Biology subjects

Chen, C.-L.

Publications and source records attributed to Chen, C.-L..

34 records · Page 2Linked to original sources

bioRxiv2021

GC content but not nucleosome positioning directly contributes to intron-splicing efficiency in Paramecium

Eukaryotic genes are interrupted by introns that must be accurately spliced from mRNA precursors. With an average length of 25 nt, the >90,000 introns of Paramecium tetraurelia stand among the shortest introns reported in eukaryotes. The mechanisms specifying the correct recognition of these tiny introns remain poorly understood. Splicing can occur co-transcriptionally and it has been proposed that chromatin structure might influence splice site recognition. To investigate the roles of nucleosome positioning in intron recognition, we determined the nucleosome occupancy along the P. tetraurelia genome. We showed that P. tetraurelia displays a regular nucleosome array with a nucleosome repeat length of [~]151 bp, amongst the smallest periodicities reported. Our analysis revealed that introns are frequently associated with inter-nucleosomal DNA, pointing to an evolutionary constraint to locate introns at the AT-rich nucleosome edge sequences. Using accurate splicing efficiency data from cells depleted for the nonsense-mediated decay effectors, we showed that introns located at the edge of nucleosomes display higher splicing efficiency than those at the centre. However, multiple regression analysis indicated that the GC content, rather than nucleosome positioning, directly contributes to intron splicing efficiency. Our data reveal a complex link between GC content, nucleosome positioning and intron evolution in Paramecium.

genomics↗

bioRxiv2020

Intrinsic disorder codes for leaps of protein expression

Serine(S)/threonine(T)-glutamine(Q) cluster domains (SCDs), polyglutamine (polyQ) tracts and polyglutamine/asparagine (polyQ/N) tracts are Q-rich motifs found in many proteins. SCDs often are intrinsically disordered regions that mediate protein phosphorylation and protein-protein interactions. PolyQ and polyQ/N tracts are structurally flexible sequences that trigger protein aggregation. We report that due to their high percentages of STQ or STQN amino acid content, four SCDs and three prion-causing Q/N-rich motifs of yeast proteins possess autonomous protein expression-enhancing activities. Since these Q-rich motifs can endow proteins with structural and functional plasticity, we suggest that they represent useful toolkits for evolutionary novelty. Comparative Gene Ontology (GO) analyses of the near-complete proteomes of 27 representative model eukaryotes reveal that Q-rich motifs prevail in proteins involved in specialized biological processes, including Saccharomyces cerevisiae RNA-mediated transposition and pseudohyphal growth, Candida albicans filamentous growth, ciliate peptidyl-glutamic acid modification and microtubule-based movement, Tetrahymena thermophila xylan catabolism and meiosis, Dictyostelium discoideum development and sexual cycles, Plasmodium falciparum infection, and the nervous systems of Drosophila melanogaster, Mus musculus and Homo sapiens. We also show that Q-rich-motif proteins are expanded massively in ten ciliates with reassigned TAAQ and TAGQ codons. Notably, the usage frequency of CAGQ is much lower in ciliates with reassigned TAAQ and TAGQ codons than in organisms with expanded and unstable Q runs (e.g., D. melanogaster and H. sapiens), indicating that the use of noncanonical stop codons in ciliates may have coevolved with codon usage biases to avoid triplet repeat disorders mediated by CAG/GTC replication slippage.

biochemistry↗

bioRxiv2020

Unscheduled origin building in S-phase upon tight CDK1 inhibition suppresses CFS instability

Genome integrity requires replication to be completed before chromosome segregation. This coordination essentially relies on replication-dependent activation of a dedicated checkpoint that inhibits CDK1, delaying mitotic onset. Under-replication of Common Fragile Sites (CFSs) however escapes surveillance, which triggers chromosome breakage. Using human cells, we asked here whether such leakage results from insufficient CDK1 inhibition under modest stresses used to destabilize CFSs. We found that tight CDK1 inhibition suppresses CFS instability. Repli-Seq and molecular combing analyses consistently showed a burst of replication initiations in mid S phase across large origin-poor domains shaped by transcription, including CFSs. Strikingly, CDC6 or CDT1 depletion or CDC7-DBF4 inhibition during the S phase prevented both extra-initiations and CFS rescue, showing that CDK1 inhibition promotes targeted and mistimed building of functional extra-origins. In addition to delay mitotic onset, checkpoint activation therefore advances replication completion of chromosome domains at risk of under-replication, two complementary roles preserving genome stability.

cell biology↗

bioRxiv2020

Multimodal Brain Age Gap as a Mediating Indicator in the Relation between Modifiable Dementia Risk Factors and Cognitive Functioning

IntroductionAs a structural proxy for evaluating brain health, neuroimaging-based brain age gap (BAG) is presumed to link the dementia risks to cognitive changes in the premorbid phase, but this remains unclear. MethodsBrain age prediction models were constructed and applied to a population-based cohort (N=371) to estimate their BAG. Further, structural equation modeling was employed to investigate the mediation effect of BAG between risk levels (assessed by 2 dementia-related risk scores) and cognitive changes (examined by 4 cognitive assessments). ResultsA higher burden of modifiable dementia risk factors was causally associated with a greater cognitive decline, and this was significantly mediated (P=0.017) by a larger multimodal BAG, which indicated an older brain. Moreover, a steeper slope (P=0.020) of association between cognitive decline and multimodal BAG was observed when individuals had higher dementia risks. DiscussionMultimodal BAG is a potential mediating indicator to reflect the changes in the pathophysiological mechanism of cognitive aging.

neuroscience↗

bioRxiv2020

Genome-Wide Mapping of Human DNA Replication by Optical Replication Mapping Supports a Stochastic Model of Eukaryotic Replication Timing

DNA replication is regulated by the location and timing of replication initiation. Therefore, much effort has been invested in identifying and analyzing the sites of human replication initiation. However, the heterogeneous nature of eukaryotic replication kinetics and the low efficiency of individual initiation site utilization in metazoans has made mapping the location and timing of replication initiation in human cells difficult. A potential solution to the problem of human replication mapping is single-molecule analysis. However, current approaches do not provide the throughput required for genome-wide experiments. To address this challenge, we have developed Optical Replication Mapping (ORM), a high-throughput single-molecule approach to map newly replicated DNA, and used it to map early initiation events in human cells. The single-molecule nature of our data, and a total of more than 2000-fold coverage of the human genome on 27 million fibers averaging ~300 kb in length, allow us to identify initiation sites and their firing probability with high confidence. In particular, for the first time, we are able to measure genome-wide the absolute efficiency of human replication initiation. We find that the distribution of human replication initiation is consistent with inefficient, stochastic initiation of heterogeneously distributed potential initiation complexes enriched in accessible chromatin. In particular, we find sites of human replication initiation are not confined to well-defined replication origins but are instead distributed across broad initiation zones consisting of many initiation sites. Furthermore, we find no correlation of initiation events between neighboring initiation zones. Although most early initiation events occur in early-replicating regions of the genome, a significant number occur in late-replicating regions. The fact that initiation sites in typically late-replicating regions have some probability of firing in early S phase suggests that the major difference between initiation events in early and late replicating regions is their intrinsic probability of firing, as opposed to a qualitative difference in their firing-time distributions. Moreover, modeling of replication kinetics demonstrates that measuring the efficiency of initiation-zone firing in early S phase suffices to predict the average firing time of such initiation zones throughout S phase, further suggesting that the differences between the firing times of early and late initiation zones are quantitative, rather than qualitative. These observations are consistent with stochastic models of initiation-timing regulation and suggest that stochastic regulation of replication kinetics is a fundamental feature of eukaryotic replication, conserved from yeast to humans.

genomics↗

bioRxiv2020

Mutant glucocorticoid receptor binding elements on Interleukin-6 promoter regulate dexamethasone effects

Glucocorticoid has been widely used as an important modulator for clinical infectious and inflammatory disease. Glucocorticoid receptor (GR) is a transcription factor belonging to the family of nuclear receptors, regulated anti-inflammatory process and the release of pro-inflammatory cytokines. Five putative GR and other transcription factor binding sites on interleukin (IL)-6 promoter were identified and dexamethasone could reduce LPS-induced IL-6 release. Among them, the mutant transcriptional factors NF-{kappa}B, AP-1, and Sp1-2 site decreased the basal and effects of lipopolysaccharide (LPS)-induced IL-6 promoter activities in different responses. GR2/3 seemed to be an important role in both basal and inducible promoter activities in LPS-induced inflammation. We concluded that the selective GR2/3 modulators may have agonistic and antagonistic combined effects and activate important signaling pathway during LPS-stimulated inflammatory process.

immunology↗

bioRxiv2019

Sustained modulation of emotion-related fibers after 8 weeks of mindfulness-based stress reduction training

An 8-week mindfulness-based stress reduction (MBSR) program enables novices to learn and practice mindfulness meditation. A longitudinal study design has been used in prior research to investigate the effect of short-term mindfulness meditation training on brain structures. Many studies have demonstrated microstructural changes in the white matter by comparing baseline measurements with measurements obtained immediately after short-term meditation training. However, these studies did not clarify the evolution of the modulated microstructures several months after mindfulness meditation practice is discontinued. Therefore, in this study, we recruited 13 novice practitioners and administered an 8-week MBSR training program. We extended the span of the longitudinal study by adding a third measurement taken approximately 6 months after the second time point. Diffusion indices derived from diffusion spectrum imaging were used to quantify the temporal changes in modulation across three time points. The analysis identified four tract bundles that were significantly modulated after the 8-week MBSR training program, namely the callosal fibers connecting the bilateral amygdalae and bilateral hippocampi, right thalamic radiation of the auditory nerve, and right uncinate fasciculus. At the third time point, at which the participants had discontinued practice for approximately 6 months, the diffusion indices of the four tract bundles still presented a significant difference compared with the baseline. Our results indicate that the modulation of microstructural properties of the white matter tract induced by the 8-week MBSR program was sustained after completion of the program and support that neuroplasticity in brain connection persists after the discontinuation of meditation training.

neuroscience↗

bioRxiv2019

SNP-CRISPR: a web tool for SNP-specific genome editing

CRISPR-Cas9 is a powerful genome editing technology in which a single guide RNA (sgRNA) confers target site specificity to achieve Cas9-mediated genome editing. Numerous sgRNA design tools have been developed based on reference genomes for humans and model organisms. However, existing resources are not optimal as genetic mutations or single nucleotide polymorphisms (SNPs) within the targeting region affect the efficiency of CRISPR-based approaches by interfering with guide-target complementarity. To facilitate identification of sgRNAs (1) in non-reference genomes, (2) across varying genetic backgrounds, or (3) for specific targeting of SNP-containing alleles, for example, disease relevant mutations, we developed a web tool, SNP-CRISPR (https://www.flyrnai.org/tools/snp_crispr/). SNP-CRISPR can be used to design sgRNAs based on public variant data sets or user-identified variants. In addition, the tool computes efficiency and specificity scores for sgRNA designs targeting both the variant and the reference. Moreover, SNP-CRISPR provides the option to upload multiple SNPs and target single or multiple nearby base changes simultaneously with a single sgRNA design. Given these capabilities, SNP-CRISPR has a wide range of potential research applications in model systems and for design of sgRNAs for disease-associated variant correction.

bioinformatics↗

bioRxiv2019

Serotonergic modulation of walking in Drosophila

To navigate complex environments, animals must generate highly robust, yet flexible, locomotor behaviors. For example, walking speed must be tailored to the needs of a particular environment: Not only must animals choose the correct speed and gait, they must also rapidly adapt to changing conditions, and respond to sudden and surprising new stimuli. Neuromodulators, particularly the small biogenic amine neurotransmitters, allow motor circuits to rapidly alter their output by changing their functional connectivity. Here we show that the serotonergic system in the vinegar fly, Drosophilamelanogaster, can modulate walking speed in a variety of contexts and in response to sudden changes in the environment. These multifaceted roles of serotonin in locomotion are differentially mediated by a family of serotonergic receptors with distinct activities and expression patterns.

neuroscience↗

bioRxiv2019

TRANSCRIPTION-MEDIATED ORGANIZATION OF THE REPLICATION INITIATION PROGRAM ACROSS LARGE GENES SETS UP COMMON FRAGILE SITES GENOME-WIDE

Common Fragile Sites (CFSs) are chromosome regions prone to breakage under replication stress, known to drive chromosome rearrangements during oncogenesis. Most CFSs nest in large expressed genes, suggesting that transcription elicits their instability but the underlying mechanisms remained elusive. Analyses of genome-wide replication timing of human lymphoblasts here show that stress-induced delayed/under-replication is the hallmark of CFSs. Extensive genome-wide analyses of nascent transcripts, replication origin positioning and fork directionality reveal that 80% of CFSs nest in large transcribed domains poor in initiation events, thus replicated by long-traveling forks. In contrast to formation of sequence-dependent fork barriers or head-on transcription-replication conflicts, traveling-long in late S phase explains CFS replication features. We further show that transcription inhibition during the S phase, which excludes the setting of new replication origins, fails to rescue CFS stability. Altogether, results show that transcription-dependent suppression of initiation events delays replication of large gene body, committing them to instability.

genomics↗

bioRxiv2019

High Spatial Resolution Ambient Ionization Mass Spectrometry Imaging Using Microscopy Image Fusion

Mass spectrometry imaging (MSI) using ambient ionization technique enables a direct chemical investigation of biological samples with minimal sample pretreatment. However, detailed morphological information of the sample is often lost due to its limited spatial resolution. In this study, predictive high-resolution molecular imaging was produced by the fusion of ambient ionization MSI with optical microscopy of routine hematoxylin and eosin (H&E) staining produces. Specifically, desorption electrospray ionization (DESI) and nanospray desorption electrospray ionization (nanoDESI) mass spectrometry are employed to visualize lipid and protein species on mice tissue sections. The resulting molecular distributions obtained by ambient ionization MSI-microscopy fusion are verified with matrix-assisted laser desorption ionization time-of-flight (MALDI-TOF) MSI and immunohistochemistry (IHC) staining. Label-free molecular imaging with 5-m spatial resolution can be acquired using DESI and nanoDESI, whereas the typical spatial resolution of ambient ionization MSI is ~100 m. In this regard, sharpened molecular histology of tissue sections is achieved, providing complementary references to the pathology. Such a multimodality integration enables the discovery of potential tumor biomarkers. After image fusion, more than a dozen of potential biomarkers that could be used to determine the tumor margins on a metastatic mouse lung tissue section and Luminal B breast tumor tissue section are identified.

bioinformatics↗

bioRxiv2019

Dense neuronal reconstruction through X-ray holographic nano-tomography

Elucidating the structure of neuronal networks provides a foundation for understanding how the nervous system processes information to generate behavior. Despite technological breakthroughs in visible light and electron microscopy, imaging dense nanometer-scale neuronal structures over millimeter-scale tissue volumes remains a challenge. Here, we demonstrate that X-ray holographic nano-tomography is capable of imaging large tissue volumes with sufficient resolution to disentangle dense neuronal circuitry in Drosophila melanogaster and mammalian central and peripheral nervous tissue. Furthermore, we show that automatic segmentation using convolutional neural networks enables rapid extraction of neuronal morphologies from these volumetric datasets. The technique we present allows rapid data collection and analysis of multiple specimens, and can be used correlatively with light microscopy and electron microscopy on the same samples. Thus, X-ray holographic nano-tomography provides a new avenue for discoveries in neuroscience and life sciences in general.

neuroscience↗

bioRxiv2019

Two different pathways for initiation of Trichoderma reesei Rad51-only meiotic recombination

BackgroundMeiotic recombination is mainly, but not exclusively, initiated by Spo11-induced double strand breaks (DSBs) in some sexual eukaryotes. DSBs are repaired by one or two RecA-like recombinases (ubiquitous Rad51 and meiosis-specific Dmc1). In yeast and mammals, Dmc1 is superior to Rad51 in tolerating mismatched sequences during highly polymorphic hybrid meiosis. The mechanisms underlying Rad51-only meiotic recombination remain less studied.\n\nResultsThe Rad51-only filamentous fungus Trichoderma reesei has only one spo11 gene. Removal of spo11 from T. reesei genome does not affect normal sexual development, meiosis or chromosome synapsis, but results in decrease of interhomolog recombination products to 70%, crossover homeostasis and lower genetic interference. Our results also suggest that T. reesei Rad51, like yeast and mammalian Dmc1 (but not Rad51), can tolerate mismatched sequences during meiotic recombination. Moreover, Topoisomerase II might act redundantly (and predominantly) with Spo11 to initiate meiotic recombination.\n\nConclusionWe suggest that T. reesei is an emerging model for studying Spo11-independent and Rad51-only meiosis.

genetics↗

bioRxiv2019

Patterned cell death sculpts functional and adaptive neural networks in flies

Changes to the structure and function of neural networks are thought to underlie the evolutionary adaptation of animal behaviours. Among the many developmental phenomena that generate change programmed cell death appears to play a key role. We show that cell death occurs continuously throughout insect neurogenesis and happens soon after neurons are born. Focusing on two dipterans which have lost flight during evolution we reveal that reductions in populations of flight interneurons are likely caused by increased cell death during development. Mimicking an evolutionary role for increasing cell numbers, we artificially block programmed cell death in the medial neuroblast lineage in Drosophila melanogaster, which results in the production of undead neurons with complex arborisations and distinct neurotransmitter identities. Activation of these undead neurons and recordings of neural activity in behaving animals demonstrate that they are functional. Our findings suggest that the evolutionary modulation of death-based patterning could generate novel network configurations.

neuroscience↗

bioRxiv2018

Imaging neural activity in the ventral nerve cord of behaving adult Drosophila

To understand neural circuits that control limbs, one must measure their activity during behavior. Until now this goal has been challenging, because the portion of the nervous system that contains limb premotor and motor circuits is largely inaccessible to large-scale recording techniques in intact, moving animals - a constraint that is true for both vertebrate and invertebrate models. Here, we introduce a method for 2-photon functional imaging from the ventral nerve cord of behaving adult Drosophila melanogaster. We use this method to reveal patterns of activity across nerve cord populations during grooming and walking and to uncover the functional encoding of moonwalker ascending neurons (MANs), moonwalker descending neurons (MDNs), and a novel class of locomotion-associated descending neurons. This new approach enables the direct investigation of circuits associated with complex limb movements.

neuroscience↗

bioRxiv2017

Trichoderma reesei Complete Genome Sequence, Repeat-Induced Point Mutation And Partitioning Of CAZyme Gene Clusters

Trichoderma reesei (Ascomycota, Pezizomycotina) QM6a is a model fungus for a broad spectrum of physiological phenomena, including plant cell wall degradation, industrial production of enzymes, light responses, conidiation, sexual development, polyketide biosynthesis and plant-fungal interactions. The genomes of QM6a and its high-enzyme producing mutants have been sequenced by second-generation-sequencing methods and are publicly available from the Joint Genome Institute (JGI). While these genome sequences have offered useful information for genomic and transcriptomic studies, their limitations and especially their short read lengths make them poorly suited for some particular biological problems, including assembly, genome-wide determination of chromosome architecture and genetic modification or engineering. We integrated Pacific Biosciences and Illumina sequencing platforms for the highest-quality genome assembly yet achieved, revealing seven telomere-to-telomere chromosomes (34,922,528 bp; 10877 genes) with 1630 newly-predicted genes and >1.5 Mb of new sequences. Most new sequences are located on AT-rich blocks, including 7 centromeres, 14 subtelomeres and 2329 interspersed AT-rich blocks. The seven QM6a centromeres separately consist of 24 conserved repeats and 37 putative centromere-encoded genes. These findings open up a new perspective for future centromere and chromosome architecture studies. Next, we demonstrate that sexual crossing readily induced cytosine-to-thymine point mutations on both tandem and unlinked duplicated sequences. We also show by bioinformatic analysis that Trichoderma reesei has evolved a robust repeat-induced point mutation (RIP) system to accumulate AT-rich sequences, with longer AT-rich blocks having more RIP mutations. The widespread distribution of AT-rich blocks correlates genome-wide partitions with gene clusters, explaining why clustering of genes has been reported to not influence gene expression in Trichoderma reesei. Compartmentation of ancestral gene clusters by AT-rich blocks might promote flexibilities that are evolutionarily advantageous in this fungus soil habitats and other natural environments. Our analyses, together with the complete genome sequence, provide a better blueprint for biotechnological and industrial applications.

genomics↗