Biology subjects

Kang, Y.

Publications and source records attributed to Kang, Y..

At least 91 records · Page 5Linked to original sources

bioRxiv2021

Targeting glucose metabolism sensitizes pancreatic cancer to MEK inhibition

Pancreatic ductal adenocarcinoma (PDAC) is almost universally lethal. A critical unmet need exists to explore essential susceptibilities in PDAC and identify druggable targets for tumor maintenance. This is especially challenging in the context of PDAC, in which activating mutations of KRAS oncogene (KRAS*) dominate the genetic landscape. By using an inducible KrasG12D-driven p53 deficient PDAC mouse model (iKras model), we demonstrate that RAF-MEK-MAPK signaling is the major effector for oncogenic Kras-mediated tumor maintenance. However, MEK inhibition has minimal therapeutic effect as single agent for PDAC both in vitro and in vivo. Although MEK inhibition partially downregulates the transcription of glycolysis genes, it surprisingly fails to suppress the glycolysis flux in PDAC cell, which is a major metabolism effector of oncogenic KRAS. Accordingly, In vivo genetic screen identified multiple glycolysis genes as potential targets that may sensitize tumor cells to MAPK inhibition. Furthermore, inhibition of glucose metabolism with low dose 2-deoxyglucose (2DG) in combination with MEK inhibitor dramatically induces apoptosis in KrasG12D-driven PDAC cell in vitro, inhibits xenograft tumor growth and prolongs the overall survival of genetically engineered mouse PDAC model. Molecular and metabolism analyses indicate that co-targeting glycolysis and MAPK signaling results in apoptosis via induction of lethal ER stress. Together, our work suggests that combinatory inhibition of glycolysis and MAPK pathway may serve as an alternative approach to target KRAS-driven PDAC.

cancer biology↗

bioRxiv2021

ConVarT: a search tool for orthologous variants: A method and server for functional inference of human genetic variants

The availability of genetic variants, together with phenotypic annotations from model organisms, facilitates comparing these variants with equivalent variants in humans. However, existing databases and search tools do not make it easy to scan for equivalent variants, namely "matching variants" (MatchVars) between humans and other organisms. Therefore, we developed an integrated search engine called ConVarT (http://www.convart.org/) for matching variants between humans, mice, and C. elegans. ConVarT incorporates annotations (including phenotypic and pathogenic) into variants, and these previously unexploited phenotypic MatchVars from mice and C. elegans can give clues about the functional consequence of human genetic variants. Our analysis shows that many phenotypic variants in different genes from mice and C. elegans, so far, have no counterparts in humans, and thus, can be useful resources when evaluating a relationship between a new human mutation and a disease.

genetics↗

bioRxiv2021

Histone H3K4me1 and H3K27ac play roles in nucleosome depletion and eRNA transcription, respectively, at enhancers

Histone H3K4me1 and H3K27ac are enhancer specific modifications and are required for enhancers to activate transcription of target genes. However the reciprocal effects of these histone modifications on each other and their roles in enhancers are not clear. Here to comparatively analyze the role of these modifications, we inhibited H3K4me1 and H3K27ac by deleting SET domain of histone methyltransferases MLL3 and MLL4 and HAT domain of histone acetyltransferase p300, respectively, in erythroid K562 cells. The loss of H3K4me1 reduced H3K27ac at the {beta}-globin enhancer LCR HSs, but H3K27ac reduction did not affect H3K4me1. This unequal relationship between two modifications was revealed in putative enhancers by genome-wide analysis using ChIP-seq. Histone H3 depletion at putative enhancers was weakened by the loss of H3K4me1 but not by the loss of H3K27ac. Chromatin remodeling complexes were recruited into the {beta}-globin LCR HSs in a H3K4me1-dependent manner. In contrast, H3K27ac was required for enhancer RNA (eRNA) transcription, and H3K4me1 was not enough for it. Forced H3K27ac induced eRNA transcription without affecting H3K4me1 at the {beta}-globin LCR HSs. These results indicate that H3K4me1 and H3K27ac affect each other in different ways and play more direct roles in nucleosome depletion and eRNA transcription, respectively, at enhancers.

molecular biology↗

bioRxiv2020

Predicting which genes will respond to perturbations of a TF: TF-independent properties of genes are major determinants of their responsiveness

BackgroundThe ability to predict which genes will respond to perturbation of a TFs activity serves as a benchmark for our systems-level understanding of transcriptional regulatory networks. In previous work, machine learning models have been trained to predict static gene expression levels in a given sample by using data from the same or similar conditions, including data on TF binding locations, histone marks, or DNA sequence. We report on a different challenge - training machine learning models that can predict which genes will respond to perturbation of a TF without using any data from the perturbed cells. ResultsExisting TF location data (ChIP-Seq) from human K562 cells have no detectable utility for predicting which genes will respond to perturbation of the TF, but data obtained by newer methods in yeast cells are useful. TF-independent features of genes, including their pre-perturbation expression level and expression variation, are very useful for predicting responses to TF perturbations. This shows that some genes are poised to respond to TF perturbations and others are resistant, shedding significant light on why it has been so difficult to predict responses from binding locations. Certain histone marks (HMs), including H3K4me1 and H3K4me3, have some predictive power, especially when downstream of the transcription start site. In human, the predictive power of HMs is much less than that of gene expression level and variation. Code is available at https://github.com/yiming-kang/TFPertRespExplainer. ConclusionsSequence-based or epigenetic properties of genes strongly influence their tendency to respond to direct TF perturbations, partially explaining the oft-noted difficulty of predicting responsiveness from TF binding location data. These molecular features are largely reflected in and summarized by the genes expression level and expression variation.

systems biology↗

bioRxiv2020

Evaluating the reproducibility of single-cell gene regulatory network inference algorithms

Networks are powerful tools to represent and investigate biological systems. The development of algorithms inferring regulatory interactions from functional genomics data has been an active area of research. With the advent of single-cell RNA-seq data (scRNA-seq), numerous methods specifically designed to take advantage of single-cell datasets have been proposed. However, published benchmarks on single-cell network inference are mostly based on simulated data. Once applied to real data, these benchmarks take into account only a small set of genes and only compare the inferred networks with an imposed ground-truth. Here, we benchmark four single-cell network inference methods based on their reproducibility, i.e. their ability to infer similar networks when applied to two independent datasets for the same biological condition. We tested each of these methods on real data from three biological conditions: human retina, T-cells in colorectal cancer, and human hematopoiesis. GENIE3 results to be the most reproducible algorithm, independently from the single-cell sequencing platform, the cell type annotation system, the number of cells constituting the dataset, or the thresholding applied to the links of the inferred networks. In order to ensure the reproducibility and ease extensions of this benchmark study, we implemented all the analyses in scNET, a Jupyter notebook available at https://github.com/ComputationalSystemsBiology/scNET.

systems biology↗

bioRxiv2020

The effect of Basal core promoter and pre-core mutations on HBV replication and persistence in mice

The appearance of the BCP or Pre-C mutations, which reduce or abolish HBeAg production, could increase HBV replication. The remove of the HBeAg often lead to a vigorous immune response, which has an important role in HBV related fulminant outcome. In this study, BCP mutations and Pre-C mutations were separately introduced by site-directed mutagenesis in the same genetic background of an HBV infectious clone, to determine the effect of these mutations per se on replication. BCP and Pre-C mutations increased HBV replication both in vitro and in vivo. HBV could persist in mice injected with wild type HBV infectious clone for about 7 weeks. However, HBV could persist about 5 weeks in mice injected with BCP HBV infectious clone, and 3 weeks only in mice injected with Pre-C HBV infectious clone. HBV related CD8+ CTL response in BCP HBV infectious clone injected mice only slightly increased, but significantly increased in Pre-C HBV infectious clone injected mice compared with that in wild type HBV infectious clone injected mice. The population of Tregs significantly increased in liver but not in spleen of mice injected with Pre-C HBV infectious clone. In summary, we demonstrate that HBeAg plays an important role in suppressing the CTL response, which is related with increasing the frequency of Tregs in mouse. Lack of HBeAg expression leads to the partial loss of immune tolerance.

microbiology↗

bioRxiv2020

Mutating PINK1 gene by paired truncated sgRNA/Cas9-D10A in Cynomolgus Monkey

Mutations of PINK1 cause early-onset Parkinsons disease (PD) with selective neurodegeneration in humans. However,current PINK1 knockout mouse and pig models are unable to recapitulate the typical neurodegenerative phenotypes observed in PD patients, indicating that it is essential to generate PINK1 disease models in non-human primates (NHPs) that are highly close to humans, to investigate the unique function of PINK1 in primate brains. Paired single guide RNA (sgRNA)/Cas9-D10A nickases and truncated sgRNA/Cas9, both enabling the reduction of the off-target effect without apparently compromising the on-target editing, are two optimized strategies in applying the CRISPR/Cas9 system to establish disease animal models. Here, we combined the two strategies and injected Cas9-D10A mRNA and two truncated sgRNAs into one-cell-stage cynomolgus zygotes to target PINK1 gene. We show precise and efficient gene editing of the target site in the three new born cynomolgus monkeys. The frame shift mutations of PINK1 in mutant fibroblasts leaded to the reduction of the mRNA, However, western-blot and immunofluorescencestaining confirmed the PINK1 proteinlevels werecomparable to that in the wild-type fibroblasts. We further reprogramed mutant fibroblast into induced pluripotent stem cells (iPSCs) and they show similar ability of differentiation into dopamine (DA) neurons. Taken together, our results showed that coinjection of Cas9-D10A nickase mRNA and sgRNA into one-cell-stage cynomolgus embryos enable the generation of human disease models in NHPs and the target editing by paired truncated sgRNA/Cas9-D10A in PINK1 gene exon 2 did not impact the protein expression.

genetics↗

bioRxiv2020

Human cerebrospinal fluids have no microbiome but can contain potential pathogens

Cerebrospinal fluids circulating human central nervous system have long been considered aseptic in healthy individuals, because normally the blood-brain barrier protects against microbial invasions. However, this dogma has been questioned by several reports that microbes were identified in human brains, raising the question whether a microbial community is present in cerebrospinal fluids of healthy individuals without neurological diseases. Here, we collected and analyzed metagenomic and metatranscriptomic sequencing data of cerebrospinal fluid specimens from a cohort of 23 pregnant women aged between 23 and 40 and one-to-one matched contamination controls. From data analysis of 116 specimens of eight different types, we detected 619 nonredundant microbial taxa which were dominated by bacteria (75%) and viruses (24%). In cerebrospinal fluids metagenomic samples, a total of 76 redundant species were detected including four (one nonredundant) eukaryota taxa, eleven (four nonredundant) bacteria, and 61 (21 nonredundant) viruses that were mostly bacteriophages. Metagenomic data analysis found no significant difference between cerebrospinal fluid specimens and negative controls in terms of microbial species diversity. In addition, no active or viable microbiome were present in the cerebrospinal fluid samples after subtracting microbes detected in contamination controls. In conclusion, we found no strong evidence that colonized microbial community exist in the cerebrospinal fluids of healthy individuals. IMPORTANCEMicrobiome are prevalent throughout human bodies with profound health implications. However, it remains unclear whether a microbiome is present and active in human cerebrospinal fluids that are long considered aseptic given the blood-brain barrier. Here, we applied unbiased metagenomic and metatranscriptomic sequencing to detect microbiome in cerebrospinal fluids collected from a cohort of 23 pregnant women with matched controls. By analyzing 116 specimens of eight types, no strong evidence was found to support a presence of colonized microbiome in the cerebrospinal fluids. Our findings have profound implications to human immunity against neurological infections and disorders, providing a guide for disease diagnostics, prevention and therapeutics in clinical settings.

microbiology↗

bioRxiv2020

DDR1-INDUCED NEUTROPHIL EXTRACELLULAR TRAPS DRIVE PANCREATIC CANCER METASTASIS

Pancreatic ductal adenocarcinoma (PDAC) tumors are characterized by a desmoplastic reaction and dense collagen that is known to promote cancer progression. A central mediator of pro-tumorigenic collagen signaling is the receptor tyrosine kinase discoid domain receptor 1 (DDR1). DDR1 is a critical driver of a mesenchymal and invasive cancer cell PDAC phenotype. Previous studies have demonstrated that genetic or pharmacologic inhibition of DDR1 prevents PDAC tumorigenesis and metastasis. Here, we investigated whether DDR1 signaling has cancer cell non-autonomous effects that promote PDAC progression and metastasis. We demonstrate that collagen-induced DDR1 activation in cancer cells is a major stimulus for CXCL5 production, resulting in the recruitment of tumor-associated neutrophils (TANs), the formation of neutrophil extracellular traps (NETs) and subsequent cancer cell invasion and metastasis. Moreover, we have identified that collagen-induced CXCL5 production was mediated by a DDR1-PKC{theta}-SYK-NF{kappa}B signaling cascade. Together, these results highlight the critical contribution of collagen I-DDR1 interaction in the formation of an immune microenvironment that promotes PDAC metastasis. SummaryDeng et al find that collagen signaling via DDR1 on human pancreatic cancer cells drives production and release of the cytokine, CXCL5, into systemic circulation. CXCL5 then triggers infiltration of neutrophils into the tumor where they promote cancer cell progression.

cancer biology↗

bioRxiv2020

Structure of voltage-modulated sodium-selective NALCN-FAM155A channel complex

Resting membrane potential determines the excitability of the cell and is essential for the cellular electrical activities. NALCN channel mediates sodium leak currents, which positively tune the resting membrane potential towards depolarization. NALCN channel is involved in many important neurological processes and is implicated in a spectrum of neurodevelopmental diseases. Despite its functional importance, the mechanisms of ion permeation and voltage-modulation for NALCN channel remain elusive. Here, we report the cryo-EM structure of rat NALCN and mouse FAM155A complex to 2.7 [A] resolution. The structure reveals detailed interactions between NALCN and extracellular cysteine-rich domain of FAM155A. The non-canonical architecture of NALCN selectivity filter dictates its sodium selectivity and calcium block. The asymmetric arrangement of two functional voltage-sensors confers the modulation by membrane potential. Moreover, mutations found in human diseases were mapped to the domain-domain interfaces or the pore domain of NALCN, intuitively suggesting their pathological mechanisms.

biophysics↗

bioRxiv2020

Essential amnion signals for primate primitive streak formation resolved by scRNA map

Essential genes for murine embryonic development can demonstrate a disparate phenotype in human cohorts. By generating a transcriptional atlas containing >30,000 cells from postimplantation non-human primate embryos, we discovered that ISL1, a gene with a well-established role in cardiogenesis, controls a gene regulatory network in primate amnion. CRISPR/Cas9-targeting of ISL1 resulted in non-human primate embryos which did not yield viable offspring, demonstrating that ISL1 is critically required in primate embryogenesis. On a cellular level, mutant ISL1 embryos displayed a failure in mesoderm formation due to reduced BMP4 signaling from the amnion. Via loss of function and rescue studies in human embryonic stem cells we confirmed a similar role of ISL1 in human in vitro derived amnion. This study highlights the importance of the amnion as a signaling center during primate mesoderm formation and demonstrates the potential of in vitro primate model systems to dissect the genetics of early human embryonic development.

developmental biology↗

bioRxiv2020

GATA-1-dependent histone H3K27ac mediates erythroid cell-specific interaction between CTCF sites

CTCF sites interact with each other in the chromatin environment, establishing domains. Our previous study showed that interaction between CTCF sites is cell type-specific around the {beta}-globin locus and is dependent on erythroid specific activator GATA-1. To find out molecular mechanisms of the cell type-specific interaction, we directly inhibited GATA-1 binding to the {beta}-globin enhancers by deleting its binding motifs and found that histone H3K27 acetylation (H3K27ac) was decreased at CTCF sites surrounding the {beta}-globin locus, even though CTCF binding itself was maintained at the sites. Forced H3K27ac by TSA treatment or CBP/p300 KD affected the interactions between CTCF sites around the {beta}-globin locus. Analysis of public ChIA-PET data revealed that H3K27ac is higher at CTCF sites forming short chromatin interactions than long interactions. The short interactions contain erythropoiesis-associated genes and GATA-1 binding sites in erythroid K562 cells. Depletion of GATA-1 reduced H3K27ac at CTCF sites near erythroid enhancers. These results indicate that GATA-1-dependent histone H3K27ac at neighboring CTCF sites mediates erythroid specific chromatin interaction between them.

molecular biology↗

bioRxiv2020

Structural basis for human TRPC5 channel inhibition by two distinct inhibitors

TRPC5 channel is a non-selective cation channel that participates diverse physiological processes. Human TRPC5 inhibitors show promise in the treatment of anxiety disorder, depression and kidney disease. Despite the high relevance of TRPC5 to human health, its inhibitor binding pockets have not been fully characterized due to the lack of structural information, which greatly hinders structure-based drug discovery. Here we show cryo-EM structures of human TRPC5 in complex with two distinct inhibitors, namely clemizole and HC-070, to the resolution of 2.7 [A]. Based on the high-quality cryo-EM maps, we uncover the different binding pockets and detailed binding modes for these two inhibitors. Clemizole binds inside the voltage sensor-like domain of each subunit, while HC-070 binds close to the ion channel pore and is wedged between adjacent subunits. Both of them exert the inhibitory function by stabilizing the ion channel in a closed state. These structures provide templates for further design and optimization of inhibitors targeting human TRPC5.

biophysics↗

bioRxiv2020

Comprehensive Chromosome End Remodeling During Programmed DNA Elimination

Germline and somatic genomes are typically the same in a multicellular organism. However, in some organisms including the parasitic nematode Ascaris, programmed DNA elimination leads to a reduced somatic genome compared to germline cells. Previous work on the parasitic nematode Ascaris demonstrated that programmed DNA elimination encompasses high fidelity chromosomal breaks at specific genome locations and loss of specific genome sequences including a major tandem repeat of 120 bp and ~1,000 germline-expressed genes. However, the precise chromosomal locations of the 120 bp repeats, the breaks regions, and the eliminated genes remained unknown. Here, we used PacBio long-read sequencing and chromosome conformation capture (Hi-C) to obtain fully assembled chromosomes of Ascaris germline and somatic genomes, enabling a complete chromosomal view of DNA elimination. Surprisingly, we found that all 24 germline chromosomes undergo comprehensive chromosome end remodeling with DNA breaks in their subtelomeric regions and loss of distal sequences including the telomeres at both chromosome ends. All new Ascaris somatic chromosome ends are recapped by de novo telomere healing. We provide an ultrastructural analysis of DNA elimination and show that Ascaris eliminated DNA is incorporated into many double membrane-bound structures, similar to micronuclei, during telophase of a DNA elimination mitosis. These micronuclei undergo dynamic changes including loss of active histone marks and localize to the cytoplasm following daughter nuclei formation and cytokinesis where they form autophagosomes. Comparative analysis of nematode chromosomes suggests that germline chromosome fusions occurred forming Ascaris sex chromosomes that become independent chromosomes following DNA elimination breaks in somatic cells. These studies provide the first chromosomal view and define novel features and functions of metazoan programmed DNA elimination.

genomics↗

bioRxiv2020

A quasi-paired cohort strategy reveals the impaired detoxifying function of microbes in the gut of autistic children

Growing evidence suggests that autism spectrum disorder (ASD) is highly associated with dysbiosis in the gut microbiome. However, results of metagenome-based microbiome studies are not always consistent due to great individual diversity that overwhelms disease-associated alterations. Here, we proposed a novel analysis strategy--quasi-paired cohort and applied it to a metagenomic study of ASD microbiomes. By comparing the paired samples of ASD and neurotypical subjects, we identified significant deficiencies in ASD children in detoxifying enzymes and pathways, which showed strong correlations to mitochondrial damage. Diagnostic models with these detoxifying enzymes accurately discriminated ASD individuals from controls, and the dysfunction score inferred from the model increased with the clinical rating scores of ASD. Conclusively, our findings suggest a previously undiscovered mechanism in which impaired microbial detoxification leads to toxicant accumulation and mitochondrion damage contributes to the pathogenesis of ASD. This novel mechanism points to future therapeutic strategies of rebuilding microbial detoxification for ASD.

microbiology↗

bioRxiv2020

The TIM22 complex regulates mitochondrial one-carbon metabolism by mediating the import of Sideroflexins

The Acylglycerol Kinase (AGK) is a mitochondrial lipid kinase that contributes to protein biogenesis as a subunit of the TIM22 complex at the inner mitochondrial membrane. Mutations in AGK cause Sengers syndrome, an autosomal recessive condition characterized by congenital cataracts, hypertrophic cardiomyopathy, skeletal myopathy and lactic acidosis. We undertook proteomic profiling of Sengers patient fibroblasts and an AGKKO cell line to map the proteomic changes that ensue upon AGK dysfunction. This uncovered extensive remodelling of mitochondrial one-carbon metabolism enzymes and showed that inner membrane serine transporters, Sideroflexins (SFXNs), are novel substrates of the TIM22 complex. Deletion of SFXN1 recapitulates the remodelling of one-carbon metabolism observed in Sengers patient cells. Proliferation of cells lacking AGK is perturbed in the absence of exogenous serine and rescuable through addition of formate, highlighting the dysregulation of one carbon metabolism as a key molecular feature in the biology of Sengers syndrome.

cell biology↗

bioRxiv2020

Inhibition mechanism of human sterol O-acyltransferase 1 by competitive inhibitor

Sterol O-acyltransferase 1 (SOAT1) is an endoplasmic reticulum (ER) resident, multi-transmembrane enzyme that belongs to the membrane-bound O-acyltransferase (MBOAT) family 1. It catalyzes the esterification of cholesterol to generate cholesteryl esters for cholesterol storage 2. SOAT1 is a target to treat several human diseases 3. However, its structure and mechanism remain elusive since its discovery. Here, we report the structure of human SOAT1 (hSOAT1) determined by cryo-EM. hSOAT1 is a tetramer consisted of a dimer of dimer. The structure of hSOAT1 dimer at 3.5 [A] resolution reveals that the small molecule inhibitor CI-976 binds inside the catalytic chamber and blocks the accessibility of the active site residues H460, N421 and W420. Our results pave the way for future mechanistic study and rational drug design of SOAT1 and other mammalian MBOAT family members.

biochemistry↗

bioRxiv2019

Surface association induces cytotoxic alkyl-quinolones in Pseudomonas aeruginosa

Surface attachment, an early step in the colonization of multiple host environments, activates the virulence of the human pathogen P. aeruginosa. However, the signaling pathways and downstream toxins specifically induced by surface association to stimulate P. aeruginosa virulence are not fully understood. Here, we demonstrate that alkyl-quinolone (AQ) secondary metabolites are rapidly induced upon surface association and represent a major class of surface-dependent cytotoxins. AQ cytotoxicity is direct and independent of other AQ functions like quorum sensing or PQS-specific activities like iron sequestration. Furthermore, the regulation of AQ production can explain the surface-dependent virulence regulation of the quorum sensing receptor, LasR, and the pilin-associated candidate mechanosensor, PilY1. PilY1 regulates surface-induced AQ production by repressing the AlgR-AlgZ two-component system. AQs also contribute to the known cytotoxicity of secreted outer membrane vesicles. These findings collectively explain previously mysterious aspects of virulence regulation and provide new avenues for the development of anti-infectives.

microbiology↗