Biology subjects

Kim, A.

Publications and source records attributed to Kim, A..

90 records · Page 5Linked to original sources

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

Spiny mice (Acomys) exhibit attenuated hallmarks of aging and rapid cell turnover after UV exposure in the skin epidermis

The study of long-lived and regenerative animal models has revealed diverse protective responses to stressors such as aging and tissue injury. Spiny mice (Acomys) are a unique mammalian model of skin regeneration, but their response to other types of physiological skin damage have not been investigated. In this study, we examine how spiny mice skin responds to acute UVB damage or chronological aging compared to non-regenerative C57Bl/6 mice (M. musculus). We find that, compared to M. musculus, the skin epidermis in A. cahirinus experiences a similar UVB-induced increase in basal cell proliferation but exhibits increased epidermal turnover. Notably, A. cahirinus uniquely form a suprabasal layer co-expressing Keratin 14 and Keratin 10 after UVB exposure concomitant with reduced epidermal inflammatory signaling and reduced markers of DNA damage. In the context of aging, old M. musculus animals exhibit typical hallmarks including epidermal thinning, increased inflammatory signaling and senescence. However, these age-related changes are absent in old A. cahirinus skin. Overall, we find that A. cahirinus have evolved novel responses to skin damage that reveals new aspects of its regenerative phenotype.

cell biology↗

bioRxiv2020

Genoppi: an open-source software for robust and standardized integration of proteomic and genetic data

Combining genetic and cell-type-specific proteomic datasets can lead to new biological insights and therapeutic hypotheses, but a technical and statistical framework for such analyses is lacking. Here, we present an open-source computational tool called Genoppi that enables robust, standardized, and intuitive integration of quantitative proteomic results with genetic data. We used Genoppi to analyze sixteen cell-type-specific protein interaction datasets of four proteins (TDP-43, MDM2, PTEN, and BCL2) involved in cancer and neurological disease. Through systematic quality control of the data and integration with published protein interactions, we show a general pattern of both cell-type-independent and cell-type-specific interactions across three cancer and one human iPSC-derived neuronal type. Furthermore, through the integration of proteomic and genetic datasets in Genoppi, our results suggest that the neuron-specific interactions of these proteins are mediating their genetic involvement in neurodevelopmental and neurodegenerative diseases. Importantly, our analyses indicate that human iPSC-derived neurons are a relevant model system for studying the involvement of TDP-43 and BCL2 in amyotrophic lateral sclerosis.

genetics↗

bioRxiv2020

Clusters of bacterial RNA polymerase are biomolecular condensates that assemble through liquid-liquid phase separation

Once described as mere "bags of enzymes", bacterial cells are in fact highly organized, with many macromolecules exhibiting non-uniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in E. coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and suggests that this process serves as a universal mechanism for intracellular organization across the tree of life. SignificanceBacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid-liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquid-liquid phase separation is a universal mechanism for intracellular organization that extends across the tree of life.

cell biology↗

bioRxiv2020

Genomic and transcriptomic analyses reveal a tandem amplification unit of 11 genes and mutations of mismatch repair genes in methotrexate-resistant HT-29 cells

DHFR gene amplification is present in methotrexate (MTX)-resistant colon cancer cells and acute lymphoblastic leukemia. However, little is known about DHFR gene amplification due to difficulties in quantifying amplification size and recognizing the repetitive rearrangements involved in the process. In this study, we have proposed an integrative framework to characterize the amplified region by using a combination of single-molecule real time sequencing, next-generation optical mapping, and chromosome conformation capture (Hi-C). Amplification of the DHFR gene was optimized to generate homogenously amplified patterns. The amplification units of 11 genes, from the DHFR gene to the ATP6AP1L gene position on chromosome 5 (~2.2Mbp), and a twenty-fold tandemly amplified region were verified using long-range genome and RNA sequencing data. In doing so, a novel inversion at the start and end positions of the amplified region as well as frameshift insertions in most of the MSH and MLH genes were detected. These might stimulate chromosomal breakage and cause the dysregulation of mismatch repair pathways. Using Hi-C technology, high adjusted interaction frequencies were detected on the amplified unit and unsuspected position on 5q, which could have a complex network of spatial contacts to harbor gene amplification. Characterizing the tandem gene-amplified unit and genomic variants as well as chromosomal interactions on intra-chromosome 5 can be critical in identifying the mechanisms behind genomic rearrangements. These findings may give new insight into the mechanisms underlying the amplification process and evolution of drug resistance.

genomics↗

bioRxiv2020

AVP-induced counter-regulatory glucagon is diminished in type 1 diabetes

Insulin-induced hypoglycemia is a major barrier to the treatment of type-1 diabetes (T1D). Accordingly, it is important that we understand the mechanisms regulating the circulating levels of glucagon - the bodys principal blood glucose-elevating hormone which is secreted from alpha-cells of the pancreatic islets. Varying glucose over the range of concentrations that occur physiologically between the fed and fuel-deprived states (from 8 to 4 mM) has no significant effect on glucagon secretion in isolated islets (in vitro) and yet associates with dramatic changes in plasma glucagon in vivo. The identity of the systemic factor(s) that stimulates glucagon secretion remains unknown. Here, we show that arginine-vasopressin (AVP), secreted from the posterior pituitary, stimulates glucagon secretion. Glucagon-secreting alpha-cells express high levels of the vasopressin 1b receptor gene (Avpr1b). Activation of AVP neurons in vivo increased circulating copeptin (the C-terminal segment of the AVP precursor peptide, a stable surrogate marker of AVP) and increased blood glucose; effects blocked by pharmacological antagonism of either the glucagon receptor or vasopressin 1b receptor. AVP also mediates the stimulatory effects of hypoglycemia produced by exogenous insulin and 2-deoxy-D-glucose on glucagon secretion. We show that the A1/C1 neurons of the medulla oblongata drive AVP neuron activation in response to insulin-induced hypoglycemia. Exogenous injection of AVP in vivo increased cytoplasmic Ca2+ in alpha-cells (implanted into the anterior chamber of the eye) and glucagon release. Hypoglycemia also increases circulating levels of AVP in humans and this hormone stimulates glucagon secretion from isolated human islets. In patients with T1D, hypoglycemia failed to increase both plasma copeptin and glucagon levels. These findings suggest that AVP is a physiological systemic regulator of glucagon secretion and that this mechanism becomes impaired in T1D.

cell biology↗

bioRxiv2019

FLASH irradiation enhances the therapeutic index of abdominal radiotherapy in mice

Peritoneal metastases are the leading cause of morbidity and mortality in ovarian cancer. Despite current surgery, chemotherapy and targeted therapies, the majority of patients diagnosed with advanced epithelial ovarian cancer develop recurrent disease and overall survival rates remain poor. It is known that ovarian cancer is a radiosensitive tumor. Historically, total abdominal irradiation (TAI) was used as an effective postsurgical adjuvant therapy in the management of chemotherapy sensitive and resistant ovarian cancer. However, TAI fell out of favor due to high toxicity, particularly of the gastrointestinal tract. We have developed a preclinical irradiation platform that allows for total abdominal ultrahigh dose rate FLASH irradiation. We demonstrate that TAI-FLASH reduces radiation-induced intestinal injury in both healthy and tumor-bearing mice compared to conventional dose rate (CONV) irradiation. Single high dose TAI-FLASH reduced mortality from gastrointestinal syndrome, spared gut function and epithelial integrity, and decreased cell death in crypt base columnar cells. Importantly, FLASH and CONV irradiation had similar efficacy in the reduction of ovarian cancer peritoneal metastases. These findings suggest that FLASH irradiation may be an effective strategy to enhance the therapeutic index of radiotherapy for the treatment of metastatic ovarian cancer in women.

cancer biology↗

bioRxiv2019

Regulation of BMP Signaling by O-GlcNAcylation

Precise regulation of signal transduction is critical throughout organismal life, both for embryonic development and for adult homeostasis. To ensure proper spatio-temporal signal transduction, Bone Morphogenetic Protein (BMP) signaling pathways, like all other signaling pathways, are regulated by both agonists and antagonists. Here, we report identification of a previously unrecognized method of signal antagonism for Dpp (Decapentaplegic), a Drosophila BMP family member. We demonstrate that the BMP type I receptor Saxophone (Sax) functions as a Dpp receptor in the Drosophila embryonic epidermis, but that its activity is normally inhibited by the O-linked glycosyltransferase Super sex combs (Sxc). In wild-type embryos, inhibition of Saxophone (Sax) activity in the epidermis marks the BMP type I receptor Thickveins (Tkv) as the sole conduit for Dpp. In contrast, in sxc mutants, the Dpp signal is transduced by both Tkv and Sax, and elevated Dpp signaling induces errors in embryonic development that lead to embryonic death. We also demonstrate that Sax is the O-glycosylated target of Sxc and that O-glycosylation of Sax can be modulated by dietary sugar. Together, these findings link fertility to nutritive environment and point to Sax (activin receptor-like kinase 2 [ACVR1 or ALK2]) signaling as the nutrient-sensitive branch of BMP signaling. O_FIG O_LINKSMALLFIG WIDTH=199 HEIGHT=200 SRC="FIGDIR/small/784629v2_ufig1.gif" ALT="Figure 1"> View larger version (51K): org.highwire.dtl.DTLVardef@1737655org.highwire.dtl.DTLVardef@15de1ddorg.highwire.dtl.DTLVardef@978c76org.highwire.dtl.DTLVardef@186b60e_HPS_FORMAT_FIGEXP M_FIG Graphical Abstract C_FIG

developmental biology↗

bioRxiv2019

Hair-bearing human skin generated entirely from pluripotent stem cells

The skin is important for regulating bodily fluid retention and temperature, guarding against external stresses, and mediating touch and pain sensation. The skin is also susceptible to damage from burns, diseases, or genetic defects, which affect nearly one billion people worldwide1,2. For the advancement of skin regenerative therapies, it remains challenging to construct new skin with hair follicles and nerves in tissue cultures and in bioengineered skin grafts3-8. Here, we report an organoid culture system that generates complex skin from human pluripotent stem cells. We use step-wise modulation of the TGF{beta} and FGF signalling pathways to co-induce cranial epithelial cells and neural crest cells within a spherical cell aggregate. During 4-5 months incubation, we observe the emergence of a cyst-like skin organoid composed of stratified epidermis, fat-rich dermis, and pigmented hair follicles equipped with sebaceous glands. A network of sensory neurons and Schwann cells form nerve-like bundles that target Merkel cells in organoid hair follicles, mimicking human touch circuitry. Single-cell RNA sequencing and direct comparison to foetal specimens suggest that skin organoids are equivalent to human facial skin in the second-trimester of development. Moreover, we show that skin organoids produce planar hair-bearing skin when grafted on nude mice. Together, our results demonstrate the self-assembly of nearly complete skin tissue in vitro that can be used to reconstitute skin in vivo. We anticipate that our skin organoid model will be foundational to future studies of human skin development, disease modelling, or reconstructive surgery.

developmental biology↗

bioRxiv2019

Temporal control of inhibition via muscarinic acetylcholine receptor signaling generates ON and OFF selectivity in a simple visual circuit

ON and OFF selectivity in visual processing is encoded by parallel pathways that respond to either increments or decrements of light. Despite lacking anatomical features to support split channels, Drosophila larvae effectively perform visually-guided behaviors. To understand principles guiding visual computation in this simple circuit, we focus on the physiological properties and behavioral relevance of larval visual interneurons and elucidate their functions in visual processing. We find that the ON vs. OFF discrimination in the larval visual circuit emerges through light-elicited cholinergic signaling that depolarizes the cholinergic interneuron (cha-lOLP) and hyperpolarizes the glutamatergic interneuron (glu-lOLP). Genetic studies further indicate that muscarinic acetylcholine receptor (mAchR)/Go signaling in glu-lOLP separates the ON and OFF signals through temporal delays, the disruption of which strongly impacts both physiological responses of downstream projection neurons and dark-induced pausing behavior. Together, our studies identify cellular and molecular substrates for OFF detection in the larval visual circuit and suggests temporal control of inhibition functions as an effective strategy in generating ON and OFF selectivity without anatomical segregation.

neuroscience↗

bioRxiv2018

Epigenomic landscape of the human pathogen Clostridium difficile

Clostridioides difficile is a leading cause of health care-associated infections. Although significant progress has been made in the understanding of its genome, the epigenome of C. difficile and its functional impact has not been systematically explored. Here, we performed the first comprehensive DNA methylome analysis of C. difficile using 36 human isolates and observed great epigenomic diversity. We discovered an orphan DNA methyltransferase with a well-defined specificity whose corresponding gene is highly conserved across our dataset and in all ~300 global C. difficile genomes examined. Inactivation of the methyltransferase gene negatively impacted sporulation, a key step in C. difficile disease transmission, consistently supported by multi-omics data, genetic experiments, and a mouse colonization model. Further experimental and transcriptomic analysis also suggested that epigenetic regulation is associated with cell length, biofilm formation, and host colonization. These findings open up a new epigenetic dimension to characterize medically relevant biological processes in this critical pathogen. This work also provides a set of methods for comparative epigenomics and integrative analysis, which we expect to be broadly applicable to bacterial epigenomics studies.

genomics↗

bioRxiv2018

In vivo gene expression analyses provide unique insights on P. vivax gametocytogenesis and chloroquine response

Studies of gene expression have provided insights on the regulation of Plasmodium parasites. However, few studies have targeted P. vivax, the cause of one third of all human malaria cases outside Africa, due to the lack of in vitro culture system and the difficulties associated with studying clinical samples. Here, we describe robust RNA-seq profiles of P. vivax parasites generated directly from infected patient blood. Gene expression deconvolution analysis reveals that most parasite mRNAs derive from trophozoites and that the asynchronicity of P. vivax infections is unlikely to confound gene expression studies. We also show that gametocyte genes form two clusters of co-regulated genes, possibly indicating the independent regulation of male and female gametocytogeneses. Finally, despite a large effect on parasitemia, we find that chloroquine does not alter trophozoite gene expression. Overall, our study highlights the biological knowledge that can be gathered by directly studying P. vivax patient infections.\n\nImportancePlasmodium vivax is the second most common cause of human malaria worldwide but, since it cannot be cultured in the laboratory, its biology remains poorly understood. In this study, we describe the analysis of the parasite gene expression profiles generated from 26 patient infections. We show that the proportion of male and female parasites varies greatly among infections, suggesting that they are independently regulated. We also compare the gene expression profiles of the same infections before and after treatment with chloroquine, a common antimalarial, and show that the drug efficiently kills most P. vivax parasites but appears to have little effect on one specific parasite stage, the trophozoites, in contrast with the effect of the drug on P. falciparum. Overall, our study exemplifies the biological insights that can be gained from applying modern genomic tools to study this difficult human pathogen.

microbiology↗

bioRxiv2018

Integrated Clinical and Omics Approach to Rare Diseases: Novel Genes and Oligogenic Inheritance in Holoprosencephaly

PurposeHoloprosencephaly (HPE) is a pathology of forebrain development characterized by high phenotypic and locus heterogeneity. Seventeen genes are known so far in HPE but the understanding of its genetic architecture remains to be refined. Here, we investigated the oligogenic nature of HPE resulting from accumulation of variants in different relevant genes.\n\nMethodsExome data from 29 patients diagnosed with HPE and 51 relatives from 26 unrelated families were analyzed. Standard variant classification approach was improved with a gene prioritization strategy based on clinical ontologies and gene co-expression networks. Clinical phenotyping and exploration of cross-species similarities were further performed on a family-by-family basis.\n\nResultsWe identified 232 rare deleterious variants in HPE patients representing 180 genes significantly associated with key pathways of forebrain development including Sonic Hedgehog (SHH) and Primary Cilia. Oligogenic events were observed in 10 families and involved novel HPE genes including recurrently mutated genes (FAT1, NDST1, COL2A1 and SCUBE2) and genes implicated in cilia function.\n\nConclusionsThis study reports novel HPE-relevant genes and reveals the existence of oligogenic cases resulting from several mutations in SHH-related genes. It also underlines that integrating clinical phenotyping in genetic studies will improve the identification of causal variants in rare disorders.

genetics↗

bioRxiv2017

Pharmacological manipulation of olfactory bulb granule cell excitability modulates beta oscillations: Testing a model

The mammalian olfactory bulb (OB) generates gamma (40 - 100 Hz) and beta (15 - 30 Hz) oscillations of the local field potential (LFP). Gamma oscillations arise at the peak of inhalation supported by dendrodendritic interactions between glutamatergic mitral cells (MCs) and GABAergic granule cells (GCs). Beta oscillations occur in response to odorants in learning or odor sensitization paradigms, but their generation mechanism and function are still poorly understood. When centrifugal inputs to the OB are blocked, beta oscillations disappear, but gamma oscillations persist. Centrifugal input targets primarily GABAergic interneurons in the GC layer (GCL) and regulates GC excitability, which suggests a causal link between beta oscillations and GC excitability. Previous modeling work from our laboratory predicted that convergence of excitatory/inhibitory inputs onto MCs and centrifugal inputs onto GCs can increase GC excitability sufficiently to drive beta oscillations primarily through voltage dependent calcium channel (VDCC) mediated GABA release, independently of NMDA channels. We test this model by examining the influence of NMDA and muscarinic acetylcholine receptors on GC excitability and beta oscillations. Intrabulbar scopolamine (muscarinic antagonist) infusion decreased or completely suppressed odor-evoked beta in response to a strong stimulus, but increased beta power in response to a weak stimulus, as predicted by our model. Piriform cortex (PC) beta power was unchanged. Oxotremorine (muscarinic agonist) tended to suppress all oscillations, probably from over-inhibition. APV, an NMDA receptor antagonist, suppressed gamma oscillations selectively (in OB and PC), lending support to the models prediction that beta oscillations can be supported by VDCC mediated currents.\n\nNew and NoteworthyO_LIOlfactory bulb beta oscillations rely on granule cell excitability.\nC_LIO_LIReducing granule cell excitability with scopolamine reduces high volatilityinduced beta power but increases low volatility-induced beta power.\nC_LIO_LIPiriform cortex beta oscillations maintain power when olfactory bulb beta power is low, and the system maintains beta band coherence.\nC_LI

neuroscience↗

bioRxiv2017

Detecting cancer vulnerabilities through gene networks under purifying selection in 4,700 cancer genomes

Large-scale cancer sequencing studies have uncovered dozens of mutations critical to cancer initiation and progression. However, a significant proportion of genes linked to tumor propagation remain hidden, often due to noise in sequencing data confounding low frequency alterations. Further, genes in networks under purifying selection (NPS), or those that are mutated in cancers less frequently than would be expected by chance, may play crucial roles in sustaining cancers but have largely been overlooked. We describe here a statistical framework that identifies genes that have a first order protein interaction network significantly depleted for mutations, to elucidate key genetic contributors to cancers. Not reliant on and thus, unbiased by, the gene of interests mutation rate, our approach has identified 685 putative genes linked to cancer development. Comparative analysis indicates statistically significant enrichment of NPS genes in previously validated cancer vulnerability gene sets, while further identifying novel cancer-specific candidate gene targets. As more tumor genomes are sequenced, integrating systems level mutation data through this network approach should become increasingly useful in pinpointing gene targets for cancer diagnosis and treatment.

bioinformatics↗

bioRxiv2017

A unified web platform for network-based analyses of genomic data

Functional genomics networks are widely used to identify unexpected pathway relationships in large genomic datasets. However, it is challenging to quantitatively compare the signal-to-noise ratio of different networks, the biology they describe, and to identify the optimal network to interpret a particular genetic dataset. Via GeNets users can train a machine-learning model (Quack) to make such comparisons; and they can execute, store, and share analyses of genetic and RNA sequencing datasets.

genomics↗

bioRxiv2017

Fine-mapping of genetic loci driving spontaneous clearance of hepatitis C virus infection

Approximately three quarters of acute HCV infections evolve to a chronic state, while one quarter are spontaneously cleared. Genetic predispositions strongly contribute to the development of chronicity. We have conducted a genome-wide association study to identify genomic variants underlying HCV spontaneous clearance using Immunochip in European and African ancestries. We confirmed two previously reported significant associations, in the IL28B/IFNL41,2 and MHC regions, with spontaneous clearance in the European population. We further fine-mapped the MHC association to a region of about 50 kilo base pairs, down from 1 mega base pairs in the previous study. Additional analyses suggested that the association in the MHC locus might be significantly stronger for virus subtype 1a than 1b, suggesting that viral subtype may have influenced the genetic mechanism underlying the clearance of HCV.

immunology↗

bioRxiv2017

Genoppi: A web application for interactive integration of experimental proteomics results with genetic datasets

SummaryIntegrating protein-protein interaction experiments and genetic datasets can lead to new insight into the cellular processes implicated in diseases, but this integration is technically challenging. Here, we present Genoppi, a web application that integrates quantitative interaction proteomics data and results from genome-wide association studies or exome sequencing projects, to highlight biological relationships that might otherwise be difficult to discern. Written in R, Python and Bash script, Genoppi is a user-friendly framework easily deployed across Mac OS and Linux distributions.\n\nAvailabilityGenoppi is open source and available at https://github.com/lagelab/Genoppi\n\nContactaprilkim@broadinstitute.org and lage.kasper@mgh.harvard.edu

bioinformatics↗