Biology subjects

Chen, Y.-J.

Publications and source records attributed to Chen, Y.-J..

47 records · Page 3Linked to original sources

bioRxiv2019

Distinct life histories impact dikaryotic genome evolution in the rust fungus Puccinia striiformis causing stripe rust in wheat

Stripe rust of wheat, caused by the obligate biotrophic fungus Puccinia striiformis f. sp. tritici, is a major threat to wheat production world-wide with an estimated yearly loss of US $1 billion. The recent advances in long-read sequencing technologies and tailored-assembly algorithms enabled us to disentangle the two haploid genomes of Pst. This provides us with haplotype-specific information at a whole-genome level. Exploiting this novel information, we perform whole genome comparative genomics of two P. striiformis f. sp. tritici isolates with contrasting life histories. We compare one isolate of the old European lineage (PstS0), which has been asexual for over 50 years, and a Warrior isolate (PstS7 lineage) from a novel incursion into Europe in 2011 from a sexual population in the Himalayan region. This comparison provides evidence that long-term asexual evolution leads to genome expansion, accumulation of transposable elements, and increased heterozygosity at the single nucleotide, structural and allele levels. At the whole genome level, candidate effectors are not compartmentalized and do not exhibit reduced levels of synteny. Yet we were able to identify two subsets of candidate effector populations. About 70% of candidate effectors are invariant between the two isolates while 30% are hypervariable. The latter might be involved in host adaptation on wheat and explain the different phenotypes of the two isolates. Overall this detailed comparative analysis of two haplotype-aware assemblies of P. striiformis f. sp. tritici are the first steps in understanding the evolution of dikaryotic rust fungi at a whole genome level.

evolutionary biology↗

bioRxiv2019

Genome-wide, integrative analysis implicates circular RNA dysregulation in autism and the corresponding circular RNA-microRNA-mRNA regulatory axes

Circular RNAs (circRNAs), a class of long non-coding RNAs, are known to be enriched in mammalian brain and neural tissues. While the effects of regulatory genetic variants on gene expression in autism spectrum disorder (ASD) have been widely reported, the role of circRNAs in ASD remains largely unknown. Here, we performed genome-wide circRNA expression profiling in post-mortem brains from individuals with ASD and controls and identified 60 circRNAs and three co-regulated modules that were perturbed in ASD. By integrating circRNA, microRNA, and mRNA dysregulation data derived from the same cortex samples, we identified 8,170 ASD-associated circRNA-microRNA-mRNA interactions. Putative targets of the axes were enriched for ASD risk genes and genes encoding inhibitory postsynaptic density (PSD) proteins, but not for genes implicated in monogenetic forms of other brain disorders or genes encoding excitatory PSD proteins. This result reflects the previous observation that ASD-derived organoids exhibit overproduction of inhibitory neurons. We further confirmed that some ASD risk genes (NLGN1, STAG1, HSD11B1, VIP, and UBA6) were indeed regulated by an upregulated circRNA (circARID1A) via sponging a downregulated microRNA (miR-204-3p) in human neuronal cells. We provided a systems-level view of landscape of circRNA regulatory networks in ASD cortex samples. We also provided multiple lines of evidence for the functional role of ASD for circRNA dysregulation and a rich set of ASD-associated circRNA candidates and the corresponding circRNA-miRNA-mRNA axes, particularly those involving ASD risk genes. Our findings thus support a role for circRNA dysregulation and the corresponding circRNA-microRNA-mRNA axes in ASD pathophysiology.

bioinformatics↗

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

Experimental Assessment of PCR Specificity and Copy Number for Reliable Data Retrieval in DNA Storage

Synthetic DNA has been gaining momentum as a potential storage medium for archival data storage1-9. Digital information is translated into sequences of nucleotides and the resulting synthetic DNA strands are then stored for later individual file retrieval via PCR7-9 (Fig. 1a). Using a previously presented encoding scheme9 and new experiments, we demonstrate reliable file recovery when as few as 10 copies per sequence are stored, on average. This results in density of about 17 exabytes/g, nearly two orders of magnitude greater than prior work has shown6. Further, no prior work has experimentally demonstrated access to specific files in a pool more complex than approximately 106 unique DNA sequences9, leaving the issue of accurate file retrieval at high data density and complexity unexamined. Here, we demonstrate successful PCR random access using three files of varying sizes in a complex pool of over 1010 unique sequences, with no evidence that we have begun to approach complexity limits. We further investigate the role of file size on successful data recovery, the effect of increasing sequencing coverage to aid file recovery, and whether DNA strands drop out of solution in a systematic manner. These findings substantiate the robustness of PCR as a random access mechanism in complex settings, and that the number of copies needed for data retrieval does not compromise density significantly.\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=111 SRC=\"FIGDIR/small/565150_fig1.gif\" ALT=\"Figure 1\">\nView larger version (29K):\norg.highwire.dtl.DTLVardef@4b79a7org.highwire.dtl.DTLVardef@11fca45org.highwire.dtl.DTLVardef@18b830org.highwire.dtl.DTLVardef@e47101_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFigure 1.C_FLOATNO (a) A high-level representation of the DNA data storage pipeline. (b) (Left) The bar chart depicts contents of the initial, undiluted pool. (Right) The illustration shows the serial nature of subsequent dilutions. Mean copy number refers to the mean number of copies of each files unique sequences as determined by qPCR (Supplemental Section 1). One serial dilution used water as the diluent in each step; the other used a solution of 150Nmers to dilute the pool to much greater complexity. (c) Details of how the samples were diluted. Note that the dilution steps were identical regardless of diluent. The smallest percent of pool accessed is calculated by dividing the size of the smallest file by the number of unique sequences in the 1 {micro}L of solution used for PCR random access. This percentage refers to the 150N diluent pool since the small file in the water diluent pool is a constant 0.13%.\n\nC_FIG

synthetic biology↗

bioRxiv2019

Quantifying Molecular Bias in DNA Data Storage

DNA has recently emerged as an attractive medium for future digital data storage because of its extremely high information density and potential longevity. Recent work has shown promising results in developing proof-of-principle prototype systems. However, very uneven (biased) sequencing coverage distributions have been reported, which indicates inefficiencies in the storage process and points to optimization opportunities. These deviations from the average coverage in oligonucleotide copy distribution result in sequence drop-out and make error-free data retrieval from DNA more challenging. The uneven copy distribution was believed to stem from the underlying molecular processes, but the interplay between these molecular processes and the copy number distribution has been poorly understood until now. In this paper, we use millions of unique sequences from a DNA-based digital data archival system to study the oligonucleotide copy unevenness problem and show that two important sources of bias are the synthesis process and the Polymerase Chain Reaction (PCR) process. By mapping the sequencing coverage of a large complex oligonucleotide pool back to its spatial distribution on the synthesis chip, we find that significant bias comes from array-based oligonucleotide synthesis. We also find that PCR stochasticity is another main driver of oligonucleotide copy variation. Based on these findings, we develop a statistical model for each molecular process as well as the overall process and compare the predicted bias with our experimental data. We further use our model to explore the trade-offs between synthesis bias, storage physical density and sequencing redundancy, providing insights for engineering efficient, robust DNA data storage systems.

synthetic biology↗

bioRxiv2018

Two Chloroflexi classes independently evolved the ability to persist on atmospheric hydrogen and carbon monoxide

Bacteria within aerated environments often exist within a variety of dormant forms. In these states, bacteria endure adverse environmental conditions such as organic carbon starvation by decreasing metabolic expenditure and using alternative energy sources. In this study, we investigated the energy sources that facilitate the persistence of the environmentally widespread but understudied bacterial phylum Chloroflexi. A transcriptome study revealed that Thermomicrobium roseum (class Chloroflexia) extensively remodels its respiratory chain upon entry into stationary phase due to organic carbon limitation. Whereas primary dehydrogenases associated with heterotrophic respiration were downregulated, putative operons encoding enzymes involved in molecular hydrogen (H2), carbon monoxide (CO), and sulfur compound oxidation were significantly upregulated. Gas chromatography and microsensor experiments were used to show that T. roseum aerobically respires H2 and CO at a range of environmentally relevant concentrations to sub-atmospheric levels. Phylogenetic analysis suggests that the enzymes mediating atmospheric H2 and CO oxidation, namely group 1h [NiFe]-hydrogenases and type I carbon monoxide dehydrogenases, are widely distributed in Chloroflexi genomes and have been acquired on at least two occasions through separate horizontal gene transfer events. Consistently, we confirmed that the sporulating isolate Thermogemmatispora sp. T81 (class Ktedonobacteria) also oxidises atmospheric H2 and CO during persistence. This study provides the first axenic culture evidence that atmospheric CO supports bacterial persistence and reports the third phylum to be experimentally shown to mediate the biogeochemically and ecologically important process of atmospheric H2 oxidation. This adds to the growing body of evidence that atmospheric trace gases serve as dependable energy sources for the survival of dormant microorganisms.

microbiology↗

bioRxiv2018

Glucose intake hampers PKA-regulated HSP90 chaperone activity

Aging is an intricate phenomenon associated with the gradual loss of physiological functions, and both nutrient sensing and proteostasis control lifespan. Although multiple approaches have facilitated the identification of candidate genes that govern longevity, the molecular mechanisms that link aging pathways are still elusive. Here, we conducted a quantitative mass spectrometry screen and identified all phosphorylation/dephosphorylation sites on yeast proteins that significantly responded to calorie restriction, a well-established approach to extend lifespan. Functional screening of 135 potential regulators uncovered that Ids2 is activated by PP2C under CR and inactivated by PKA under glucose intake. ids2{Delta} or ids2 phosphomimetic cells displayed heat sensitivity and lifespan shortening. Ids2 serves as a co-chaperone to form a complex with Hsc82 or the redundant Hsp82, and phosphorylation of Ids2 impedes its association with chaperone HSP90. Thus, PP2C and PKA orchestrate glucose sensing and protein folding to enable cells to maintain protein quality for sustained longevity.

cell biology↗

bioRxiv2017

EB1 binding provides a diffusion trap mechanism regulating STIM1 localization and Ca2+ signaling

The endoplasmic reticulum (ER) Ca2+ sensor STIM1 forms oligomers and translocates to ER-plasma membrane (PM) junctions to activate store-operated Ca2+ entry (SOCE) following ER Ca2+ depletion. STIM1 also directly interacts with end binding protein 1 (EB1) at microtubule (MT) plus-ends and resembles comet-like structures during time-lapse imaging. Nevertheless, the role of STIM1-EB1 interaction in regulating SOCE remains unresolved. Using live-cell imaging combined with pharmacological perturbation and a reconstitution approach, we revealed that EB1 binding constitutes a diffusion trap mechanism restricting STIM1 targeting to ER-PM junctions. We further showed that STIM1 oligomers retain EB1 binding ability in ER Ca2+-depleted cells. EB1 binding delayed the translocation of STIM1 oligomers to ER-PM junctions and recaptured STIM1 to prevent excess SOCE and ER Ca2+ overload. Thus, the counterbalance of EB1 binding and PM targeting of STIM1 shapes the kinetics and amplitude of local SOCE in regions with growing MTs, and contributes to precise spatiotemporal regulation of Ca2+ signaling crucial for cellular functions and homeostasis.\n\nSummarySTIM1 activates store-operated Ca2+ entry (SOCE) by translocating to endoplasmic reticulum-plasma membrane junctions. Chang et al. revealed that STIM1 localization and SOCE are regulated by a diffusion trap mechanism mediated by STIM1 binding to EB1 at growing microtubule ends.

cell biology↗

bioRxiv2017

Rapid, modular, and cost-effective generation of donor DNA constructs for CRISPR-based gene knock-in

CRISPR-based gene knock-in at endogenous sites is desirable in multiple fields such as quantitative studies of signal transduction pathways and gene regulation, synthetic biology, and disease modeling. Contrasting the knock-out procedure, a key step of CRISPR knock-in procedure relies on the homology-directed repairing (HDR) process that requires a donor construct as repair template. Therefore, it is desirable to generate a series of donor DNA constructs efficiently and cost-effectively. In this study, we developed a general Gibson assembly procedure that combines strengths of a Modular Overlap-Directed Assembly with Linkers (MODAL) strategy and a restriction enzyme based hierarchical framework. This procedure also allows fusing sgRNAs to the constructs for enhanced homology-directed repairing efficiency. Experimental tests on multiple constructs achieved from 3-8 folds of increase in assembly efficiency to high yield of constructs that failed to make with conventional Gibson assembly. The modularized procedure is simple, fast and cost-effective while making multiple constructs, and a computer package is provided for customized design.

molecular biology↗

bioRxiv2017

Cells Interpret Temporal Information From TGF-β Through A Nested Relay Mechanism

The detection and transmission of the temporal quality of intracellular and extracellular signals is an essential cellular mechanism. It remains largely unexplored how cells interpret the duration information of a stimulus. In this paper, through an integrated quantitative and computational approach we demonstrate that crosstalk among multiple TGF-{beta} activated pathways forms a relay from SMAD to GLI1 that initializes and maintains SNAILl expression, respectively. This transaction is smoothed and accelerated by another temporal switch from elevated cytosolic GSK3 enzymatic activity to reduced nuclear GSK3 enzymatic activity. The intertwined network places SNAIL1 as a key integrator of information from TGF-{beta} signaling subsequently distributed through upstream divergent pathways; essentially cells generate a transient or sustained expression of SNAIL1 depending on TGF-{beta} duration. Other signaling pathways may use similar network structure to encode duration information.

systems biology↗

bioRxiv2017

Scaling up DNA data storage and randomaccess retrieval

Current storage technologies can no longer keep pace with exponentially growing amounts of data. 1 Synthetic DNA offers an attractive alternative due to its potential information density of ~ 1018 B/mm3, 107 times denser than magnetic tape, and potential durability of thousands of years.2 Recent advances in DNA data storage have highlighted technical challenges, in particular, coding and random access, but have stored only modest amounts of data in synthetic DNA. 3,4,5 This paper demonstrates an end-to-end approach toward the viability of DNA data storage with large-scale random access. We encoded and stored 35 distinct files, totaling 200MB of data, in more than 13 million DNA oligonucleotides (about 2 billion nucleotides in total) and fully recovered the data with no bit errors, representing an advance of almost an order of magnitude compared to prior work. 6 Our data curation focused on technologically advanced data types and historical relevance, including the Universal Declaration of Human Rights in over 100 languages,7 a high-definition music video of the band OK Go,8 and a CropTrust database of the seeds stored in the Svalbard Global Seed Vault.9 We developed a random access methodology based on selective amplification, for which we designed and validated a large library of primers, and successfully retrieved arbitrarily chosen items from a subset of our pool containing 10.3 million DNA sequences. Moreover, we developed a novel coding scheme that dramatically reduces the physical redundancy (sequencing read coverage) required for error-free decoding to a median of 5x, while maintaining levels of logical redundancy comparable to the best prior codes. We further stress-tested our coding approach by successfully decoding a file using the more error-prone nanopore-based sequencing. We provide a detailed analysis of errors in the process of writing, storing, and reading data from synthetic DNA at a large scale, which helps characterize DNA as a storage medium and justify our coding approach. Thus, we have demonstrated a significant improvement in data volume, random access, and encoding/decoding schemes that contribute to a whole-system vision for DNA data storage.

bioengineering↗