechanosensitive channel genes save for MscL whose expression is tuned across three orders of magnitude through modifications of the Shine-Dalgarno sequence. While theoretical models suggest that only a few MscL channels would be needed to alleviate even large changes in osmotic pressure, we find that between 500 and 700 channels per cell are needed to convey upwards of 80% survival. This number agrees with the average MscL copy number measured in wild-type E. coli cells through proteomic studies and quantitative Western blotting. Furthermore, we observe zero survival events in cells with less than 100 channels per cell. This work opens new questions concerning the contribution of other mechanosensitive channels to survival as well as regulation of their activity.

biophysics↗

bioRxiv2018

Estimating parasite load dynamics to reveal novel resistance mechanisms to human malaria

Improved methods are needed to identify host mechanisms which directly protect against human infectious diseases in order to develop better vaccines and therapeutics1,2. Pathogen load determines the outcome of many infections3, and is a consequence of pathogen multiplication rate, duration of the infection, and inhibition or killing of pathogen by the host (resistance). If these determinants of pathogen load could be quantified then their mechanistic correlates might be determined. In humans the timing of infection is rarely known and treatment cannot usually be withheld to monitor serial changes in pathogen load and host response. Here we present an approach to overcome this and identify potential mechanisms of resistance which control parasite load in Plasmodium falciparum malaria. Using a mathematical model of longitudinal infection dynamics for orientation, we made individualized estimates of parasite multiplication and growth inhibition in Gambian children at presentation with acute malaria and used whole blood RNA-sequencing to identify their correlates. We identified novel roles for secreted proteases cathepsin G and matrix metallopeptidase 9 (MMP9) as direct effector molecules which inhibit P. falciparum growth. Cathepsin G acts on the erythrocyte membrane, cleaving surface receptors required for parasite invasion, whilst MMP9 acts on the parasite. In contrast, the type 1 interferon response and expression of CXCL10 (IFN-{gamma}-inducible protein of 10 kDa, IP-10) were detrimental to control of parasite growth. Natural variation in iron status and plasma levels of complement factor H were determinants of parasite multiplication rate. Our findings demonstrate the importance of accounting for the dynamic interaction between host and pathogen when seeking to identify correlates of protection, and reveal novel mechanisms controlling parasite growth in humans. This approach could be extended to identify additional mechanistic correlates of natural- and vaccine-induced immunity to malaria and other infections.

systems biology↗

bioRxiv2018

Glucocorticoid-mediated Aβ and SCG10 upregulation evoke microtubule dysfunction and memory deficits

We investigated glucocorticoid, a major risk factor of Alzheimers disease, promoted microtubule instability that culminates in memory deficits. Mice group exposed to corticosteroid had reduced trafficking of AMPAR1/2 and mitochondria into the synapse due to microtubule destabilization, which finally impaired cognitive function. Furthermore, cortisol reduced microtubule stability through the mitochondria glucocorticoid receptor (GR)-dependent pathway in SH-SY5Y cells. Cortisol translocated the Hsp70-bound GR into mitochondria before stimulating ER-mitochondria interaction via increasing GR-Bcl-2 complex. Subsequently, A{beta} was produced since {gamma}-secretase activity was upregulated by increased ER-mitochondria connectivity. Mitochondrial Ca2+ influx was also elevated due to ER-mitochondria bridging, resulting in activation of mTOR pathway. Subsequent autophagy inhibition failed to remove A{beta} and led to its accumulation. Moreover, selective autophagy through ubiquitination of SCG10 was suppressed. We eventually showed that both elevated A{beta} and SCG10 levels drive cells to fail trafficking AMPAR1/2 and mitochondria into the cell terminus. In conclusion, glucocorticoid regulates ER-mitochondria coupling, which evokes A{beta} generation and SCG10 upregulation. Subsequent microtubule destabilization leads to memory impairment through failure of AMPAR1/2 or mitochondria transport into cell periphery.

neuroscience↗

bioRxiv2018

An Integrin-Independent Kaposi’s Sarcoma-Associated Herpesvirus Infection Mechanism

Host receptor usage by KSHV has been best studied using primary microvascular endothelial and fibroblast cells, although the virus infects a wide variety of cell types in culture and in natural infections. In these two infection models, KSHV adheres to the cell though heparan sulfate (HS) binding, then interacts with a complex of EphA2, xct, and integrins 3{beta}1, V{beta}3, V{beta}5 to catalyze viral entry. We dissected this receptor complex at the genetic level with CRISPR-Cas9 to precisely determine receptor usage in two epithelial cell lines. Surprisingly, we discovered an infection mechanism that requires HS and EphA2 but is independent of V- and {beta}1-family integrin expression. Furthermore, infection appears to be independent of the EphA2 intracellular domain. We also demonstrated while two other endogenous Eph receptors were dispensable for KSHV infection, transduced EphA4 and EphA5 significantly enhanced infection of cells lacking EphA2.\n\nIMPORTANCEOur data reveals an integrin-independent route of KSHV infection and suggests that multiple Eph receptors besides EphA2 can promote and regulate infection. Since integrins and Eph receptors are large protein families with diverse expression patterns across cells and tissues, we propose that KSHV may engage with several proteins from both families in different combinations to negotiate successful entry into diverse cell types.

microbiology↗

bioRxiv2017

Integrated pathogen load and dual transcriptome analysis of systemic host-pathogen interactions in severe malaria

The pathogenesis of severe Plasmodium falciparum malaria is incompletely understood. Since the pathogenic stage of the parasite is restricted to blood, dual RNA-sequencing of host and parasite transcripts in blood can reveal their interactions at a systemic scale. Here we identify human and parasite gene expression associated with severe disease features in Gambian children. Differences in parasite load explained up to 99% of differential expression of human genes but only a third of the differential expression of parasite genes. Co-expression analyses showed a remarkable co-regulation of host and parasite genes controlling translation, and host granulopoiesis genes uniquely co-regulated and differentially expressed in severe malaria. Our results indicate that high parasite load is the proximal stimulus for severe P. falciparum malaria, that there is an unappreciated role for many parasite genes in determining virulence, and hint at a molecular arms-race between host and parasite to synthesise protein products.

systems biology↗

bioRxiv2017

Massive Mining of Publicly Available RNA-seq Data from Human and Mouse

RNA-sequencing (RNA-seq) is currently the leading technology for genome-wide transcript quantification. While the volume of RNA-seq data is rapidly increasing, the currently publicly available RNA-seq data is provided mostly in raw form, with small portions processed non- uniformly. This is mainly because the computational demand, particularly for the alignment step, is a significant barrier for global and integrative retrospective analyses. To address this challenge, we developed all RNA-seq and ChIP-seq sample and signature search (ARCHS4), a web resource that makes the majority of previously published RNA-seq data from human and mouse freely available at the gene count level. Such uniformly processed data enables easy integration for downstream analyses. For developing the ARCHS4 resource, all available FASTQ files from RNA-seq experiments were retrieved from the Gene Expression Omnibus (GEO) and aligned using a cloud-based infrastructure. In total 137,792 samples are accessible through ARCHS4 with 72,363 mouse and 65,429 human samples. Through efficient use of cloud resources and dockerized deployment of the sequencing pipeline, the alignment cost per sample is reduced to less than one cent. ARCHS4 is updated automatically by adding newly published samples to the database as they become available. Additionally, the ARCHS4 web interface provides intuitive exploration of the processed data through querying tools, interactive visualization, and gene landing pages that provide average expression across cell lines and tissues, top co-expressed genes, and predicted biological functions and protein-protein interactions for each gene based on prior knowledge combined with co-expression. Benchmarking the quality of these predictions, co-expression correlation data created from ARCHS4 outperforms co-expression data created from other major gene expression data repositories such as GTEx and CCLE.\n\nARCHS4 is freely accessible at: http://amp.pharm.mssm.edu/archs4

bioinformatics↗

bioRxiv2017

Joint Profiling Of Chromatin Accessibility, DNA Methylation And Transcription In Single Cells

Parallel single-cell sequencing protocols represent powerful methods for investigating regulatory relationships, including epigenome-transcriptome interactions. Here, we report a novel single-cell method for parallel chromatin accessibility, DNA methylation and transcriptome profiling. scNMT-seq (single-cell nucleosome, methylation and transcription sequencing) uses a GpC methyltransferase to label open chromatin followed by bisulfite and RNA sequencing. We validate scNMT-seq by applying it to differentiating mouse embryonic stem cells, finding links between all three molecular layers and revealing dynamic coupling between epigenomic layers during differentiation.

genomics↗

bioRxiv2016

Parkin knockout inhibits neuronal development via regulation of proteasomal degradation of p21

PARK2 encodes for the E3 ubiquitin ligase parkin and iimplicates in the development of Parkinsons disease (PD). Although the neuroprotective role of parkin is well known, the mechanism of parkins function in neural stem differentiation is not clear. Co-expressions network analysis showed that SNAP25 and BDNF were positively correlated with parkin, but negatively correlated with p21 in human patient brain. Therefore, we investigated a link between the ubiquitin E3 ligase parkin and proteasomal degradation of p21 for the control of neural stem cell differentiation. We discovered that p21 directly binds with parkin and is ubiquitinated by parkin resulting in the loss of cell differentiation ability. Tranfection of p21 shRNA in PARK2 KO mice significantly rescued the differentiation efficacy as well as SNAP25 and BDNF expression. We also defined the decreased p21 ubiquitination and differentiation ability were reversed after treatment with JNK inhibitor, SP600125 in PARK2 KO mice derived neural stem cells. Thus, the present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.\n\nSummary statementThe present study indicated that parkin knockout inhibits neural stem cell differentiation by JNK-dependent proteasomal degradation of p21.

cell biology↗

bioRxiv2016

Tracking the embryonic stem cell transition from ground state pluripotency

Mouse embryonic stem (ES) cells are locked into self-renewal by shielding from inductive cues. Release from this ground state in minimal conditions offers a system for delineating developmental progression from naive pluripotency. Here we examined the initial transition of ES cells. The population behaves asynchronously. We therefore exploited a short-half-life Rex1::GFP reporter to isolate cells either side of exit from naive status. Extinction of ES cell identity in single cells is acute. It occurs only after near-complete elimination of naive pluripotency factors, but precedes appearance of lineage specification markers. Cells newly departed from the ES cell state exhibit global transcriptome features consistent with features of early post-implantation epiblast and distinct from primed epiblast. They also exhibit a genome-wide increase in DNA methylation, intermediate between early and late epiblast. These findings are consistent with the proposition that naive cells transition to a discrete formative phase of pluripotency preparatory to lineage priming.\n\nHighlightsO_LIThe Rex1 destabilized GFP reporter demarcates naive pluripotency.\nC_LIO_LIExit from the naive state is asynchronous in the population.\nC_LIO_LITransition is relatively acute in individual cells and precedes lineage priming.\nC_LIO_LITranscriptome and DNA methylome reflect events in the pre-gastrulation embryo.\nC_LI

developmental biology↗