Biology subjects

Wan, J.

Publications and source records attributed to Wan, J..

103 records · Page 6Linked to original sources

bioRxiv2019

Inducible overexpression of zebrafish microRNA-722 suppresses chemotaxis of human neutrophil like cells

Neutrophil migration is essential for battling against infections but also drives chronic inflammation. Since primary neutrophils are terminally differentiated and not genetically tractable, leukemia cells such as HL-60 are differentiated into neutrophil-like cells to study mechanisms underlying neutrophil migration. However, constitutive overexpression or inhibition in this cell line does not allow the characterization of the genes that affect the differentiation process. Here we apply the tet-on system to induce the expression of zebrafish miR-722 in differentiated HL-60. Overexpression of miR-722 reduced the mRNA level of genes in the chemotaxis and inflammation pathways, including RAC2. Consistently, cell migration is significantly inhibited upon induced miR-722 overexpression. Together, miR-722 is an evolutionarily conserved suppressor for neutrophil migration and signaling in zebrafish and human.

immunology↗

bioRxiv2019

MicroRNA Overexpression screen reveals a noncanonical role of CDK2 in regulating neutrophil migration

Neutrophil migration is essential for inflammatory responses to kill pathogens, however it also causes tissue injury. To discover novel therapeutic targets that modulate neutrophil migration, we performed a neutrophil-specific microRNA overexpression screen in zebrafish, and identified eight microRNAs as potent suppressors of neutrophil migration. Among those, miR-199 decreases neutrophil chemotaxis in zebrafish and human neutrophil-like cells. Intriguingly, in terminally differentiated neutrophils, miR-199 alters the cell cycle-related pathways and directly suppresses cyclin-dependent kinase 2 (cdk2), whose known activity is restricted to cell cycle progression and cell differentiation. Inhibiting CDK2, but not DNA replication, disrupts cell polarity and chemotaxis of zebrafish neutrophils. Chemotaxis of primary human neutrophils are also reduced by CDK2 inhibition. Furthermore, miR-199 overexpression or CDK2 inhibition significantly improves the outcome of lethal systemic inflammation challenges in zebrafish. Together, our results reveal previously unknown functions of miR-199 and CDK2 in regulating neutrophil migration and provide new directions in alleviating systemic inflammation.\n\nOne Sentence SummarymiR-199 directly suppresses cdk2 expression, neutrophil chemotaxis and systemic inflammation.

immunology↗

bioRxiv2019

Amplification-free Library Preparation Improves Quality of Hi-C Analysis

PCR amplification of Hi-C libraries introduces unusable duplicates and results in a biased representation of chromatin interactions. We present a simplified, fast, and economically efficient Hi-C library preparation procedure that generates sufficient non-amplified ligation products for deep sequencing from 30 million Drosophila cells. Comprehensive analysis of the resulting data indicates that amplification-free Hi-C preserves higher complexity of chromatin interaction and lowers sequencing depth dramatically for the same number of unique paired reads. For human cells which has a large genome, this method recovers an amount of ligated fragments enough for direct high-throughput sequencing without amplification on as low as 250 thousand of cells. Comparison with published in situ Hi-C on millions of human cells reveals that amplification introduces distance-dependent amplification bias, which results in increasing background noise level against genomic distance. With amplification bias avoided, our method may produce a chromatin interaction network more faithfully reflecting the real three-dimensional genomic architecture.

genomics↗

bioRxiv2019

OsLUGL involved in floral development through regulating auxin level and OsARFs expression in rice (Oryza sative L.)

Specification of floral organ identity is critical for floral morphology and inflorescence architecture. Floral organ identity in plants is controlled by floral homeotic A/B/C/D/E-class genes. Although multiple genes regulate floral organogenesis our understanding of the regulatory network remains fragmentary. Here, we characterized rice floral organ gene KAIKOUXIAO (KKX), mutation of which produces an uncharacteristic open hull, abnormal seed, and semi-sterility. KKX encodes a putative LEUNIG-like (LUGL) transcriptional co-repressor. OsLUGL is preferentially expressed in young panicles and its protein can interact with OsSEU, which functions were reported as an adaptor for LEUNIG. OsLUGL-OsSEU functions together as a transcriptional regulatory complex to control organ identity specification through regulation of MADS-box genes. During this process, SEP3 (such as OsMADS8) and AP1 (such as OsMADS18) serve as the DNA-binding partner of OsLUGL-OsSEU complex. Further studies indicated that OsMADS8 and OsMADS18 could bind the promoter of OsGH3-8 and regulate its expression. The altered expression of OsGH3-8 caused the increased auxin level and the decreased expression of OsARFs. Overall, our results demonstrate a possible pathway whereby OsLUGL-OsSEU-OsAP1-OsSEP3 complex as a transcriptional regulator by targeting the promoter of OsGH3-8 directly, affecting auxin level, OsARFs expression level and thereby influencing floral development. These findings provide a valuable insight into the molecular functions of OsLUGL in rice floral development. HighlightOsLUGL through forming OsLUGL-OsSEU-OsAP1-OsSEP3 complex regulate OsGH3-8 expression, and regulate auxin level and OsARFs expression indirectly. This work is a new insight to floral development molecular mechanism.

genetics↗

bioRxiv2018

Microbial communities across a hillslope-riparian transect shaped by proximity to the stream, groundwater table, and weathered bedrock

Watersheds are important suppliers of freshwater for human societies. Within mountainous watersheds, microbial communities impact water chemistry and element fluxes as water from precipitation events discharges through soils and underlying weathered rock, yet there is limited information regarding the structure and function of these communities. Within the East River, CO watershed, we conducted a depth-resolved, hillslope to riparian zone transect study to identify factors that control how microorganisms are distributed and their functions. Metagenomic and geochemical analyses indicate that distance from the East River and proximity to groundwater and underlying weathered shale strongly impact microbial community structure and metabolic potential. Riparian zone microbial communities are compositionally distinct from all hillslope communities. Bacteria from phyla lacking isolated representatives consistently increase in abundance with increasing depth, but only in the riparian zone saturated sediments did we find Candidate Phyla Radiation bacteria. Riparian zone microbial communities are functionally differentiated from hillslope communities based on their capacities for carbon and nitrogen fixation and sulfate reduction. Selenium reduction is prominent at depth in weathered shale and saturated riparian zone sediments. We anticipate that the drivers of community composition and metabolic potential identified throughout the studied transect will predict patterns across the larger watershed hillslope system.

microbiology↗

bioRxiv2018

Keystone mutualists can facilitate transition between alternative ecosystem states in the soil

Symbioses between plants and microbial organisms can fundamentally alter the structure of ecosystems, from their species diversity to rates of nutrient cycling. Yet, many aspects of how differences in the prevalence of microbial symbioses arise are unclear. This is a key knowledge gap, as if co-variation in plant and microbial distributions are primarily determined by extrinsic abiotic factors then symbioses should exert little independent control over ecosystems. To examine the potential for alternative symbiotic communities to arise under similar conditions we examined biogeochemical cycling and microbial community structure in a coastal landscape where historical patterns of vegetation transition are known, allowing us to eliminate abiotic determinism. We found that alternative states in microbial community structure and ecosystem processes emerged under different plant species. Greenhouse studies further demonstrated that plant selection of symbiotic microbes is central to emergence of these alternative states and occurs independent of soil abiotic conditions. Moreover, we provide evidence that transition between states may be highly dependent on the presence of a small set of ruderal symbionts that are rare in mature systems but may act as keystone mutualists. Because differences between these alternative states can be directly linked to plant-microbe symbioses, independent of initial conditions, our results suggesting that biotic feedbacks between keystone symbiotic microbes and plants play a foundational role in the diversity and function of soils.

ecology↗

bioRxiv2018

SGC-GAK-1: a chemical probe for cyclin G associated kinase (GAK)

We describe SGC-GAK-1 (11), a potent, selective, and cell-active inhibitor of cyclin G associated kinase (GAK), together with a structurally-related negative control SGC-GAK-1N (14). SGC-GAK-1 is highly selective in a kinome-wide screen, but cellular engagement assays defined RIPK2 as a collateral target. We identified 18 as a potent inhibitor of RIPK2 lacking GAK activity. Together, the chemical probe set of 11, 14, and 18 can be used to interrogate the cellular biology of GAK inhibition.

cell biology↗

bioRxiv2018

Axon Guidance Signaling Modulates Epithelial to Mesenchymal Transition in Stem Cell-Derived Retinal Pigment Epithelium

The critical role of epithelial to mesenchymal transition (EMT) in embryonic development, malignant transformation, and tumor progression has been well studied in normal and cancerous tissues and cells. Interestingly, EMT has also been reported to play a key role in the early progression of several retinal degenerative diseases, including scarring associated proliferative vitro-retinopathy (PVR), choroidal neo-vascularization induced \"wet\" age-related macular degeneration (AMD) and diabetic retinopathy (DR). Despite these studies, many questions remain unexplored regarding EMT-associated retinal pigment epithelium (RPE) degeneration and dysfunction. We hypothesize that RPE cells undergo EMT prior to cell death during the progression of atrophic \"dry\" AMD. Utilizing human stem cell-derived RPE (hRPE) as a model to study RPE EMT, we optimized two independent but complementary RPE EMT induction systems: 1) enzymatic dissociation of hRPE monolayer cultures and 2) co-treatment of hRPE monolayer cultures with transforming growth factor beta (TGF-{beta}) and the inflammatory cytokine, tumor necrosis factor alpha (TNF-). To further understand the molecular mechanisms of RPE EMT regulation, we performed an RNA-Sequencing (RNA-Seq) time course examination across 48 hours beginning with EMT induction. Our transcriptome profiling provides a comprehensive quantification of dynamic signaling events and associated biological pathways underlying RPE EMT and reveals an intriguing significance for widespread dysregulation of multiple axon guidance molecules in this process.

genomics↗

bioRxiv2018

Specific Eph Receptor-Cytoplasmic Effector Signaling Mediated by SAM-SAM Domain Interactions

The Eph receptor tyrosine kinase (RTK) family is the largest subfamily of RTKs playing critical roles in many developmental processes such as tissue patterning, neurogenesis and neuronal circuit formation, angiogenesis, etc. How the 14 Eph proteins, via their highly similar cytoplasmic domains, can transmit diverse and sometimes opposite cellular signals upon engaging ephrins is a major unresolved question. Here we systematically investigated the bindings of each SAM domain of Eph receptors to the SAM domains from SHIP2 and Odin, and uncover a highly specific SAM-SAM interaction-mediated cytoplasmic Eph-effector binding pattern. Comparative X-ray crystallographic studies of several SAM-SAM heterodimer complexes, together with biochemical and cell biology experiments, not only revealed the exquisite specificity code governing Eph/effector interactions but also allowed us to identify SAMD5 as a new Eph binding partner. Finally, these Eph/effector SAM heterodimer structures can explain numerous Eph SAM mutations identified in patients suffering from cancers and other diseases.

biophysics↗

bioRxiv2017

WSL5, a pentatricopeptide repeat protein, is essential for chloroplast biogenesis in rice under cold stress

AbstactChloroplasts play an essential role in plant growth and development, and cold has a great effect on chloroplast development. Although many genes or regulators involved in chloroplast biogenesis and development have been isolated and characterized, identification of novel components associated with cold is still lacking. In this study, we reported the functional characterization of white stripe leaf 5 (wsl5) mutant in rice. The mutant developed white-striped leaves during early leaf development and was albinic when planted under cold stress. Genetic and molecular analysis revealed that WSL5 encodes a novel chloroplast-targeted pentatricopeptide repeat protein. RNA-seq analysis showed that expression of nuclear-encoded photosynthetic genes in the mutant was significantly repressed, and expression of many chloroplast-encoded genes was also significantly changed. Notably, the WSL5 mutation caused defects in editing of rpl2 and atpA, and in splicing of rpl2 and rps12. Chloroplast ribosome biogenesis was impaired under cold stress. We propose that WSL5 is required for normal chloroplast development in rice under cold stress.

genetics↗

bioRxiv2017

Lhx2 regulates temporal changes in chromatin accessibility and transcription factor binding in retinal progenitor cells.

Retinal progenitor cells (RPCs) pass through multiple stages of developmental competence, where they successively acquire and lose the ability to generate individual cell subtypes. To identify the transcriptional regulatory networks that control these transitions, we conducted epigenomic and transcriptomic profiling of early and late-stage RPCs and observed a developmentally dynamic landscape of chromatin accessibility. Open chromatin regions that showed stage-specificity, as well as those shared by early and late-stage RPCs, were selectively targeted by the homeodomain factor Lhx2, which is expressed throughout retinal neurogenesis but also regulates many stage-specific processes in RPCs. Stage-specific Lhx2 binding sites were frequently associated with target sites for transcription factors that are preferentially expressed in either early or late-stage RPCs, and which were predicted to possess pioneer activity. Lhx2 loss of function in RPCs led to a loss of chromatin accessibility at both direct Lhx2 target sites and more broadly across the genome, as well as a loss of binding by transcription factors associated with stage-specific Lhx2 target sites. These findings demonstrate a central role for Lhx2 in control of chromatin accessibility in RPCs, and identify transcription factors that may guide stage-specific target site selection by Lhx2.\n\nSummaryLhx2 is a central regulator of chromatin accessibility in retinal progenitor cells, and interacts with stage-specific transcription factors to regulate genes that are dynamically expressed during retinal neurogenesis.

neuroscience↗

bioRxiv2017

Foxd1 is required for terminal differentiation of anterior hypothalamic neuronal subtypes.

Although the hypothalamus functions as a master homeostat for many behaviors, little is known about the transcriptional networks that control its development. To investigate this question, we analyzed mice deficient for the Forkhead domain transcription factor Foxd1. Foxd1 is selectively expressed in neuroepithelial cells of the prethalamus and hypothalamus prior to the onset of neurogenesis, and is later restricted to neural progenitors of the prethalamus and anterior hypothalamus. During early stages of neurogenesis, we observed that Foxd1-deficient mice showed reduced expression of Six3 and Vax1 in anterior hypothalamus, but overall patterning of the prethalamus and hypothalamus is unaffected. After neurogenesis is complete, however, a progressive reduction and eventual loss of expression of molecular markers of the suprachiasmatic, paraventricular and periventricular hypothalamic is observed. These findings demonstrate that Foxd1 acts in hypothalamic progenitors to allow sustained expression of a subset of genes selectively expressed in mature neurons of the anterior hypothalamus.\n\nHighlightsO_LIFoxd1 is broadly expressed in neuroepithelial cells of the hypothalamus and prethalamus.\nC_LIO_LIFoxd1 mutants show severe defects in anterior hypothalamic development, although prethalamic development is only modestly affected.\nC_LIO_LILoss of Foxd1 results does not affect initial patterning of the hypothalamus, but leads to a progressive loss of expression of markers specific to neurons of the suprachiasmatic, paraventricular and periventricular nuclei.\nC_LIO_LIFoxd1 regulates expression of multiple transcription factors expressed in developing anterior hypothalamus.\nC_LI

developmental biology↗

bioRxiv2017

Myosin IIA interacts with the spectrin-actin membrane skeleton to control red blood cell membrane curvature and deformability

The biconcave disc shape and deformability of mammalian red blood cells (RBCs) relies upon the membrane skeleton, a viscoelastic network of short, membrane-associated actin filaments (F-actin) cross-linked by long, flexible spectrin tetramers. Nonmuscle myosin II (NMII) motors exert force on diverse F-actin networks to control cell shapes, but a function for NMII contractility in the 2D spectrin-F-actin network in RBCs has not been tested. Here, we show that RBCs contain membrane skeleton-associated NMIIA puncta, identified as bipolar filaments by super-resolution fluorescence microscopy. NMIIA association with the membrane skeleton is ATP-dependent, consistent with NMIIA motor domains binding to membrane skeleton F-actin and contributing to membrane mechanical stability. In addition, the NMIIA heavy and light chains are phosphorylated in vivo in RBCs, indicating active regulation of NMIIA motor activity and filament assembly, while reduced heavy chain phosphorylation of membrane skeleton-associated NMIIA indicates assembly of stable filaments at the membrane. Treatment of RBCs with blebbistatin, an inhibitor of NMII motor activity, decreases the number of NMIIA filaments associated with the membrane and enhances local, nanoscale membrane oscillations, suggesting decreased membrane tension. Blebbistatin-treated RBCs also exhibit elongated shapes, loss of membrane curvature, and enhanced deformability, indicating a role for NMIIA contractility in promoting membrane stiffness and maintaining RBC biconcave disc cell shape. As structures similar to the RBC membrane skeleton are conserved in many metazoan cell types, these data demonstrate a general function for NMII in controlling specialized membrane morphology and mechanical properties through contractile interactions with short F-actin in spectrin-F-actin networks.\n\nSignificance statementThe biconcave disc shape and deformability of the mammalian RBC is vital to its circulatory function, relying upon a 2D viscoelastic spectrin-F-actin network attached to the membrane. A role for myosin II (NMII) contractility in generating tension in this network and controlling RBC shape has never been tested. We show that NMIIA forms phosphorylated bipolar filaments in RBCs, which associate with F-actin at the membrane. NMIIA motor activity is required for interactions with the spectrin-F-actin network, and regulates RBC biconcave shape and deformability. These results provide a novel mechanism for actomyosin force generation at the plasma membrane, and may be applicable to other cell types such as neurons and polarized epithelial cells with a spectrin-F-actin-based membrane skeleton.

cell biology↗