Biology subjects

Ren, G.

Publications and source records attributed to Ren, G..

24 records · Page 2Linked to original sources

bioRxiv2019

Exploring the Changing Landscape of Cell-to-Cell Variation After CTCF Knockdown via Single Cell RNA-seq

BackgroundCCCTC-Binding Factor (CTCF), also known as 11-zinc finger protein, participates in many cellular processes, including insulator activity, transcriptional regulation and organization of chromatin architecture. Based on single cell flow cytometry and single cell RNA-FISH analyses, our previous study showed that deletion of CTCF binding site led to a significantly increase of cellular variation of its target gene. However, the effect of CTCF on genome-wide landscape of cell-to-cell variation is unclear. ResultsWe knocked down CTCF in EL4 cells using shRNA, and conducted single cell RNA-seq on both wild type (WT) cells and CTCF-Knockdown (CTCF-KD) cells using Fluidigm C1 system. Principal component analysis of single cell RNA-seq data showed that WT and CTCF-KD cells concentrated in two different clusters on PC1, indicating gene expression profiles of WT and CTCF-KD cells were systematically different. Interestingly, GO terms including regulation of transcription, DNA binding, Zinc finger and transcription factor binding were significantly enriched in CTCF-KD-specific highly variable genes, indicating tissue-specific genes such as transcription factors were highly sensitive to CTCF level. The dysregulation of transcription factors potentially explain why knockdown of CTCF lead to systematic change of gene expression. In contrast, housekeeping genes such as rRNA processing, DNA repair and tRNA processing were significantly enriched in WT-specific highly variable genes, potentially due to a higher cellular variation of cell activity in WT cells compared to CTCF-KD cells. We further found cellular variation-increased genes were significantly enriched in down-regulated genes, indicating CTCF knockdown simultaneously reduced the expression levels and increased the expression noise of its regulated genes. ConclusionsTo our knowledge, this is the first attempt to explore genome-wide landscape of cellular variation after CTCF knockdown. Our study not only advances our understanding of CTCF function in maintaining gene expression and reducing expression noise, but also provides a framework for examining gene function.

genomics↗

bioRxiv2019

Biocontrol of PGPR strain Bacillus amyloliquefaciens Ba168 against Phytophthora nicotianae on tobacco

Tobacco black shank (TBS) caused by Phytophthora nicotianae is destructive to almost all kinds of tobacco cultivars and is widespread in many tobacco-planted countries. Here, an isolated plant growth-promoting rhizobacteria (PGPR) strain Ba168 is promise in biocontrol of TBS. In vitro assays disclosed a strong P. nicotianae suppression activity and the field utilization potential (FUP) by characterized the crude extract of culture filtrates of Ba168. P. nicotianaes growth was inhibited by the crude extract at minimum inhibitory concentration (MIC) of 5l/mL. Extracellular conductivity, pH and the wet, dry weight of P. nicotianaes mycelia, were significantly different after treated with different concentrations of the crude extract and the deformity and perforation of treated P. nicotianaes hyphae can be observed in scanning electron microscope (SEM) analysis. Proteome characterizations of the crude extract were used as supplementary proofs that further evaluated FUP of Ba168. We then identified strain Ba168 as B. amyloliquefaciens by its genetic and phenotypic characteristics. Field assays comparatively evaluated TBS control efficacy of these PGPRs and agrochemicals. Pooling analysis of the results showed that the biocontrol efficacy of Ba168 preparation is only lower than Mixture of Propamocarb hydrochloride and Azoxystrobin (MPA) but better than other tested subjects. Although the existence of differences in biocontrol efficacy, PGPR preparations effectively reduced the disease index of tobacco.\n\nImportanceThis work demonstrates the promising biocontrol potential of B. amyloliquefaciens Ba168 and highlights the positive roles of PGPR in suppression of this soil-borne disease.

microbiology↗

bioRxiv2019

Identification of PKC -dependent phosphoproteins in mouse retina

Adjusting to a wide range of light intensities is an essential feature of retinal rod bipolar cell (RBC) function. While persuasive evidence suggests this modulation involves phosphorylation by protein kinase C-alpha (PKC), the targets of PKC phosphorylation in the retina have not been identified. PKC activity and phosphorylation in RBCs was examined by immunofluorescence confocal microscopy using a conformation-specific PKC antibody and antibodies to phosphorylated PKC motifs. PKC activity was dependent on light and expression of TRPM1, and RBC dendrites were the primary sites of light-dependent phosphorylation. PKC-dependent retinal phosphoproteins were identified using a phosphoproteomics approach to compare total protein and phosphopeptide abundance between phorbol ester-treated wild type and PKC knockout (PKC-KO) mouse retinas. Phosphopeptide mass spectrometry identified over 1100 phosphopeptides in mouse retina, with 12 displaying significantly greater phosphorylation in WT compared to PKC-KO samples. The differentially phosphorylated proteins fall into the following functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein). Two strongly differentially expressed phosphoproteins, BORG4 and TPBG, were localized to the synaptic layers of the retina, and may play a role in PKC-dependent modulation of RBC physiology. Data are available via ProteomeXchange with identifier PXD012906.\n\nSignificanceRetinal rod bipolar cells (RBCs), the second-order neurons of the mammalian rod visual pathway, are able to modulate their sensitivity to remain functional across a wide range of light intensities, from starlight to daylight. Evidence suggests that this modulation requires the serine/threonine kinase, PKC, though the specific mechanism by which PKC modulates RBC physiology is unknown. This study examined PKC phosophorylation patterns in mouse rod bipolar cells and then used a phosphoproteomics approach to identify PKC-dependent phosphoproteins in the mouse retina. A small number of retinal proteins showed significant PKC-dependent phosphorylation, including BORG4 and TPBG, suggesting a potential contribution to PKC-dependent modulation of RBC physiology.\n\nHighlightsO_LIPKC is a major source of phosphorylation in retinal RBC dendrites and its activity in RBCs is light dependent.\nC_LIO_LIProteins showing differential phosphorylation between phorbol ester-treated wild type and PKC-KO retinas belong to the following major functional groups: cytoskeleton/trafficking (4 proteins), ECM/adhesion (2 proteins), signaling (2 proteins), transcriptional regulation (3 proteins), and homeostasis/metabolism (1 protein).\nC_LIO_LIThe PKC-dependent phosphoproteins, BORG4 and TPBG, are present in the synaptic layers of the retina and may be involved in PKC-dependent modulation of RBC physiology.\nC_LI

neuroscience↗

bioRxiv2019

Expression and distribution of Trophoblast Glycoprotein in the mouse retina

We recently identified the leucine-rich repeat adhesion protein, trophoblast glycoprotein (TPBG), as a novel PKC-dependent phosphoprotein in retinal rod bipolar cells (RBCs). Since TPBG has not been thoroughly examined in the retina, this study characterizes the localization and expression patterns of TPBG in the developing and adult mouse retina using two antibodies, one against the N-terminal, leucine-rich domain and the other against the C-terminal PDZ-interacting motif. Both antibodies labeled dendrites and synaptic terminals of RBCs, as well as the cell bodies and dendrites of an uncharacterized class of amacrine cell. In transfected HEK293 cells, TPBG was localized to the plasma membrane and intracellular membranes and was associated with the tips of thin filopodia-like membrane projections. TPBG immunofluorescence in RBCs detected with the C-terminal antibody was strongly dependent on the activity state of the adult retina, with less labeling in dark-adapted compared to light-adapted retina, and less labeling in light-adapted PKC knockout and TRPM1 knockout retinas compared to wild type, despite no change in total TPBG detected by immunoblotting. These results suggest that the C-terminal epitope is blocked in the dark-adapted and knockout retinas compared to light-adapted wild type retinas, possibly through interaction with a PDZ domain protein. During development, TPBG expression increases dramatically just prior to eye opening with a time course closely correlated with that of TRPM1 expression. In the retina, leucine-rich repeat proteins like TPBG have been implicated in the development and maintenance of functional bipolar cell synapses, and TPBG may play a similar role in RBCs.

neuroscience↗

bioRxiv2018

3D images of Neuronal Adhesion Molecule Contactin-2 Reveal an Unanticipated Two-State Architecture

Contactins (CNTNs) are important cell adhesion molecules that mediate neuronal and axoglial contacts, and lesions in these molecules are linked to neuropsychiatric disorders. The extracellular domain of CNTNs contains six Ig domains and four FNIII domains. Crystal structures have shown that Ig1-Ig4 forms a horseshoe-shaped headpiece, in which the N-terminal domains might fold back on the C-terminal domains to form molecular super-U shaped architecture. The arrangement of these domains has been controversial, which may due to the structural dynamics and conformation heterogeneity of the protein. Here, we used a single-molecule 3D imaging method, individual-particle electron tomography (IPET), to study the extracellular domain of CNTN2 that forms monomers with a broad spectrum of conformations, and obtained 60 three-dimensional (3D) reconstructions. In addition to the known horseshoe-shaped headpiece, ~75% headpieces unexpectedly adopt an open (elongated) or a semi-open conformations contributed to our understanding about structural dynamics. The ectodomains formed curve but not double-back in any uniform way, with an averaged molecular dimension of ~255 [A]. The first-time demonstration of the dynamic nature and conformational preferences of the full-length CNTN2 ectodomain suggest that the headpiece exists in equilibrium in the closed or not-closed states. The important architecture may provide a structural platform for protein partners to influence this balance regulating the function of CNTN2. Encoding the ability of this neural adhesion molecule to form both homomers with itself, as well as recruit different protein partner to neuronal and axoglial contact points play the key role in mediating cell-cell interactions.

biochemistry↗

bioRxiv2017

Fibroblasts from metastatic sites induce broad-spectrum drug desensitization via modulation of mitochondrial priming

Due to tumor heterogeneity, most believe that effective treatments should be tailored to the features of an individual tumor or tumor subclass. It is still unclear what information should be considered for optimal disease stratification, and most prior work focuses on tumor genomics. Here, we focus on the tumor micro-environment. Using a large-scale co-culture assay optimized to measure drug-induced cell death, we identify tumor-stroma interactions that modulate drug sensitivity. Our data show that the chemo-insensitivity typically associated with aggressive subtypes of breast cancer is not cell intrinsic, but rather a product of tumor-fibroblast interactions. Additionally, we find that fibroblast cells influence tumor drug response in two distinct and divergent manners, which were predicable based on the anatomical origin from which the fibroblasts were harvested. These divergent phenotypes result from modulation of \"mitochondrial priming\" of tumor cells, caused by secretion of inflammatory cytokines, such as IL6 and IL8, from stromal cells.

cancer biology↗