Biology subjects

Zhang, Q.

Publications and source records attributed to Zhang, Q..

At least 559 records · Page 31Linked to original sources

bioRxiv2021

Lipid chaperone LBP-8 coordinates with nuclear factors to promote longevity in Caenorhabditis elegans

Eukaryotic cells are composed of a variety of organelles. Their coordination plays crucial roles in cellular homeostasis and organism longevity and is mediated by proteins with the ability to transport between different organelles. In Caenorhabditis elegans, LBP-8 is a pro-longevity lipid chaperone that can localize to both lysosomes and the nucleus. Here we profiled LBP-8s binding partners using immunoprecipitation and mass spectrometry. From the 45 identified candidates, we discovered four nuclear factors that are required for the LBP-8-induced longevity. Among them, RPC-2, an RNA Polymerase III core subunit, is also necessary for the nuclear localization of LBP-8. Moreover, we have screened nuclear transport machinery components, and revealed the requirement of the nuclear import, not export, for the LBP-8 longevity effects. Together, these results suggest that the lipid chaperone LBP-8 relies on specific nuclear factors to retain in the nucleus and regulate longevity.

cell biology↗

bioRxiv2021

Scallop2 enables accurate assembly of multiple-end RNA-seq data

Transcript assembly (i.e., to reconstruct the full-length expressed transcripts from RNA-seq data) has been a critical but yet unsolved step in RNA-seq analysis. Modern RNA-seq protocols can produce paired-/multiple-end RNA-seq reads, where information is available that two or more reads originate from the same transcript. The long-range constraints implied in these paired-/multiple-end reads can be much beneficial in correctly phasing the complicated spliced isoforms. However, there often exist gaps among individual ends, which may even contain junctions, making the efficient use of such constraints algorithmically challenging. Here we introduce Scallop2, a new reference-based transcript assembler optimized for multiple-end (including paired-end) RNA-seq data. Scallop2 uses an algorithmic frame-work that first represents reads from the same molecule as the so-called multiple-end phasing paths in the context of a splice graph, then "bridges" each multiple-end phasing path into a long, single-end phasing path, and finally decomposes the splice graph into paths (i.e., transcripts) guided by the bridged phasing paths. An efficient bridging algorithm is designed to infer the true path connecting two consecutive ends following a novel formulation that is robust to sequencing errors and transcript noises. By observing that failing to bridge two ends is mainly due to incomplete splice graphs, we propose a new method to determine false starting/ending vertices of the splice graphs which has been showed efficient in reducing false positive rate. Evaluations on both (multiple-end) single-cell RNA-seq datasets from Smart-seq3 protocol and Illumina paired-end RNA-seq samples demonstrate that Scallop2 vastly outperforms recent assemblers including StringTie2, Scallop, and CLASS2 in assembly accuracy.

bioinformatics↗

bioRxiv2021

Extensive germline-somatic interplay drives prostate cancer through HNF1B co-option of TMPRSS2-ERG

Genome-wide association studies have identified 270 loci conferring risk for prostate cancer (PCa), yet the underlying biology and clinical impact remain to be investigated. Here we observe an enrichment of transcription factor genes including HNF1B within PCa risk-associated regions. While focused on the 17q12/HNF1B locus, we find a strong eQTL for HNF1B and multiple potential causal variants involved in the regulation of HNF1B expression in PCa. An unbiased genome-wide co-expression analysis reveals PCa-specific somatic TMPRSS2-ERG fusion as a transcriptional mediator of this locus and the HNF1B eQTL signal is ERG fusion status dependent. We investigate the role of HNF1B and find its involvement in several pathways related to cell cycle progression and PCa severity. Furthermore, HNF1B interacts with TMPRSS2-ERG to co-occupy large proportion of genomic regions with a remarkable enrichment of additional PCa risk alleles. We finally show that HNF1B co-opts ERG fusion to mediate mechanistic and biological effects of the PCa risk-associated locus 17p13.3/VPS53/FAM57A/GEMIN4. Taken together, we report an extensive germline-somatic interaction between TMPRSS2-ERG fusion and genetic variations underpinning PCa risk association and progression.

cancer biology↗

bioRxiv2021

The Lambda variant of SARS-CoV-2 has a better chance than the Delta variant to escape vaccines

The newly emerging variants of SARS-CoV-2 from India (Delta variant) and South America (Lambda variant) have led to a higher infection rate of either vaccinated or unvaccinated people. We found that sera from Pfizer-BioNTech vaccine remain high reactivity toward the receptor binding domain (RBD) of Delta variant while it drops dramatically toward that of Lambda variant. Interestingly, the overall titer of antibodies of Pfizer-BioNTech vaccinated individuals drops 3-fold after 6 months, which could be one of major reasons for breakthrough infections, emphasizing the importance of potential third boost shot. While a therapeutic antibody, Bamlanivimab, decreases binding affinity to Delta variant by ~20 fold, it fully lost binding to Lambda variant. Structural modeling of complexes of RBD with human receptor, Angiotensin Converting Enzyme 2 (ACE2), and Bamlanivimab suggest the potential basis of the change of binding. The data suggest possible danger and a potential surge of Lambda variant in near future.

immunology↗

bioRxiv2021

A universal approach for integrating super large-scale single-cell transcriptomes by exploring gene rankings

Advancement in single-cell RNA sequencing leads to exponential accumulation of single-cell expression data. However, there is still lack of tools that could integrate these unlimited accumulation of single-cell expression data. Here, we presented a universal approach iSEEEK for integrating super large-scale single-cell expression via exploring expression rankings of top-expressing genes. We developed iSEEEK with 13.7 million single-cells. We demonstrated the efficiency of iSEEEK with canonical single-cell downstream tasks on five heterogenous datasets encompassing human and mouse samples. iSEEEK achieved good clustering performance benchmarked against well-annotated cell labels. In addition, iSEEEK could transfer its knowledge learned from large-scale expression data on new dataset that was not involved in its development. iSEEEK enables identification of gene-gene interaction networks that are characteristic of specific cell types. Our study presents a simple and yet effective method to integrate super large-scale single-cell transcriptomes and would facilitate translational single-cell research from bench to bedside.

bioinformatics↗

bioRxiv2021

Emergence and Enhancement of Ultrasensitivity through Posttranslational Modulation of Protein Stability

Signal amplification converts a linear input to a steeply sigmoid output and is central to cellular functions. One canonical signal amplifying motif is zero-order ultrasensitivity through the posttranslational modification (PTM) cycle signaling proteins. The functionality of this signaling motif has been examined conventionally by supposing that the total amount of the protein substrates remains constant. However, covalent modification of signaling proteins often results in changes in their stability, which affects the abundance of the protein substrates. Here we use a mathematical model to explore the signal amplification properties in such scenarios. Our simulations indicate that PTM-induced protein stabilization brings the enzymes closer to saturation, and as a result, ultrasensitivity may emerge or is greatly enhanced, with a steeper sigmoidal response of higher magnitude and generally longer response time. In cases where PTM destabilizes the protein, ultrasensitivity can be regained through changes in the activities of the involved enzymes or from increased protein synthesis. Interestingly, ultrasensitivity is not limited to modified or unmodified protein substrates; the total protein substrate can also exhibit ultrasensitivity. It is conceivable that cells use inducible protein stabilization as a way to boost signal amplification while saving energy by keeping the protein substrate at low basal conditions.

systems biology↗

bioRxiv2021

Exon-Skipping Antisense Oligonucleotides for Cystic Fibrosis Therapy

Mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene cause cystic fibrosis (CF), and the CFTR-W1282X nonsense mutation causes a severe form of CF. Although Trikafta and other CFTR-modulation therapies benefit most CF patients, targeted therapy for patients with the W1282X mutation is lacking. The CFTR-W1282X protein has residual activity, but is expressed at a very low level due to nonsense-mediated mRNA decay (NMD). NMD-suppression therapy and read-through therapy are actively being researched for CFTR nonsense mutants. NMD suppression could increase the mutant CFTR mRNA, and read-through therapies may increase the levels of full-length CFTR protein. However, these approaches have limitations and potential side effects: because the NMD machinery also regulates the expression of many normal mRNAs, broad inhibition of the pathway is not desirable; and read-through drugs are inefficient, partly because the mutant mRNA template is subject to NMD. To bypass these issues, we pursued an exon-skipping antisense oligonucleotide (ASO) strategy to achieve gene-specific NMD evasion. A cocktail of two splice-site-targeting ASOs induced the expression of CFTR mRNA without the PTC-containing exon 23 (CFTR-{Delta}ex23), which is an in-frame exon. Treatment of human bronchial epithelial cells with this cocktail of ASOs that target the splice sites flanking exon 23 results in efficient skipping of exon 23 and an increase in CFTR-{Delta}ex23 protein. The splice-switching ASO cocktail increases the CFTR-mediated chloride current in human bronchial epithelial cells. Our results set the stage for developing an allele-specific therapy for CF caused by the W1282X mutation.

molecular biology↗

bioRxiv2021

Mathematical Modeling Reveals Quantitative Properties of KEAP1-NRF2 Signaling

In response to oxidative and electrophilic stresses, cells launch an NRF2-mediated transcriptional antioxidant program. The activation of NRF2 depends on a redox sensor, KEAP1, which acts as an E3-ligase adaptor to promote the ubiquitination and degradation of NRF2. While a great deal has been learned about this redox duo, its quantitative signaling properties are still largely unexplored. In the present study, we examined these properties including response time, half-life, maximal activation, and response steepness (ultrasensitivity) of NRF2, through mathematical modeling. The models describe, with increasing complexity, the reversible binding of KEAP1 dimer and NRF2 via the ETGE and DLG motifs, NRF2 production, KEAP1-dependent and independent NRF2 degradation, and perturbations by different classes of NRF2 activators. Our simulations revealed that at the basal condition, NRF2 molecules are largely sequestered by KEAP1, with the KEAP1-NRF2 complex distributed comparably in either an ETGE-bound only (open) state or an ETGE and DLG dual-bound (closed) state, corresponding to the unlatched and latched configurations of the conceptual hinge-latch model. With two-step ETGE binding, the open and closed states operate in cycle mode at the basal condition and transition to equilibrium mode at stressed conditions. Class I-V, electrophilic NRF2 activators, which modify redox-sensing cysteine residues of KEAP1, shift the balance to a closed state that is unable to degrade NRF2 effectively. When total NRF2 accumulates to a level that nearly saturates existing KEAP1, ultrasensitive NRF2 activation, i.e., a steep rise in the free NRF2 level, can occur. The ultrasensitivity results from two simultaneous mechanisms, zero-order degradation mediated by DLG binding and protein sequestration (molecular titration) mediated by ETGE binding. These response characteristics of class I-V activators do not require disruption of DLG binding to unlatch the KEAP1-NRF2 complex. In comparison, class VI NRF2 activators, which directly compete with NRF2 for KEAP1 binding, cause a shift to the unlatched, open state of KEAP1-NRF2 complex and ultimately its complete dissociation (unhinged), resulting in a fast release of free NRF2 followed by stabilization. Although class VI activators may induce free NRF2 to higher levels, ultrasensitivity is lost due to lower free KEAP1 and thus its NRF2-sequestering effect. Stress-induced nuclear NRF2 accumulation is enhanced when basal nuclear NRF2 turnover constitutes a small load of NRF2 production. Our simulation further demonstrated that optimal abundances of cytosolic and nuclear KEAP1 exist to maximize ultrasensitivity. In summary, by simulating the dual role of KEAP1 in repressing NRF2, i.e., sequestration and promoting degradation, our mathematical modeling provides key novel quantitative insights into the signaling properties of the KEAP1-NRF2 system, which may help with the design of novel classes of NRF2 activators and inhibitors and understanding of the toxic actions of environmental oxidative stressors.

systems biology↗

bioRxiv2021

Ectopic cervical thymi and no thymic involution until midlife in naked mole-rats

Immunosenescence is a hallmark of aging and manifests as increased susceptibility to infection, autoimmunity, and cancer in the elderly. One component of immunosenescence is thymic involution, age-associated shrinkage of the thymus, observed in all vertebrates studied to date. The naked mole-rat (Heterocephalus glaber) has become an attractive animal model in aging research due to its extreme longevity and resistance to disease. Here we show that naked mole rats display no thymic involution up to 11 years of age. Furthermore, we found large ectopic cervical thymi in addition to the canonical thoracic thymus, both being identical in their cell composition. The developmental landscape in naked mole-rat thymi revealed overt differences from the murine T cell compartment, most notably a decrease of CD4+/CD8+ double-positive cells and lower abundance of cytotoxic effector T cells. Our observations suggest that naked mole rats display a delayed immunosenescence. Therapeutic interventions aimed at reversing thymic aging remain limited, underscoring the importance of understanding the cellular and molecular mechanisms behind a sustained immune function in the naked mole rat.

immunology↗

bioRxiv2021

Model-guided engineering of DNA sequences with predictable site-specific recombination rates

Site-specific recombination (SSR) is an important tool in genome editing and gene circuit design. However, its applications are limited by the inability to simply and predictably tune SSR reaction rates across orders of magnitude. Facile rate manipulation can in principle be achieved by modifying the nucleotide sequence of the DNA substrate of the recombinase, but the design principles for rationally doing so have not been elucidated. To enable predictable tuning of SSR reaction kinetics via DNA sequence, we developed an integrated experimental and computational method to parse individual nucleotide contributions to the overall reaction rate, which we used to analyze and engineer the DNA attachment sequence attP for the inversion reaction mediated by the serine recombinase Bxb1. A quantitative PCR method was developed to measure the Bxb1 reaction rate in vitro. Then, attP sequence libraries were designed, selected, and sequenced to inform a machine-learning model, which revealed that the Bxb1 reaction rate can be accurately represented assuming independent contributions of nucleotides at key positions. Next, we used the model to predict the performance of DNA site variants in reaction rate assays both in vitro and in Escherichia coli, with flipping rates ranging from 0.01- to 10-fold that of the wild-type attP sequence. Finally, we demonstrate that attP variants with predictable DNA recombination rates can be used in concert to achieve kinetic control in gene circuit design by coordinating the coexpression of two proteins in both their relative proportion and their total amount. Our high-throughput, data-driven method for rationally tuning SSR reaction rates through DNA sequence modification enhances our understanding of recombinase function and expands the synthetic biology toolbox. Graphical abstract O_FIG_DISPLAY_L [Figure 1] M_FIG_DISPLAY C_FIG_DISPLAY

synthetic biology↗

bioRxiv2021

Regeneration of humoral immunity from pluripotent stem cells by defined transcription factors

Regeneration of humoral immunity from pluripotent stem cells (PSCs) is a crucial aim in translational medicine. However, reconstitution of complete, sustained, and functional B lymphopoiesis from PSCs has not yet been developed. Here, we successfully achieved regenerative B lymphopoiesis in B-cell deficient animals transplanted with PSC-derived hematopoietic progenitors (iHPCs) guided by synergistic expression of Runx1, Hoxa9, and Lhx2. Upon transplantation, the iHPCs immediately gave rise to pro/pre-B cells in recipients bone marrow, which were able to further differentiate into the entire B cell lineages, including innate B-1a, B-1b, MZ B cells, as well as adaptive FO B cells. In responding to antigen stimuli, the regenerative B cells produced adaptive humoral immune responses, sustained a prolonged antigen-specific antibody production, and formed immune-memory. Particularly, the regenerative B cells in spleen showed developing patterns of immunoglobulin chain-switch and hyper-mutation via a cross-talk with the host T follicular helper cells, which eventually formed T cell-dependent humoral responses. This study provides de novo evidence that B lymphopoiesis can be regenerated from PSCs via a HSC-independent approach, which provides insights into treating B-cell related humoral deficiencies using PSCs as unlimited cell resource.

immunology↗

bioRxiv2021

A pair of transporters controls mitochondrial Zn2+ levels to maintain mitochondrial homeostasis

Zn2+ is required for the activity of many mitochondrial proteins, which regulate mitochondrial dynamics, apoptosis and mitophagy. However, it is not understood how the proper mitochondrial Zn2+ level is achieved to maintain mitochondrial homeostasis. Using Caenorhabditis elegans, we reveal here that a pair of mitochondrion-localized transporters controls the mitochondrial level of Zn2+. We demonstrate that SLC-30A9/ZnT9 is a mitochondrial Zn2+ exporter. Loss of SLC-30A9 leads to mitochondrial Zn2+ accumulation, which damages mitochondria, impairs animal development and shortens the life span. We further identify SLC-25A25/SCaMC-2 as an important regulator of mitochondrial Zn2+ import. Loss of SLC-25A25 suppresses the abnormal mitochondrial Zn2+ accumulation and defective mitochondrial structure and functions caused by loss of SLC-30A9. Moreover, we reveal that the endoplasmic reticulum contains the Zn2+ pool from which mitochondrial Zn2+ is imported. These findings establish the molecular basis for controlling the correct mitochondrial Zn2+ levels for normal mitochondrial structure and functions. SummaryZn2+ is a trace ion essential for the function of many mitochondrial proteins. It is not known how mitochondrial Zn2+ levels are regulated. Ma at al. identify transporters that mediate mitochondrial Zn2+ export and import to maintain mitochondrial homeostasis.

cell biology↗

bioRxiv2021

ACVR1 antibodies exacerbate heterotopic ossification in fibrodysplasia ossificans progressiva (FOP) by activating FOP-mutant ACVR1

Fibrodysplasia ossificans progressiva (FOP) is a rare genetic disorder whose most debilitating pathology is progressive and cumulative heterotopic ossification (HO) of skeletal muscles, ligaments, tendons, and fascia. FOP is caused by mutations in the type I BMP receptor gene ACVR1, which enable ACVR1 to utilize its natural antagonist, Activin A, as an agonistic ligand. The physiological relevance of this property is underscored by the fact that HO in FOP is exquisitely dependent on activation of FOP-mutant ACVR1 by Activin A, an effect countered by inhibition of Activin A via monoclonal antibody treatment. Hence, we surmised that ACVR1 antibodies that block activation of ACVR1 by ligand should also inhibit HO in FOP and provide an additional therapeutic option for this condition. Therefore, we generated ACVR1 monoclonal antibodies that block ACVR1s activation by its ligands. Surprisingly, in vivo, these ACVR1 antibodies stimulate HO and activate signaling of FOP-mutant ACVR1. This property is restricted to FOP-mutant ACVR1 and results from ACVR1 antibody-mediated dimerization of ACVR1. Conversely, wild type ACVR1 is inhibited by ACVR1 antibodies. These results uncover an additional novel property of FOP-mutant ACVR1 and indicate that ACVR1 antibodies should not be considered as therapeutics for FOP.

biochemistry↗

bioRxiv2021

Network expansion of genetic associations defines a pleiotropy map of human cell biology

Proteins that interact within molecular networks tend to have similar functions and when perturbed influence the same organismal traits. Interaction networks can be used to expand the list of likely trait associated genes from genome-wide association studies (GWAS). Here, we used improvements in SNP-to-gene mapping to perform network based expansion of trait associated genes for 1,002 human traits showing that this recovers known disease genes or drug targets. The similarity of network expansion scores identifies groups of traits likely to share a common genetic basis as well as the biological processes underlying this. We identified 73 pleiotropic gene modules linked to multiple traits that are enriched in genes involved in processes such as protein ubiquitination and RNA processing. We show examples of modules linked to human diseases enriched in genes with pathogenic variants found in patients or relevant mouse knock-out phenotypes and can be used to map targets of approved drugs for repurposing opportunities. Finally, we illustrate the use of the network expansion scores to study genes at inflammatory bowel disease (IBD) GWAS loci, and implicate IBD-relevant genes with strong functional and genetic support.

genetics↗

bioRxiv2021

Structural and functional characterization of ubiquitin variant inhibitors for the JAMM-family deubiquitinases STAMBP and STAMBPL1

Ubiquitination is one of the most crucial post-translational protein modifications involved in a myriad of biological pathways. This process is reversed by deubiquitinases (DUBs) that deconjugate the ubiquitin (Ub) moiety or poly-Ub chains from substrates. In the past decade, tremendous efforts have been focused on targeting DUBs for drug discovery. However, most chemical compounds with inhibitory activity for DUBs suffer from mild potency and low selectivity. To overcome these obstacles, we developed a phage display-based protein engineering strategy for generating Ub variant (UbV) inhibitors and have previously successfully applied it to the Ub-specific protease (USP) family of cysteine proteases. In this work, we leveraged the UbV platform to target STAMBP, a member of the JAB1/MPN/MOV34 (JAMM) metalloprotease family of DUB enzymes. We identified two UbVs (UbVSP.1 and UbVSP.3) that bind to STAMBP with high affinity but differ in their selectivity for the closely related paralog STAMBPL1. We determined the STAMBPL1-UbVSP.1 complex structure by X-ray crystallography, revealing hotspots of the tight JAMM-UbV interaction. Finally, we show that UbVSP.1 and UbVSP.3 are potent inhibitors of the STAMBP isopeptidase activity, far exceeding the reported small-molecule inhibitor BC-1471. This work demonstrates that UbV technology is suitable to develop tool molecules for metalloproteases. These tools can be used to understand the cellular function of JAMM family DUBs.

biochemistry↗

bioRxiv2021

TaBln1 negatively regulates wheat resistance to stripe rust by reducing Ca2+ influx

Blufensin1 (Bln1) has been identified as a negative regulator of basal defense mechanisms that is unique to the cereal grain crops barley, wheat, and rice. However, the molecular mechanisms through which Blufensin1 regulates the wheat immune response are poorly understood. In this study, we found that TaBln1 is significantly induced by Puccinia striiformis f. sp. tritici (Pst) virulent race CYR31 infection. Knockdown the expression of TaBln1 by virus-induced gene silencing reduced Pst growth and development, and enhanced the host defense response. In addition, TaBln1 was found to physically interact with TaCaM3 on the plasma membrane. Silencing TaCaM3 with virus-induced gene silencing increased fungal infection areas and sporulation and reduced wheat resistance to the Pst CYR23 and CYR31. Moreover, we found that the TaCaM3 transcription level could be induced by treatment with chitin but not flg22. Silencing TaCaM3 decreased the Ca2+ influx induced by chitin, but silencing TaBln1 increased the Ca2+ influx in vivo using a non-invasive micro-test technique. Taken together, we identified the wheat negative regulator TaBln1, which interacts with TaCaM3 to impair Ca2+ influx and inhibits plant defenses. One-sentence summaryTaBln1 negatively regulate wheat resistance to stripe rust possibly due to the interaction with TaCaM3 on the plasma membrane, which impairs the calcium influx modulated by TaCaM3.

pathology↗

bioRxiv2021

Native MOWChIP-seq: Genome-wide profiles of key protein bindings reveal functional differences among various brain regions

Genome-wide profiling of interactions between genome and various functional proteins is critical for understanding regulatory processes involved in development and diseases. Conventional assays require a large number of cells and high-quality data on tissue samples are scarce. Here we optimized a low-input chromatin immunoprecipitation followed by sequencing (ChIP-seq) technology for profiling RNA polymerase II (Pol II), transcription factor (TF), and enzyme binding at the genome scale. The new approach, termed native MOWChIP-seq, produces high-quality binding profiles using 1000-50,000 cells. We used the approach to examine the binding of Pol II and two TFs (EGR1 and MEF2C) in cerebellum and prefrontal cortex of mouse brain and found that their binding profiles are highly reflective of the functional differences between the two brain regions. Our analysis reveals the potential for linking genome-wide TF or Pol II profiles with neuroanatomical origins of brain cells.

genomics↗

bioRxiv2021

Local CpG density affects the trajectory of age-associated epigenetic changes

DNA methylation is an epigenetic mark associated with gene repression and genome stability. Its pattern in the genome is disrupted with age and these changes can be used to statistically predict age with epigenetic clocks. Rates of aging inferred from these clocks correlate with human health. However, the molecular mechanisms underpinning age-associated DNA methylation changes are unknown. Local DNA sequence plays a strong role in programming DNA methylation levels at individual loci independently of age, but its influence on age-associated DNA methylation changes is unknown. We analysed longitudinal human DNA methylation trajectories at 345,895 CpGs from 600 individuals aged between 67 and 80 to understand the factors responsible for age-associated epigenetic changes at individual CpGs in the genome. We show that changes in methylation with age are especially apparent at 8,322 low CpG density loci. Using SNP data from the same individuals we demonstrate that DNA methylation trajectories are affected by local sequence polymorphisms at 1,487 loci with low CpG density. More generally, we find that local CpG density is a strong determinant of a CpGs methylation trajectory and that CpGs located in low CpG density regions are particularly prone to change. Overall, our results demonstrate that local DNA sequence influences age-associated DNA methylation changes in humans in vivo. We suggest that this occurs because interactions between CpGs reinforce maintenance of methylation patterns in CpG dense regions.

genetics↗