Biology subjects

Zhao, J.

Publications and source records attributed to Zhao, J..

At least 451 records · Page 25Linked to original sources

bioRxiv2021

Infer disease-associated microbial biomarkers based on metagenomic and metatranscriptomic data

The identification of disease-associated microbial characteristics is crucial for disease diagnosis and therapy. However, the heterogeneity, high dimensionality, and large amounts of microbial data present tremendous challenges for the discovery of key microbial features. In this paper, we present IDAM, a novel computational method for disease-associated gene module inference from metagenomic and metatranscriptomic data. This method integrates gene context conservation (uber-operon) and regulatory mechanisms (gene co-expression patterns) to explore gene modules associated with specific phenotypes using a mathematical graph model, without relying on prior meta-data. We applied IDAM to publicly available datasets from inflammatory bowel disease, melanoma, type 1 diabetes mellitus, and irritable bowel syndrome and demonstrated the superior performance of IDAM in disease-associated characteristics inference compared to popular tools. We also showed high reproducibility of the inferred gene modules of IDAM using independent cohorts with inflammatory bowel disease. We believe that IDAM can be a highly advantageous method for exploring disease-associated microbial characteristics. The source code of IDAM is freely available at https://github.com/OSU-BMBL/IDAM.

bioinformatics↗

bioRxiv2021

Interfered Chromosome Pairing Promotes Meiosis Instability of Autotetraploid Arabidopsis by High Temperatures

Alterations of environmental temperature affect multiple meiosis processes in flowering plants. Polyploid plants derived from whole genome duplication (WGD) have enhanced genetic plasticity and tolerance to environmental stress, but meanwhile face a challenge for organization and segregation of doubled chromosome sets. In this study, we investigated the impact of increased environmental temperature on male meiosis in autotetraploid Arabidopsis thaliana. Under low to mildly-increased temperatures (5-28{degrees}C), irregular chromosome segregation universally takes place in synthesized autotetraploid Columbia-0 (Col-0). Similar meiosis lesions occur in autotetraploid rice (Oryza sativa L.) and allotetraploid canola (Brassica napus cv. Westar), but not in evolutionary-derived hexaploid wheat (Triticum aestivum). As temperature increases to extremely high, chromosome separation and tetrad formation are severely disordered due to univalent formation caused by suppressed crossing-over. We found a strong correlation between tetravalent formation and successful chromosome pairing, both of which are negatively correlated with temperature elevation, suggesting that increased temperature interferes with crossing-over prominently by impacting homolog pairing. Besides, we showed that loading irregularities of axis proteins ASY1 and ASY4 co-localize on the chromosomes of syn1 mutant, and the heat-stressed diploid and autotetraploid Col-0, revealing that heat stress affects lateral region of synaptonemal complex (SC) by impacting stability of axis. Moreover, we showed that chromosome axis and SC in autotetraploid Col-0 are more sensitive to increased temperature than that of diploid Arabidopsis. Taken together, our study provide evidence suggesting that WGD without evolutionary and/or natural adaption negatively affects stability and thermal tolerance of meiotic recombination in Arabidopsis thaliana.

plant biology↗

bioRxiv2021

Renal Interstitial Cells Promote Nephron Regeneration by Secreting Prostaglandin E2

In organ regeneration, progrnitor and stem cells reside in their native microenvironment, which provides dynamic physical and chemical cues essential to their survival, proliferation and differentiation. However, what kind of cells provide a native microenvironment for renal progenitor cells has not been clarified. Here, single-cell sequencing of zebrafish kidney revealed that fabp10 was a marker of renal interstitial cells (RICs), and the Tg(fabp10a:GFP) transgenic line can specifically label RICs in zebrafish kidney. The formation of RICs and nephrons are closely accompanied during nephron regeneration. RICs form a network to wrap the renal progenitor cell aggregates. RICs in close contact with cell aggregates express cyclooxygenase 2 and secrete prostaglandin 2 (PGE2). Inhibiting PGE2 production prevented nephrogenesis by reducing the proliferation and differentiation of progenitor cell aggregates. PGE2 promoted maturation of the nephron by activating the WNT signaling pathway in progenitor cell aggregates in cooperation with Wnt4a. These findings suggest that RICs provide a necessary microenvironment for rapid nephrogenesis during nephron regeneration.

developmental biology↗

bioRxiv2021

Detection of Multiple Types of Cancer Driver Mutations Us-ing Targeted RNA Sequencing in NSCLC

Currently, DNA and RNA are used separately to capture different types of gene mutations. DNA is commonly used for the detection of SNVs, indels and CNVs; RNA is used for analysis of gene fusion and gene expression. To perform both DNA sequencing (DNA-seq) and RNA-seq, material is divided into two copies, and two different procedures are required for sequencing. Due to overconsumption of samples and experimental process complexity, it is necessary to create an experimental method capable of analyzing SNVs, indels, fusions and expression. We developed an RNA-based hybridization capture panel targeting actionable driver oncogenes in solid tumors and corresponding sample preparation and bioinformatics workflows. Analytical validation with an RNA standard reference containing 16 known fusion mutations and 6 SNV mutations demonstrated a detection specificity of 100.0% [95% CI 88.7%~100.0%] for SNVs and 100.0% [95% CI 95.4%~100.0%] for fusions. The targeted RNA panel achieved a 0.73-2.63 copies/ng RNA lower limit of detection (LOD) for SNVs and 0.21-6.48 copies/ng RNA for fusions. Gene expression analysis revealed a correlation greater than 0.9 across all 15 cancer-related genes between the RNA-seq results and targeted RNA panel. Among 1253 NSCLC FFPE tumor samples, multiple mutation types were called from DNA- and RNA-seq data and compared between the two assays. The DNA panel detected 103 fusions and 21 METex14 skipping events; 124 fusions and 26 METex14 skipping events were detected by the target RNA panel; 21 fusions and 4 METex14 skipping events were only detected by the target RNA panel. Among the 173 NSCLC samples negative for targetable mutations by DNA-seq, 15 (15/173, 8.67%) showed targetable gene fusions that may change clinical decisions with RNA-seq. In total, 226 tier I and tier II missense variants for NSCLC were analyzed at genomic (DNA-seq) and transcriptomic (RNA-seq) levels. The positive percent agreement (PPA) was 97.8%, and the positive predictive value (PPV) was 98.6%. Interestingly, variant allele frequencies were generally higher at the RNA level than at the DNA level, suggesting relatively dominant expression of mutant alleles. PPA was 97.6% and PPV 99.38% for EGFR 19del and 20ins variants. We also explored the relationship of RNA expression with gene copy number and protein expression. The RPKM of EGFR transcripts assessed by the RNA panel showed a linear relationship with copy number quantified by the DNA panel, with an R of 0.8 in 1253 samples. In contrast, MET gene expression is regulated in a more complex manner. In IHC analysis, all 3+ samples exhibited higher RPKM levels; IHC level of 2+ and below showed lower RNA expression. Parallel DNA- and RNA-seq and systematic analysis demonstrated the accuracy and robustness of the RNA sequencing panel in identifying multiple types of variants for cancer therapy. Contact: zhaojia0327@126.com

molecular biology↗

bioRxiv2021

Assembly status transition offers an avenue for activity modulation of a supramolecular enzyme

Nature has evolved many supramolecular proteins assembled in certain, sometimes even seemingly oversophisticated, morphological manners. The rationale behind such evolutionary efforts is often poorly understood. Here we provide atomic-resolution insights into how the dynamic building of a structurally complex enzyme with higher-order symmetry offers amenability to intricate allosteric regulation. We have established the functional coupling between enzymatic activity and protein morphological states of glutamine synthetase (GS), an old multi-subunit enzyme essential for cellular nitrogen metabolism. Cryo-EM structure determination of GS in both the catalytically active and inactive assembly states allows us to reveal an unanticipated self-assembly-induced dynamics-driven allosteric paradigm, in which the remote interactions between two subcomplex entities significantly rigidify the otherwise structurally fluctuating active sites, thereby regulating activity. We further show in vivo evidences that how the enzyme morphology transitions could be modulated by cellular factors on demand. Collectively, our data present an example of how assembly status transition offers an avenue for allosteric modulation, and sharpens our mechanistic understanding of allostery, dynamics, cooperativity, and other complex functional and regulatory properties of supramolecular enzymes.

biochemistry↗

bioRxiv2021

Covalent inhibition of endoplasmic reticulum chaperone GRP78 disconnects the transduction of ER stress signals to inflammation and lipid accumulation in diet-induced obese mice

Targeting endoplasmic reticulum (ER) stress, inflammation and metabolic dysfunctions may halt the pathogenesis of obesity and thereby reduce the prevalence of diabetes, cardiovascular disesases and cancers. The present study was designed to elucidate the mechnaisms by which plant-derived celastrol ameliorated inflammation and lipid accumulation in obesity. The mouse model of diet-induced obesity was induced by feeding high-fat diet for 3 months and subsequently intervented with celastrol for 21 days. Hepatic and adipose tissues were analysed for lipid accumulation, macrophage activation and biomarker expression. As result, celastrol effectively reduced body weight, suppressed ER stress, inflammation and lipogenesis while promoted hepatic lipolysis. RNA-sequencing revealed that celastrol-loaded nanomicelles restored the expression of 49 genes that regulate ER stress, inflammation and lipid metabolism. On the other hand, celastrol-PEG4-alkyne was synthesized for identifying celastrol-bound proteins in RAW264.7 macrophages. ER chaperone GRP78 was identified by proteomics approach for celastrol binding to the residue Cys41. Upon binding and conjugation, celastrol diminished the chaperone activity of GRP78 by 130-fold and reduced ER stress in palmitate-challenged cells, while celastrol analogue lacking quinone methide failed to exhibit anti-obesity effects. Thus, covalent GRP78 inhibition may induce the reprograming of ER signaling, inflammation and metabolism against diet-induced obesity.

pharmacology and toxicology↗

bioRxiv2021

Multiomics reveals the genomic, proteomic and metabolic influences of histidyl dipeptides on heart

Histidyl dipeptides, are synthesized in the heart via enzyme carnosine synthase (Carns), which facilitates glycolysis and glucose oxidation by proton buffering and attenuate ischemia and reperfusion injury. However, a composite understanding of the histidyl dipeptide mediated responses in the heart are lacking. We performed multilayer omics in the cardio specific Carns overexpressing mice, showing higher myocardial levels of histidyl dipeptides lead to extensive changes in microRNAs that could target the expression of contractile proteins and enzymes involved in {beta}-fatty acid oxidation and citric acid cycle (TCA). Similarly, global proteomics showed contractile function, fatty acid degradation and TCA cycle, pathways were enriched in the CarnsTg heart. Parallel with these changes, free fatty acids, and TCA intermediate-succinic acid were lower under aerobic and significantly attenuated under anaerobic conditions in the CarnsTg heart. Integration of multiomics data shows {beta}-fatty acid oxidation and TCA cycle exhibit correlative changes at all three levels in CarnsTg heart, suggesting histidyl dipeptides are critical regulators of myocardial structure, function and energetics.

systems biology↗

bioRxiv2021

rRNA Biogenesis Regulates Mouse 2C-like State by 3D Structure Reorganization of Peri-Nucleolar Heterochromatin

Nucleolus is the organelle for ribosome biogenesis and for sensing various types of stress. Its role in regulating stem cell fate is unclear. Here, we present multiple lines of evidence that nucleolar stress induced by interfering rRNA biogenesis can drive 2-cell stage embryo-like (2C-like) transcriptional program and induce an expanded 2C-like cell population in mouse embryonic stem (mES) cells. Mechanistically, the nucleolar integrity mediated by rRNA biogenesis maintains the normal liquid-liquid phase separation (LLPS) of nucleolus and the formation of peri-nucleolar heterochromatin (PNH). Upon rRNA biogenesis defect, the natural LLPS of nucleolus is disrupted, causing dissociation of NCL/TRIM28 complex from PNH and changes of epigenetic states and reorganization of the 3D structure of PNH, which leads to Dux, a 2C program transcription factor gene, to be released from the PNH region and activation of 2C-like program. Correspondingly, embryos with rRNA biogenesis defect are incompatible to develop from 2-cell (2C) to 4-cell embryos, with delayed repression of 2C/ERV genes and a transcriptome skewed toward earlier cleavage embryo signatures. Our results highlight that rRNA-mediated nucleolar integrity and 3D structure reshaping of PNH compartment regulates the fate transition of mES cells to 2C-like cells, and that rRNA biogenesis is a critical regulator during the 2-cell-to-4-cell transition of murine pre-implantation embryo development.

cell biology↗

bioRxiv2021

Strigo-D2 - a bio-sensor for monitoring the spatio-temporal pattern of strigolactone signaling in intact plants

Strigolactones (SLs) are a class of plant hormones modulating developmental programs in response to endogenous and exogenous stimuli and mediating biotic interactions. However, a comprehensive view on the spatio-temporal pattern of SL signaling has not been established and tools for a systematic in planta analysis do not exist. Here, we present Strigo-D2, a genetically encoded ratiometric SL signaling sensor, allowing the examination of SL signaling distribution with cellular resolution and its rapid response to altered SL levels in intact plants. By monitoring the abundance of a truncated and fluorescently labeled SUPPRESSOR OF MAX2 1-LIKE 6 (SMXL6) protein, a proteolytic target of the SL signaling machinery, we show that all cell types investigated have the capacity to respond to changes in SL levels but with very different dynamics. In particular, SL signaling is pronounced in vascular cells but low in guard cells and the meristematic region of the root. We also show that other hormones leave Strigo-D2 activity unchanged indicating that initial SL signaling steps work in isolation from other hormonal signaling pathways. Specificity and spatio-temporal resolution of Strigo-D2 underline the value of the sensor for monitoring SL signaling in a broad range of biological contexts and with highly instructive analytical depth.

plant biology↗

bioRxiv2021

The northern gene flow into southeastern East Asians inferred from genome-wide array genotyping

The population history of Southeast China remains poorly understood due to the sparse sampling of present-day populations and far less modeling with ancient genomic data. We here newly reported genome-wide genotyping data from 207 present-day Han Chinese and Hmong-Mien-speaking She people from Fujian and Taiwan, southeast China. We co-analyzed with 66 early-Neolithic to Iron-Age ancient Fujian and Taiwan individuals obtained from literature to explore the genetic continuity and admixture based on the genetic variations of high-resolution time transect. We found the genetic differentiation between northern and southern East Asians defined by a north-south East Asian genetic cline and the studied southern East Asians were clustered in the southern end of this cline. We also found that southeastern coastal continental modern East Asians harbored the genetic differentiation with other southern Tai-Kadai, Hmong-Mien, Austronesian and Austroasiatic speakers, as well as geographically close Neolithic-to-Iron Age populations, but relatedly close to post-Neolithic Yellow River ancients, which suggested the influence of southward gene flow on the modern southern coastal gene pool. Besides, we also identified one new Hmong-Mien genetic cline in East Asia with the coastal Fujian She localizing at the intersection position between Hmong-Mien and Han clines in the principal component analysis. She people show stronger genetic affinity with southern East Asian indigenous populations with the main ancestry deriving from Hanben-related populations. The southeastern Han Chinese could be modeled with the primary ancestry deriving from the group related to the Yellow River Basin millet farmers and the remaining from groups related to southeastern ancient indigenous rice farmers, which was consistent with the northern China origin of modern southeastern Han Chinese and in line with the historically and archaeologically attested southward migrations of Han people and their ancestors. Interestingly, f4-statistics and three-way admixture model results showed both coastal ancient sources related to Austronesian speakers and inland ancient sources related to Austroasiatic speakers complexed the modern observed fine-scale genetic structure here. Our estimated north-south admixture time ranges based on the decay of the linkage disequilibrium spanned from the Bronze age to historic periods, suggesting the recent large-scale population migrations and subsequent admixture participated in the formation of modern Han in Southeast Asia.

genetics↗

bioRxiv2021

Convergence of MCR-8.2 and chromosome-mediated resistance to colistin and tigecycline in an NDM-5-producing ST656 Klebsiella pneumoniae from a lung transplant patient

For infection caused by NDM-5-producing Klebsiella pneumoniae, tigecycline and colistin are the last treatment options. In this study, we characterized the first NDM-5 and MCR-8.2 co-harboring K. pneumoniae clinical isolate, combining with chromosomal gene-mediated resistance to colistin and tigecycline. The K. pneumoniae KP32558 was isolated from the bronchoalveolar lavage fluid from a lung transplant male patient. Whole genome sequencing was carried out using Illumina HiSeq sequencing platform as well as nanopore sequencing method. The K. pneumoniae KP32558 was identified as a pan-drug resistant bacteria, belonged to ST656, and harbored plasmid-encoded blaNDM-5 and mcr-8.2 genes. The blaNDM-5 gene was located on an IncX3 type plasmid, which was successfully transferred to Escherichia coli strain J53 without visible fitness cost. The mcr-8.2 gene was located on a conjugative plasmid pKP32558-2-mcr8. It had two replicons, IncFII(K) and IncQ1, harbored previously by another two mcr-8.2-carrying plasmids pMCR8_020135 and pMCR8_095845. These three plasmids were clustered into the same clade and derived from K. pneumoniae isolates of the same clonal complex, indicating that pKP32558-2-mcr8 may come from pMCR8_020135 and pMCR8_095845 related ancestor. The MIC of KP32558 for colistin was 256 mg/L, the 6 amino acid substitutions in the two-component system may involve in the high-level colistin resistance. The truncation in acrR gene, related to tigecycline resistance, was also identified. K. pneumoniae has evolved a variety of complex resistance mechanisms to the last-resort antimicrobials, close surveillance is urgently needed to monitor the prevalence of this clone.

microbiology↗

bioRxiv2021

Intrinsically disordered N-terminal domain (NTD) of p53 interacts with mitochondrial PTP regulator Cyclophilin D

Mitochondrial permeability transition pore (mPTP) plays crucial roles in cell death in a variety of diseases, including ischemia/reperfusion injury in heart attack and stroke, neurodegenerative conditions, and cancer. To date, cyclophilin D is the only confirmed component of mPTP. Under stress, p53 can translocate into mitochondria and interact with CypD, triggering necrosis and cell growth arrest. However, the molecular details of p53/CypD interaction are still poorly understood. Previously, several studies reported that p53 interacts with CypD through its DNA-binding domain (DBD). However, using surface plasmon resonance (SPR), we found that full-length p53 (FLp53) binds to CypD with KD of ~1 M, while both NTD-DBD and NTD bind to CypD at ~10 M KD (Fig. 1C and 1D). Thus, instead of DBD, NTD is the major CypD binding site on p53. NMR titration and MD simulation revealed that NTD binds CypD with broad and dynamic interfaces dominated by electrostatic interactions. NTD 20-70 was further identified as the minimal binding region for CypD interaction, and two NTD fragments, D1 (residues 22-44) and D2 (58-70), can each bind CypD with mM affinity. Our detailed biophysical characterization of the dynamic interface between NTD and CypD provides novel insights on the p53-dependent mPTP opening and drug discovery targeting NTD/CypD interface in diseases. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=123 SRC="FIGDIR/small/453429v2_fig1.gif" ALT="Figure 1"> View larger version (26K): org.highwire.dtl.DTLVardef@12a85b4org.highwire.dtl.DTLVardef@aff747org.highwire.dtl.DTLVardef@936401org.highwire.dtl.DTLVardef@8a893b_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOFig. 1.C_FLOATNO p53 binds to CypD at M affinity determined by surface plasmon resonance (SPR). (A) SPR scheme (top) and p53 construct (bottom). (B) SPR sensorgrams of the full length p53 (FLp53)-CypD interaction. The concentrations of FLp53 (from top to bottom) were 2.0, 1.0, 0.5, 0.25, and 0.13 M, respectively. (C) SPR sensorgrams of the NTD-DBD-CypD interaction. The concentrations of NTD-DBD (from top to bottom) were 50, 25, 12.5, 6.25, and 3.13 M, respectively. (D) SPR sensorgrams of the NTD-CypD interaction. The concentrations of NTD (from top to bottom) were 40, 25, 12.5, 6.25, and 3.13 M, respectively. (E) SPR sensorgrams of the interaction between CypD and NTD1-70. The concentrations of NTD1-70 (from top to bottom) were 10, 5, 2.5, 1.25, and 0.63 M, respectively. All SPR sensorgrams are fitted using 1:1 Langmuir binding model (Black curves) in BIAevaluation software 4.0.1. C_FIG

biochemistry↗

bioRxiv2021

Genomic insights into the differentiated population admixture structure and demographic history of North East Asians

North China and South Siberia, mainly populated by Altaic-speaking populations, possess extensive ethnolinguistic diversity and serve as the crossroad for the initial peopling of America and western-eastern trans-continental communication. Yet, the complex scenarios of genetic origin, population structure, and admixture history of North-East Asia remain to be fully characterized, especially for Mongolic people in China with a genome-wide perspective. Thus, we genotyped genome-wide SNPs for 510 individuals from 38 Chinese Mongolic, Tungusic, and Sinitic populations to explore the sharing alleles and haplotypes within the studied groups and following merged it with 3508 modern and ancient Eurasian individuals to reconstruct the deep evolutionary and natural selection history of northern East Asians. We identified significant substructures within Altaic-speaking populations with the primary common ancestry linked to the Neolithic northern East Asians: Western Turkic people harbored more western Eurasian ancestry; Northern Mongolic people in Siberia and eastern Tungusic people in Amur River Basin (ARB) possessed dominant Neolithic Mongolian Plateau (MP) or ARB ancestry; Southern Mongolic people in China owned obvious genetic impact from Neolithic Yellow River Basin (YRB) farmers. Additionally, we found the differentiated admixture history between western and eastern Mongolians and geographically close Northeast Hans: the former received a genetic impact from western Eurasians and the latter retained the dominant YRB and ARB Neolithic ancestry. Moreover, we demonstrated that Kalmyk people from the northern Caucasus Mountain possessed a strong genetic affinity with Neolithic MP people, supporting the hypothesis of their eastern Eurasian origin and long-distance migration history. We also illuminated that historic pastoral empires in the MP contributed considerably to the gene pool of northern Mongolic people but rarely to southern ones. We finally found natural signatures in Mongolians associated with alcohol metabolism. Generally, our results not only illuminated that complex population migration and admixture of Neolithic ancestral sources from the MP or ARB played an important role in the spread of Altaic-speaking populations and Proto-Altaic language, which partly supported the Northeast Asia-origin hypothesis, but also demonstrated that the observed multi-sources of genetic diversity contributed significantly to the modern existing extensive ethnolinguistic diversity in North-East Asia.

genomics↗

bioRxiv2021

PD-1 in hippocampal neurons regulates excitability, synaptic plasticity, and cognition

Immunotherapy using monoclonal antibodies against programmed cell death protein 1 (PD-1) demonstrated improved survival in cancer patients through immune activation. Here we show that functional PD-1 is expressed in mouse and primate hippocampal neurons and PD-1 inhibition improves cognition in physiological and pathological conditions. Mice lacking the Pdcd1 gene encoding PD-1 exhibit enhanced long-term potentiation (LTP) and learning and memory. These behavioral and cellular changes can be recapitulated by selective deletion of Pdcd1 in hippocampal excitatory neurons but not in microglia. Perfusion of mouse or nonhuman primate brain slices with anti-PD-1 antibody is sufficient to increase excitability in CA1 hippocampal neurons. Conversely, re-expression of Pdcd1 in PD-1 deficient hippocampal neurons suppresses memory and LTP. Traumatic brain injury impairs learning and memory, which is improved by intraventricular administration of anti-PD-1. These findings suggest that anti-PD-1 treatment has therapeutic potential to counteract cognitive decline. HighlightsO_LIAdult mice lacking Pdcd1 in hippocampal neurons exhibit enhanced memory and LTP C_LIO_LIAnti-PD-1 antibody treatment increases CA1 neuron excitability in brain slices of mice and primates C_LIO_LIRe-expression of Pdcd1 in PD-1 deficient hippocampal neurons impairs memory and LTP C_LIO_LICognitive deficits after traumatic brain injury are improved by anti-PD-1 treatment C_LI

neuroscience↗

bioRxiv2021

Minimal synthetic enhancers reveal control of the probability of transcriptional engagement and its timing by a morphogen gradient

How enhancers interpret morphogen gradients to generate spatial patterns of gene expression is a central question in developmental biology. Although recent studies have begun to elucidate that enhancers can dictate whether, when, and at what rate a promoter will engage in transcription, the complexity of endogenous enhancers calls for theoretical models with too many free parameters to quantitatively dissect these regulatory strategies. To overcome this limitation, we established a minimal synthetic enhancer system in embryos of the fruit 2y Drosophila melanogaster. Here, a gradient of the Dorsal activator is read by a single Dorsal binding site. By quantifying transcriptional activity using live imaging, our experiments revealed that this single Dorsal binding site is capable of regulating whether promoters engage in transcription in a Dorsal concentration-speci1c manner. By modulating binding-site aZnity, we determined that a genes decision to engage in transcription and its transcriptional onset time can be explained by a simple theoretical model where the promoter has to traverse multiple kinetic barriers before transcription can ensue. The experimental platform developed here pushes the boundaries of live-imaging in studying gene regulation in the early embryo by enabling the quanti1cation of the transcriptional activity driven by a single transcription factor binding site, and making it possible to build more complex enhancers from the ground up in the context of a dialogue between theory and experiment.

developmental biology↗

bioRxiv2021

Genome sequences of the five Sitopsis species of Aegilops and the origin of polyploid wheat B-subgenome

Bread wheat (Triticum aestivum L., BBAADD) is a major staple food crop worldwide. The diploid progenitors of the A- and D-subgenomes have been unequivocally identified, that of B however remains ambiguous and controversial but is suspected to be related to species of Aegilops, section Sitopsis. Here, we report the assembly of chromosome-level genome sequences of all five Sitopsis species, namely Ae. bicornis, Ae. longissima, Ae. searsii, Ae. sharonensis, and Ae. speltoides, as well as partial assembly of Ae. mutica genome for phylogenetic analysis. Our results support that the donor of bread wheat B-subgenome is a distinct, probably extinct, diploid species that diverged from an ancestral progenitor of the B-lineage similar to Ae. mutica and Ae. speltoides. The five Sitopsis species have variable genome sizes (4.11-5.89 Gb) with high proportions of repetitive sequences (85.99-89.81%); nonetheless, they retain high collinearity with other wheat genomes. Differences in genome size are primarily due to independent post-speciation amplification of transposons rather than to inter-specific genetic introgression. We also identified a set of Sitopsis genes pertinent to important agronomic traits that can be harnessed for wheat breeding. These resources provide a new roadmap for evolutionary and genetic studies of the wheat group. SignificanceThe origin of the B-subgenome of hexaploid bread wheat remains unknown. Here we report the assembly of chromosome-level genome sequences of all five Sitopsis species of the genus Aegilops, which are previously considered as possible direct progenitors or contributors to the B-subgenome. Our comparative genomic analyses reveal that the B-subgenome originated from an unknown, most likely extinct species phylogenetically distinct from Ae. speltoides, its extant closest relative. We also provide evidence that Ae. speltoides is neither the direct progenitor of the G-subgenome of tetraploid wheat Triticum timopheevii. The high-quality Sitopsis genomes provide novel avenues to identify new important genes for wheat breeding.

evolutionary biology↗

bioRxiv2021

Mutations in PIKFYVE cause autosomal dominant congenital cataract

Congenital cataract, an ocular disease predominantly occurring within the first decade of life, is one of the leading causes of blindness in children. Through whole exome sequencing of a Chinese family with congenital cataract, we identified a disease-causing mutation (p.G1943E) in PIKFYVE, which affecting the PIP kinase domain of the PIKfyve protein. We demonstrated that heterozygous/homozygous disruption of PIKfyve kinase domain, instead of overexpression of PIKFYVEG1943E in zebrafish mimicked the cataract defect in human patients, suggesting that haploinsufficiency, rather than dominant-negative inhibition of PIKfyve activity caused the disease. Phenotypical analysis of pikfyve zebrafish mutants revealed that loss of Pikfyve caused aberrant vacuolation (accumulation of Rab7+Lc3+ amphisomes) in lens cells, which was significantly alleviated by treatment with the V-ATPase inhibitor bafilomycin A1 (Baf-A1). Collectively, we identified PIKFYVE as a novel causative gene for congenital cataract and demonstrated the potential application of Baf-A1 in treatment of congenital cataract.

genomics↗

bioRxiv2021

Efficient tagging and purification of endogenous proteins for structural studies by single particle cryo-EM

A major bottleneck in structural biology is producing biologically relevant samples at sufficient quantities. This is particularly true for large protein assemblies where conventional techniques of gene overexpression require substantial optimization, hampering structural studies and drug development efforts. Here we describe a method combining CRISPR/Cas gene editing and fluorescence cell sorting to rapidly tag and purify endogenous human proteins from cell lines, enabling structural analysis of native proteins that are properly folded and assembled. We applied this approach to study the human proteasome from HEK cells and rapidly determined structures of major proteasomal complexes. Structures of the PA28-20S complex reveal the native subunit stoichiometry of PA28 and a distinct functional state of the complex. The efficient strategy for tagging and extracting endogenous proteins described here will enable the structural study of many challenging targets and provide more biologically relevant samples for research and therapeutic development.

molecular biology↗