Biology subjects

Chen, C.

Publications and source records attributed to Chen, C..

772 records · Page 43Linked to original sources

bioRxiv2017

Cryo-ET reveals nucleosome reorganization in condensed mitotic chromosomes in vivo

Chromosomes condense during mitosis in most eukaryotes. This transformation involves rearrangements at the nucleosome level and has consequences for transcription, but the details remain unclear. Here, we use cryo-electron tomography to determine the 3-D arrangement of nucleosomes and other large nuclear features in frozen-hydrated fission-yeast cells. Nucleosomes can form irregular clusters in both interphase and mitotic cells, but they are smaller than expected for Hi-C domains. The nucleosomes are co-mingled with two features: nucleosome-free pockets and megadalton-sized \"megacomplexes\". Compared to interphase, the nucleosomes in mitotic chromosomes pack into slightly larger clusters. However, nearest-neighbor distance analysis reveals that mitotic nucleosome clusters have the same internal packing density as in interphase. Furthermore, mitotic chromosomes contain fewer megacomplexes. This uneven chromosome condensation helps explain a longstanding enigma of mitosis: most genes are repressed but a subset is upregulated.

cell biology↗

bioRxiv2017

Conformational dynamics of Cas9 governing DNA cleavage revealed by single molecule FRET

Off-target binding and cleavage by Cas9 pose as major challenges in its applications. How conformational dynamics of Cas9 governs its nuclease activity under on- and off-target conditions remains largely unknown. Here, using intra-molecular single molecule fluorescence resonance energy transfer measurements, we revealed that Cas9 in apo, sgRNA-bound, and dsDNA/sgRNA-bound forms all spontaneously transits between three major conformational states, mainly reflecting significant conformational mobility of the catalytic HNH domain. We furthermore uncovered a surprising long-range allosteric communication between the HNH domain and RNA/DNA heteroduplex at the PAM-distal end to ensure correct positioning of the catalytic site, which demonstrated a unique proofreading mechanism served as the last checkpoint before DNA cleavage. Several Cas9 residues were likely to mediate the allosteric communication and proofreading step. Modulating interactions between Cas9 and heteroduplex at the distal end by introducing mutations on these sites provides an alternative route to improve and optimize the CRISPR/Cas9 toolbox.

biophysics↗

bioRxiv2017

Positional effects revealed in Illumina Methylation Array and the impact on analysis

With the evolution of rapid epigenetic research, Illumina Infinium HumanMethylation BeadChips have been widely used to study DNA methylation. However, in evaluating the accuracy of this method, we found that the commonly used Illumina HumanMethylation BeadChips are substantially affected by positional effects; the DNA samples location in a chip affects the measured methylation levels. We analyzed three HumanMethylation450 and three HumanMethylation27 datasets by using four methods to prove the existence of positional effects. Three datasets were analyzed further for technical replicate analysis or differential methylation CpG sites analysis. The pre- and post-correction comparisons indicate that the positional effects could alter the measured methylation values and downstream analysis results. Nevertheless, ComBat, linear regression and functional normalization could all be used to minimize such artifact. We recommend performing ComBat to correct positional effects followed by the correction of batch effects in data preprocessing as this procedure slightly outperforms the others. In addition, randomizing the sample placement should be a critical laboratory practice for using such experimental platforms. Code for our method is freely available at: https://github.com/ChuanJ/posibatch.

bioinformatics↗

bioRxiv2017

Ectopic activation of the Spindle Assembly Checkpoint reveals its biochemical design and physiological operation

Switch-like activation of the Spindle Assembly Checkpoint (SAC) is critical for accurate chromosome segregation during cell division. To determine the mechanisms that implement it, we engineered an ectopic, kinetochore-independent SAC activator, the \"eSAC\". The eSAC stimulates the SAC signaling cascade by artificially dimerizing the Mps1 kinase domain and a cytosolic KNL1 phosphodomain, the signaling scaffold in the kinetochore. Quantitative analyses and mathematical modeling of the eSAC reveal that the recruitment of multiple SAC proteins by the KNL1 phosphodomain stimulates synergistic signaling, which enables a small number of KNL1 molecules produce a disproportionately strong anaphase-inhibitory signal. However, when multiple KNL1 molecules signal concurrently, they compete for a limited cellular pool of SAC proteins. This frustrates synergistic signaling and modulates signal output. Together, these mechanisms institute automatic gain control - inverse, non-linear scaling between the signal output per kinetochore and the unattached kinetochore number, and thus enact the SAC switch.

cell biology↗

bioRxiv2017

A group of nuclear factor Y transcription factors are likely sub-functionalized in the endosperm development of rice and other monocots

Nuclear factor Y (NF-Y) is a heterotrimeric transcription factor that consists of three subunits, NF-YA, NF-YB, and NF-YC. Although NF-Ys play multiple roles in plant development, their functions during endosperm development are not well understood. In this study, we identified eight NF-Y encoding genes, including OsNF-YA8, OsNF-YB1,9, and OsNF-YC8,9,10,11,12, which predominantly express in the rice endosperm. Interestingly, the closest homologs of these OsNF-Ys are present only in the monocot species. All the genes are preferentially expressed in the endosperm, suggesting their roles in the regulation of endosperm development. A systemic analysis of the interactions between rice endosperm-preferential NF-Ys in yeast revealed that NF-YBs and NF-YCs could interact with each other. OsNF-YA8 is a recently evolved NF-YA in rice. Generally, NF-YA does not interact with NF-YB monomers in plants; however, in the present study, we found that OsNY-YA8 interacts with OsNF-YB9. Our results also indicated that the endosperm-preferential OsNF-YBs and OsNF-YCs could interact with some ethylene response factors (ERFs) of rice. Unlike the OsNF-YC8,9,10, the members of OsNF-YB1, 9 or OsNF-YC 11 and 12 showed lack of transcriptional activation when present alone. However, they displayed functional activity while in dimer form. In addition, OsNF-YB1 knockout lines showed significant changes in the seed morphology, further confirms its role in endosperm development. Our findings have provided strong evidences that the group of phylogenetically conserved NF-Ys are differentiated in monocots to regulate the endosperm development.

plant biology↗

bioRxiv2017

Characterization Of Imprinted Genes In Rice Reveals Post-Fertilization Regulation And Conservation At Some Loci Of Imprinting In Plant Species

Genomic imprinting is an epigenetic phenomenon by which certain genes display monoallelic expression in a parent-of-origin-dependent manner. Hundreds of imprinted genes have been identified from several plant species. Here we identified, with a high level of confidence, 208 imprinted candidates from rice. Imprinted genes of rice showed limited association to the transposable elements, which is contrast to the findings in Arabidopsis. Generally, imprinting of rice is conserved within species, but intraspecific variations were confirmed here. Imprinting between cultivated rice and wild rice are likely similar. The imprinted genes of rice do not show significant selective signatures overall, which suggests that domestication imposes limited evolutionary effects on genomic imprinting of rice. Though the conservation of imprinting in plants is limited, here we prove that some loci tend to be imprinted in different species. In addition, our results suggest that differential epigenetic regulation between parental alleles can be established either prior to or post-fertilization. The imprinted 24-nt small RNAs, but not the 21-nt ones, likely involve the regulation of imprinting in an opposite parental-allele targeting manner. Together, our findings suggest that regulation of imprinting can be very diverse, and genomic imprinting as well as imprinted genes have essential evolutionary and biological significance.

plant biology↗

bioRxiv2017

Natural Chromatin Is Heterogeneous And Self Associates In Vitro

The 30-nm fiber is commonly found in oligonucleosome arrays in vitro but rarely found in chromatin within nuclei. To determine how chromatin high-order structure is controlled, we used cryo-ET to study the undigested natural chromatin released from cells that do not have evidence of 30-nm fibers in vivo: picoplankton and yeast. In the presence of divalent cations, most of the chromatin from both organisms is compacted into a large mass. Rare irregular 30-nm fibers do form at the periphery of this mass, some of which include face-to-face interactions. In the absence of divalent cations, picoplankton chromatin decondenses into open zigzags. By contrast, yeast chromatin mostly remains compact with looser nucleosome packing, even after treatment with histone-deacetylase inhibitor. The 3-D configuration of natural chromatin is therefore sensitive to the local environment, but generally nonpermissive of regular motifs, even at the level of oligonucleosomes.

cell biology↗

bioRxiv2017

Agonist-Dependent And -Independent Kappa Opioid Receptor Phosphorylation Showed Distinct Phosphorylation Patterns And Resulted In Different Cellular Outcomes

We reported previously that the selective agonist U50,488H promoted phosphorylation of the mouse kappa opioid receptor (KOPR) at residues S356, T357, T363 and S369. Here, we found that agonist (U50,488H)-dependent KOPR phosphorylation at all the residues were mediated by Gi/o proteins and multiple protein kinases [GRKs2, 3, 5 and 6 and protein kinase C (PKC)]. In addition, PKC activation by phorbol ester induced agonist-independent KOPR phosphorylation. Compared with U50,488H, PKC activation promoted much higher S356/T357 phosphorylation, much lower T363 phosphorylation and similar levels of S369 phosphorylation. Following U50,488H, GRKs, but not PKC, were involved in agonist-induced KOPR internalization. In contrast, PKC activation caused a lower level of agonist-independent KOPR internalization, compared to U50,488H. U50,488H-induced activation of extracellular signal regulated kinase 1/2 (ERK1/2) was G protein-, but not {beta}-arrestin-, dependent. After U50,488H, GRK- mediated, but not PKC-mediated, KOPR phosphorylation followed by {beta}-arrestin recruitment desensitized U50,488H-induced ERK1/2 response. Therefore, agonist-dependent (GRK- and PKC-mediated) and agonist-independent (PKC-promoted) KOPR phosphorylations show distinct phosphorylation patterns, leading to diverse cellular outcomes.\n\nAbbreviations

pharmacology and toxicology↗

bioRxiv2017

A lncRNA/Lin28/Let7 Axis Coupled To DNA Methylation Fine Tunes The Dynamics Of A Cell State Transition

Execution of pluripotency requires progression from the naive status represented by mouse embryonic stem cells (ESCs) to a condition poised for lineage specification. This process is controlled at transcriptional, post-transcriptional and epigenetic levels and non-coding RNAs are contributors to this regulation complexity. Here we identify a molecular cascade initiated by a long non-coding RNA (lncRNA), Ephemeron (Epn), that modulates the dynamics of exit from naive pluripotency. Epn deletion delays the extinction of ESC identity, an effect mediated by perduring expression of the pivotal transcription factor Nanog. In the absence of Epn, Lin28a expression is reduced, resulting in an elevated level of Mirlet7g that suppresses de novo methyltransferases Dnmt3a/b. Dnmt3a/b deletion also retards exit from the ESC state, and is associated with delayed promoter methylation and slower down-regulation of Nanog. Altogether, our findings reveal a lncRNA/miRNA/DNA methylation axis that facilitates a timely stem cell state transition.

cell biology↗

bioRxiv2017

Genome Wide Association Analyses Based On Broadly Different Specifications For Prior Distributions, Genomic Windows, And Estimation Methods

A popular strategy (EMMAX) for genome wide association (GWA) analysis fits all marker effects as classical random effects (i.e., Gaussian prior) by which association for the specific marker of interest is inferred by treating its effect as fixed. It seems more statistically coherent to specify all markers as sharing the same prior distribution, whether it is Gaussian, heavy-tailed (BayesA), or has variable selection specifications based on a mixture of, say, two Gaussian distributions (SSVS). Furthermore, all such GWA inference should be formally based on posterior probabilities or test statistics as we present here, rather than merely being based on point estimates. We compared these three broad categories of priors within a simulation study to investigate the effects of different degrees of skewness for quantitative trait loci (QTL) effects and numbers of QTL using 43,266 SNP marker genotypes from 922 Duroc-Pietrain F2 cross pigs. Genomic regions were based either on single SNP associations, on non-overlapping windows of various fixed sizes (0.5 to 3 Mb) or on adaptively determined windows that cluster the genome into blocks based on linkage disequilibrium (LD). We found that SSVS and BayesA lead to the best receiver operating curve properties in almost all cases. We also evaluated approximate marginal a posteriori (MAP) approaches to BayesA and SSVS as potential computationally feasible alternatives; however, MAP inferences were not promising, particularly due to their sensitivity to starting values. We determined that it is advantageous to use variable selection specifications based on adaptively constructed genomic window lengths for GWA studies.\n\nSUMMARYGenome wide association (GWA) analyses strategies have been improved by simultaneously fitting all marker effects when inferring upon any single marker effect, with the most popular distributional assumption being normality. Using data generated from 43,266 genotypes on 922 Duroc-Pietrain F2 cross pigs, we demonstrate that GWA studies could particularly benefit from more flexible heavy-tailed or variable selection distributional assumptions. Furthermore, these associations should not just be based on single markers or even genomic windows of markers of fixed physical distances (0.5 - 3.0 Mb) but based on adaptively determined genomic windows using linkage disequilibrium information.

genetics↗

bioRxiv2017

A new organotypic model of synaptic competition reveals activity-dependent localization of C1q

Immature neural circuits undergo synaptic refinement, in which activity-dependent competition between synapses results in pruning of inappropriate connections and maintenance of appropriate ones. A longstanding question is how neuronal activity eliminates specific synapses based on their strength. The technical challenges of in vivo studies have made it difficult to identify a molecular link between decreased activity and synapse elimination. We developed an organotypic coculture model of the mouse retinogeniculate system that facilitates real-time imaging and elucidation of molecular mechanisms underlying the removal of less active synapses during synaptic competition. Using this model we show for the first time that complement component C1q is necessary for activity-dependent synaptic competition and preferentially localizes to less active, competing presynaptic inputs. In conjunction with classic in vivo and ex vivo models, this coculture model is a new tool to reveal molecular pathways underlying CNS circuit refinement.

neuroscience↗

bioRxiv2017

Contrasting determinants of mutation rates in germline and soma

Recent studies of somatic and germline mutations have led to the identification of a number of factors that influence point mutation rates, including CpG methylation, expression levels, replication timing and GC content. Intriguingly, some of the effects appear to differ between soma and germline: in particular, whereas mutation rates have been reported to decrease with expression levels in tumors, no clear effect has been detected in the germline. Distinct approaches were taken to analyze the data, however, so it is hard to know whether these apparent differences are real. To enable a cleaner comparison, we considered a statistical model in which the mutation rate of a coding region is predicted by GC content, expression levels, replication timing, and two histone repressive marks. We applied this model to both a set of germline mutations identified in exomes and to exonic somatic mutations in four types of tumors. Germline and soma share most determinants of mutations; notably, we detected an effect of expression levels on germline mutations as well as on somatic ones. However, whereas in somatic tissues, increased expression levels are associated with greater strand asymmetry and decreased mutation rates, in ovaries and testes, increased expression leads to greater strand asymmetry but increased mutation rates. This contrast points to differences in damage or repair rates during transcription in soma and germline.

evolutionary biology↗

bioRxiv2017

A lineage-related reciprocal inhibition circuitry for sensory-motor action selection

The insect central complex and vertebrate basal ganglia are forebrain centres involved in selection and maintenance of behavioural actions. However, little is known about the formation of the underlying circuits, or how they integrate sensory information for motor actions. Here, we show that paired embryonic neuroblasts generate central complex ring neurons that mediate sensory-motor transformation and action selection in Drosophila. Lineage analysis resolves four ring neuron subtypes, R1-R4, that form GABAergic inhibition circuitry among inhibitory sister cells. Genetic manipulations, together with functional imaging, demonstrate subtype-specific R neurons mediate the selection and maintenance of behavioural activity. A computational model substantiates genetic and behavioural observations suggesting that R neuron circuitry functions as salience detector using competitive inhibition to amplify, maintain or switch between activity states. The resultant gating mechanism translates facilitation, inhibition and disinhibition of behavioural activity as R neuron functions into selection of motor actions and their organisation into action sequences.

neuroscience↗

bioRxiv2017

BGDMdocker: an workflow base on Docker for analysis and visualization pan-genome and biosynthetic gene clusters of Bacterial

MotivationAt present Docker technology has received increasing level of attention throughout the bioinformatics community. However, its implementation details have not yet been mastered by most biologists and applied widely in biological researches. In order to popularizing this technology in the bioinformatics and sufficiently use plenty of public resources of bioinformatics tools (Dockerfile and image of scommunity, officially and privately) in Docker Hub Registry and other Docker sources based on Docker, we introduced full and accurate instance of a bioinformatics workflow based on Docker to analyse and visualize pan-genome and biosynthetic gene clusters of a bacteria in this article, provided the solutions for mining bioinformatics big data from various public biology databases. You could be guided step-by-step through the workflow process from docker file to build up your own images and run an container fast creating an workflow.\n\nResultsWe presented a BGDMdocker (bacterial genome data mining docker-based) workflow based on docker. The workflow consists of three integrated toolkits, Prokka v1.11, panX, and antiSMASH3.0. The dependencies were all written in Dockerfile, to build docker image and run container for analysing pan-genome of total 44 Bacillus amyloliquefaciens strains, which were retrieved from public? database. The pan-genome totally includes 172,432 gene, 2,306 Core gene cluster. The visualized pan-genomic data such as alignment, phylogenetic trees, maps mutations within that cluster to the branches of the tree, infers loss and gain of genes on the core-genome phylogeny for each gene cluster were presented. Besides, 997 known (MIBiG database) and 553 unknown (antiSMASH-predicted clusters and Pfam database) genes of biosynthesis gene clusters types and orthologous groups were mined in all strains. This workflow could also be used for other species pan-genome analysis and visualization. The display of visual data can completely duplicated as well as done in this paper. All result data and relevant tools and files can be downloaded from our website with no need to register. The pan-genome and biosynthetic gene clusters analysis and visualization can be fully reusable immediately in different computing platforms (Linux, Windows, Mac and deployed in the cloud), achieved cross platform deployment flexibility, rapid development integrated software package.\n\nAvailability and implementationBGDMdocker is available at http://42.96.173.25/bapgd/ and the source code under GPL license is available at https://github.com/cgwyx/debian_prokka_panx_antismash_biodocker.\n\nContactchenggongwyx@foxmail.com\n\nSupplementary informationSupplementary data are available at biorxiv online.

bioinformatics↗

bioRxiv2016

Tracking stem cell differentiation without biomarkers using pattern recognition and phase contrast imaging

Bio-image informatics is the systematic application of image analysis algorithms to large image datasets to provide an objective method for accurately and consistently scoring image data. Within this field, pattern recognition (PR) is a form of supervised machine learning where the computer identifies relevant patterns in groups (classes) of images after being trained on examples. Rather than segmentation, image-specific algorithms or adjustable parameter sets, PR relies on extracting a common set of image descriptors (features) from the entire image to determine similarities and differences between image classes.\n\nGross morphology can be the only available description of biological systems prior to their molecular characterization, but these descriptions can be subjective and qualitative. In principle, generalized PR can provide an objective and quantitative characterization of gross morphology, thus providing a means of computationally defining morphological biomarkers. In this study, we investigated the potential of a pattern recognition approach to a problem traditionally addressed using genetic or biochemical biomarkers. Often these molecular biomarkers are unavailable for investigating biological processes that are not well characterized, such as the initial steps of stem cell differentiation.\n\nHere we use a general contrast technique combined with generalized PR software to detect subtle differences in cellular morphology present in early differentiation events in murine embryonic stem cells (mESC) induced to differentiate by the overexpression of selected transcription factors. Without the use of reporters, or a priori knowledge of the relevant morphological characteristics, we identified the earliest differentiation event (3 days), reproducibly distinguished eight morphological trajectories, and correlated morphological trajectories of 40 mESC clones with previous micro-array data. Interestingly, the six transcription factors that caused the greatest morphological divergence from an ESC-like state were previously shown by expression profiling to have the greatest influence on the expression of downstream genes.

cell biology↗

bioRxiv2016

Millstone: Software for Multiplex Microbial Genome Analysis and Engineering

Inexpensive DNA sequencing and advances in genome editing have made computational analysis a major rate-limiting step in adaptive laboratory evolution and microbial genome engineering. We describe Millstone, a web-based platform which automates genotype comparison and visualization for projects with up to hundreds of genomic samples. To enable iterative genome engineering, Millstone allows users to design oligonucleotide libraries and create successive versions of reference genomes. Millstone is open source and easily deployable to a cloud platform, local cluster, or desktop, making it a scalable solution for any lab.

synthetic biology↗