Biology subjects

Wu, B.

Publications and source records attributed to Wu, B..

At least 145 records · Page 8Linked to original sources

bioRxiv2021

SALTS: SURFR (small noncoding RNA) And LAGOOn (long noncoding RNA) Transcriptomics Suite

The widespread utilization of high-throughput sequencing technologies has unequivocally demonstrated that eukaryotic transcriptomes consist primarily (>98%) of non-coding RNA (ncRNA) transcripts significantly more diverse than their protein-coding counterparts. ncRNAs are typically divided into two categories based on their length. (1) ncRNAs less than 200 nucleotides (nt) long are referred as small non-coding RNAs (sncRNAs) and include microRNAs (miRNAs), piwi-interacting RNAs (piRNAs), small nucleolar RNAs (snoRNAs), transfer ribonucleic RNAs (tRNAs), etc., and the majority of these are thought to function primarily in controlling gene expression. That said, the full repertoire of sncRNAs remains fairly poorly defined as evidenced by two entirely new classes of sncRNAs only recently being reported, i.e., snoRNA-derived RNAs (sdRNAs) and tRNA-derived fragments (tRFs). (2) ncRNAs longer than 200 nt long are known as long ncRNAs (lncRNAs). lncRNAs represent the 2nd largest transcriptional output of the cell (behind only ribosomal RNAs), and although functional roles for several lncRNAs have been reported, most lncRNAs remain largely uncharacterized due to a lack of predictive tools aimed at guiding functional characterizations. Importantly, whereas the cost of high-throughput transcriptome sequencing is now feasible for most active research programs, tools necessary for the interpretation of these sequencings typically require significant computational expertise and resources markedly hindering widespread utilization of these datasets. In light of this, we have developed a powerful new ncRNA transcriptomics suite, SALTS, which is highly accurate, markedly efficient, and extremely user-friendly. SALTS stands for SURFR (sncRNA) And LAGOOn (lncRNA) Transcriptomics Suite and offers platforms for comprehensive sncRNA and lncRNA profiling and discovery, ncRNA functional prediction, and the identification of significant differential expressions among datasets. Notably, SALTS is accessed through an intuitive Web-based interface, can be used to analyze either user-generated, standard next-generation sequencing (NGS) output file uploads (e.g., FASTQ) or existing NCBI Sequence Read Archive (SRA) data, and requires absolutely no dataset pre-processing or knowledge of library adapters/oligonucleotides. SALTS constitutes the first publically available, Web-based, comprehensive ncRNA transcriptomic NGS analysis platform designed specifically for users with no computational background, providing a much needed, powerful new resource capable of enabling more widespread ncRNA transcriptomic analyses. The SALTS WebServer is freely available online at http://salts.soc.southalabama.edu.

bioinformatics↗

bioRxiv2021

Genome-wide Detection of Cytosine Methylations in Plant from Nanopore sequencing data using Deep Learning

Methylation states of DNA bases can be detected from native Nanopore reads directly. At present, there are many computational methods that can detect 5mCs in CpG contexts accurately by Nanopore sequencing. However, there is currently a lack of methods to detect 5mCs in non-CpG contexts. In this study, we propose a computational pipeline which can detect 5mC sites in both CpG and non-CpG contexts of plant genomes by using Nanopore sequencing. And we sequenced two model plants Arabidopsis thaliana (A. thaliana) and Oryza sativa (O. sativa) by using Nanopore sequencing and bisulfite sequencing. The results of our proposed pipeline in the two plants achieved high correlations with bisulfite sequencing: above 0.98, 0.96, 0.85 for CpG, CHG, and CHH (H indicates A, C or T) motif, respectively. Our proposed pipeline also achieved high performance on Brassica nigra (B. nigra). Experiments also showed that our proposed pipeline can achieve high performance even with low coverage of reads. Moreover, by using Nanopore sequencing, our proposed pipeline is capable of profiling methylation of more cytosines than bisulfite sequencing.

bioinformatics↗

bioRxiv2020

Amphipathic Bax core dimer forms part of apoptotic pore wall in the mitochondrial membrane

Bax proteins form pores in the mitochondrial outer membrane to initiate apoptosis. They may embed in the cytosolic leaflet of the lipid bilayer generating tension to induce a lipid pore with radially arranged lipids forming the wall. Alternatively, they may comprise part of the pore wall. However, there is no unambiguous structural evidence for either hypothesis. Using NMR, we determine a high-resolution structure of the Bax core region that forms a dimer with the nonpolar surface covering the lipid bilayer edge and the polar surface exposed to water. The dimer tilts from the bilayer normal, not only maximizing nonpolar interactions with lipid tails but creating polar interactions between charged residues and lipid heads. Structure-guided mutations demonstrate the importance of both protein-lipid interactions in Bax pore assembly and core dimer configuration. Therefore, the Bax core dimer forms part of the proteolipid pore wall to permeabilize mitochondria.

biophysics↗

bioRxiv2020

Maximum Antigen Diversification in a Lyme Bacterial Population and Evolutionary Strategies to Overcome Pathogen Diversity

Natural populations of microbes and their hosts are engaged in an arms race in which microbes diversify to escape host immunity while hosts evolve novel immunity. This co-evolutionary process, known as the "Red Queen" hypothesis, poses a fundamental challenge to the development of broadly effective vaccines and diagnostics against a diversifying pathogen. Based on surveys of natural allele frequencies and experimental immunization of mice, we show minimal antigenic cross-reactivity among natural variants of the outer surface protein C (OspC), a dominant antigen of a Lyme Disease-causing bacterium (Borrelia burgdorferi). To overcome the challenge of OspC antigenic diversity to clinical development of preventive measures, we implemented a number of evolution-based strategies to broaden OspC immunological cross-reactivity. In particular, the centroid algorithm - a genetic algorithm to minimize sequence differences with natural variants - generated synthetic OspC analogs with the greatest promise as diagnostic and vaccine candidates against diverse Lyme pathogen strains coexisting in the Northeast United States. Mechanistically, we propose a model of runaway maximum antigen di-versification (MAD) mediated by amino-acid variations distributed across hypervariable regions on the OspC molecule. Under the MAD model, evolutionary centroids display high cross-reactivity by occupying the central void in the antigenic space excavated by diversifying natural variants. In contrast to the vaccine design based on concatenated epitopes, the centroid algorithm generates analogs of native antigens and is automated. The MAD model and evolution-inspired antigen designs have broad implications for combating diversifying pathogens driven by pathogen-host coevolution. ImportanceMicrobial pathogens rely on molecular diversity of cell surface antigens to escape host immunity. Vaccines based on one antigen variant often fail to protect the host against pathogens carrying other variants. Here we show evolution-based designs of synthetic antigens that are broadly reactive to all natural variants. The evolutionary analogs of a major surface antigen of a Lyme disease bacterium (Borrelia burgdorferi) showed promise as vaccine candidates against diverse pathogen strains coexisting in the endemic areas of Lyme disease in Northeast United States. Our evolution-based computational design is automated, generates molecular analogs of natural antigens, and opens a novel path to combating fast-evolving microbial pathogens.

microbiology↗

bioRxiv2020

RAD51AP1 mediates RAD51 activity through nucleosome interaction

RAD51 Associated Protein 1 (RAD51AP1) is a key protein in the homologous recombination DNA repair pathway (HR). Loss of RAD51AP1 leads to defective HR, genome instability and telomere erosion. RAD51AP1 physically interacts with the RAD51 recombinase and promotes RAD51-mediated capture of the donor DNA, synaptic complex assembly and displacement-loop formation when tested with synthetic, nucleosome-free DNA substrates in vitro. In cells, however, DNA is packaged into chromatin, posing an additional barrier to the complexities of the HR reaction. How RAD51AP1 functions as an HR activator in the context of chromatin has remained unclear. In this study, we show that RAD51AP1 binds to Nucleosome Core Particles (NCPs). We identified a C-terminal region in RAD51AP1 and its previously mapped DNA binding domain as critical for mediating the association between RAD51AP1 and both the NCP and the histone octamer. We show that RAD51AP1 is capable of promoting duplex DNA capture and initiating joint-molecule formation with the NCP and chromatinized template DNA, respectively. Together, our results suggest that RAD51AP1 directly assists the RAD51-mediated search of donor DNA in chromatin. We present a model, in which RAD51AP1 anchors the DNA template through affinity for its nucleosomes to the RAD51-ssDNA nucleoprotein filament.

biochemistry↗

bioRxiv2020

Novel gene-specific translation mechanism of dysregulated, chronic inflammation reveals promising, multifaceted COVID-19 therapeutics

Hyperinflammation and lymphopenia provoked by SARS-CoV-2-activated macrophages contribute to the high mortality of Coronavirus Disease 2019 (COVID-19) patients. Thus, defining host pathways aberrantly activated in patient macrophages is critical for developing effective therapeutics. We discovered that G9a, a histone methyltransferase that is overexpressed in COVID-19 patients with high viral load, activates translation of specific genes that induce hyperinflammation and impairment of T cell function or lymphopenia. This noncanonical, pro-translation activity of G9a contrasts with its canonical epigenetic function. In endotoxin-tolerant (ET) macrophages that mimic conditions which render patients with pre-existing chronic inflammatory diseases vulnerable to severe symptoms, our chemoproteomic approach with a biotinylated inhibitor of G9a identified multiple G9a-associated translation regulatory pathways that were upregulated by SARS-CoV-2 infection. Further, quantitative translatome analysis of ET macrophages treated progressively with the G9a inhibitor profiled G9a-translated proteins that unite the networks associated with viral replication and the SARS-CoV-2-induced host response in severe patients. Accordingly, inhibition of G9a-associated pathways produced multifaceted, systematic effects, namely, restoration of T cell function, mitigation of hyperinflammation, and suppression of viral replication. Importantly, as a host-directed mechanism, this G9a-targeted, combined therapeutics is refractory to emerging antiviral-resistant mutants of SARS-CoV-2, or any virus, that hijacks host responses.

systems biology↗

bioRxiv2020

Genetic composition and evolution of the prevalent Mycobacterium tuberculosis lineages 2 and 4 in the Chinese and Zhejiang Province populations

The causative agent of tuberculosis (TB) comprises seven human-adapted lineages. Human movements and host genetics are crucial to TB dissemination. We analyzed whole-genome sequencing data for a countrywide collection of 1154 isolates and a provincial collection of 1296 isolates, constructed the best-scoring maximum likelihood phylogenetic tree, conducted Bayesian evolutionary analysis to compute the most recent common ancestors of lineages 2 and 4, and assessed the antigenic diversity in human T cell epitopes by calculating pairwise dN/dS ratios. Of the 1296 Zhejiang isolates, 964 (74.38%) belonged to lineage 2 and 332 (25.62%) belonged to lineage 4. L2.2 is the most ancient sub-lineage in Zhejiang, first appearing approximately 6897 years ago (95% HDI: 6513-7298). L4.4 is the most modern sub-lineage, first appearing approximately 2217 years ago (95% HDI: 1864-2581). The dN/dS ratios revealed that the epitope and non-epitope regions of lineage 2 strains were significantly (P<0.001) more conserved than those of lineage 4. An increase in the frequency of lineage 4 may reflect its successful transmission over the last 20 years. The recent common ancestors and transmission routes of the sub-lineages are related to the entry of humans into China and Zhejiang Province.

molecular biology↗

bioRxiv2020

Leukemia stemness and co-occurring mutations drive resistance to IDH inhibitors in acute myeloid leukemia

Allosteric inhibitors of mutant IDH1 or IDH2 induce terminal differentiation of the mutant leukemic blasts and provide durable clinical responses in approximately 40% of acute myeloid leukemia (AML) patients with the mutations. However, primary resistance and acquired resistance to the drugs are major clinical issues. To understand the molecular underpinnings of clinical resistance to IDH inhibitors (IDHi), we performed multipronged genomic analyses (DNA sequencing, RNA sequencing and cytosine methylation profiling) in longitudinally collected specimens from 68 IDH1- or IDH2-mutant AML patients treated with the inhibitors. The analysis revealed that leukemia stemness is a major driver of primary resistance to IDHi, whereas selection of mutations in RUNX1/CEBPA or RAS-RTK pathway genes was the main driver of acquired resistance to IDHi, along with BCOR, homologous IDH gene, and TET2. These data suggest that targeting stemness and certain high-risk co-occurring mutations may overcome resistance to IDHi in AML.

cancer biology↗

bioRxiv2020

Parental CG and CHG methylation variation is associated with allelic-specific expression in elite hybrid rice

Heterosis refers to the superior performance of the hybrid over the inbred parental lines. Besides genetic variation, epigenetic difference between the parental lines has been suggested to be involved in heterosis. However, precise nature and extent of parental epigenome difference and reprograming in hybrids governing heterotic gene expression remain unclear. In this work, we analyzed DNA methylomes and transcriptomes of the widely cultivated and genetically studied elite hybrid rice SY63, the reciprocal hybrid, and the parental varieties ZS97 and MH63, of which the high-quality reference genomic sequences are available. We show that the parental varieties display important variation in genic methylation at CG and CHG (H=A, C, or T) sequences. Compared with the parents the hybrids display dynamic methylation variation during development. However, many parental differentially methylated regions (DMR) at CG and CHG sites are maintained in the hybrid. Only a small fraction of the DMRs display non-additive DNA methylation variation which, however, shows no overall correlation with gene expression variation. By contrast, most of the allelic-specific expression (ASE) genes in the hybrid are associated with DNA methylation and the ASE negatively correlates with allelic-specific methylation (ASM) at CHG but positively at CG sites. The results reveal a specific DNA methylation reprogramming pattern in the hybrid rice and point to a role of parental CG and CHG methylation divergence in allelic specific expression that has been associated with phenotype variation and hybrid vigor in several plant species. One sentence summaryParental CG and CHG methylation divergence is maintained in hybrid and is related to allelic specific expression associated with phenotype variation and hybrid vigor.

genetics↗

bioRxiv2020

Construction of a third NAD+ de novo biosynthesis pathway

Only two de novo biosynthetic routes to nicotinamide adenine dinucleotide (NAD+) have been described, both of which start from a proteinogenic amino acid and are tightly controlled. Here we establish a C3N pathway starting from chorismate in Escherichia coli as a third NAD+ de novo biosynthesis pathway. Significantly, the C3N pathway yielded extremely high cellular concentrations of NAD(H) in E. coli. Its utility in cofactor engineering was demonstrated by introducing the four-gene C3N module to cell factories to achieve higher production of 2,5-dimethylpyrazine and develop an efficient C3N-based whole-cell bioconversion system for preparing chiral amines. The wide distribution and abundance of chorismate in most kingdoms of life implies a general utility of the C3N pathway for modulating cellular levels of NAD(H) in versatile organisms.

synthetic biology↗

bioRxiv2020

Defects in plant immunity modulate the rates and patterns of RNA virus evolution

It is assumed that host genetic variability for susceptibility to infection necessarily conditions virus evolution. Differences in host susceptibility can either drive the virus to diversify into strains that track different defense alleles (e.g., antigenic diversity) or to infect only the most susceptible genotypes. To clarify these processes and their effect on virulence, we have studied how variability in host defense responses determine the evolutionary fate of viruses. To accomplish this, we performed evolution experiments with Turnip mosaic potyvirus in Arabidopsis thaliana mutants. Mutant plants had disruptions in infection-response signaling pathways or in genes whose products are essential for potyvirus infection. Genotypes were classified into five phenogroups according to their response to infection. In order to disentangle how host susceptibility affects virus adaptation, independent viral lineages were evolved in selected plant genotypes. Evaluating disease-related traits of the evolved lineages, we found that evolution proceeded faster in the most resistant hosts than in the most permissive ones, as expected for adaptation to a harsh environment. By sequencing the genomes of the evolved viral lineages, we found that the multifunctional protein VPg turned out to be the target of selection in most host genotypes. When all evolved viral lineages were tested for fitness in all plant genotypes used in the experiments, we found compelling evidences that generalist viruses were selected by the most restrictive plant genotypes, while permissive genotypes selected for specialist viruses. Overall, this work illustrates how different host defense signaling pathways constrain not only disease-related traits but virus evolution.

evolutionary biology↗

bioRxiv2020

Parvalbumin interneurons in the nucleus accumbens regulate impairment of risk avoidance in DISC1 transgenic mice

One strong survival instinct in animals is to approach things that are of benefit and avoid risk. In humans, a large portion of mental disorders are accompanied by cognition-related impairments including the inability to recognize potential risks. One of the most important genes involved in risk behavior is disrupted-in-schizophrenia-1 (DISC1), and animal models where this gene has some dysfunction show cognitive impairments. However, whether DISC1 mice models have an impairment in avoiding potential risks is still not fully understood. In the present study, we used DISC1-N terminal truncation (DISC1-NTM) mice to study cognitive abilities related to potential risks. We found that DISC1-NTM mice were impaired in risk avoidance on the elevated plus maze (EPM) test, and showed impairment in social preference in a three-chamber social interaction test. Staining for c-Fos following the EPM indicated that the nucleus accumbens (NAc) was associated with risk avoidance behavior in DISC1-NTM mice. Meanwhile, in vivo electrophysiological recordings showed that firing rates of fast spiking neurons (FS) in the NAc significantly decreased in DISC1-NTM mice following tamoxifen administration. In addition, theta band power was lower when mice shuttled from the safe (closed) arms to the risky (open) arms, an effect which disappeared after induction of the truncated DISC1 gene. Furthermore, we found through in vitro patch clamp recording that the frequency of action potentials stimulated by current injection was lower in parvalbumin (PV) neurons in the NAc of DISC1-NTM mice than their wild-type littermates. Risk-avoidance impairments in DISC1-NTM mice were rescued using optogenetic tools that activated NAcPV neurons. Finally, we inhibited activitiy of NAcPV neurons in PV-Cre mice, which mimicked the risk-avoidance impairment found in the DISC1-NTM mice during tests on the elevated zero maze. Taken together, our findings confirmed a cognitive impairment in DISC1-NTM mice related to risk recognition and suggests that reduced excitability of NAcPV neurons may be responsible.

neuroscience↗

bioRxiv2020

Structural and biochemical mechanisms of NLRP1 inhibition by DPP9

The nucleotide-binding domain (NBD) and leucine-rich repeat (LRR)-containing receptors (NLRs) mediate innate immunity by forming inflammasomes. Activation of the NLR protein NLRP1 requires auto-cleavage within its FIIND domain1-7. In resting cells, the dipeptidyl peptidase DPP9 interacts with NLRP1-FIIND and together with a related enzyme DPP8, suppresses spontaneous NLRP1 activation8,9. The mechanisms of DPP8/9-mediated NLRP1 inhibition, however, remain elusive. Here we provide structural and biochemical evidence demonstrating that rat NLRP1 (rNLRP1) interacts with rDPP9 in a stepwise manner to form a 2:1 complex. An auto-inhibited rNLRP1 molecule first interacts with rDPP9 via its ZU5 domain. This 1:1 rNLRP1-rDPP9 complex then captures the UPA domain of a second rNLRP1 molecule via a UPA-interacting site on DPP9 and dimeric UPA-UPA interactions with the first rNLRP1. The 2:1 rNLRP1-rDPP9 complex prevents NLRP1 UPA-mediated higher order oligomerization and maintains NLRP1 in the auto-inhibited state. Structure-guided biochemical and functional assays show that both NLRP1-binding and its enzymatic activity are required for DPP9 to suppress NLRP1, supporting guard-type activation of the NLR. Together, our data reveal the mechanism of DPP9-mediated inhibition of NLRP1 and shed light on activation of the NLRP1 inflammasome.

immunology↗

bioRxiv2020

Structural basis for distinct inflammasome complex assembly by human NLRP1 and CARD8

Nod-like receptor (NLR) proteins activate pyroptotic cell death and IL-1 driven inflammation by assembling and activating the inflammasome complex. Closely related NLR proteins, NLRP1 and CARD8 undergo unique auto-proteolysis-dependent activation and are implicated in auto-inflammatory diseases; however, the molecular mechanisms of activation are not understood. Here we report the structural basis of how the activating domains (FIINDUPA-CARD) of NLRP1 and CARD8 self-oligomerize to trigger the assembly of distinct inflammasome complexes. Recombinant FIINDUPA-CARD of NLRP1 forms a two-layered filament, with an inner core composed of oligomerized CARD domains and the outer layer consisting of FIINDUPA rings. Biochemically, oligomerized NLRP1-CARD is sufficient to drive ASC speck formation in cultured human cells via filament formation-a process that is greatly enhanced by NLRP1-FIINDUPA, which forms ring-like oligomers in vitro. In addition, we report the cryo-EM structures of NLRP1-CARD and CARD8-CARD filaments at 3.7 [A], which uncovers unique structural features that enable NLRP1 and CARD8 to discriminate between ASC and pro-caspase-1. In summary, our findings provide unique structural insight into the mechanisms of activation for human NLRP1 and CARD8, uncovering an unexpected level of specificity in inflammasome signaling mediated by heterotypic CARD domain interactions.

immunology↗

bioRxiv2020

Dynamic regulation of translation quality control associated with ribosome stalling

Translation of problematic mRNA sequences induces ribosome stalling. Collided ribosomes at the stall site are recognized by cellular quality control machinery, resulting in dissociation of the ribosome from the mRNA and subsequent degradation of the nascent polypeptide and in some organisms, decay of the mRNA. However, the timing and regulation of these processes are unclear. We developed a SunTag-based reporter to monitor translation in real time on single mRNAs harboring difficult-to-translate poly(A) stretches. This reporter recapitulates previous findings in human cells that an internal poly(A) stretch reduces protein output [~]10-fold, while mRNA levels are relatively unaffected. Long-term imaging of translation indicates that poly(A)-containing mRNAs are robustly translated in the absence of detectable mRNA cleavage. However, quantification of ribosome density reveals a [~]3-fold increase in the number of ribosomes on poly(A)-containing mRNAs compared to a control mRNA, consistent with queues of many stalled ribosomes. Using single-molecule harringtonine runoff experiments, we observe the resolution of these queues in real-time by the cellular quality control machinery, and find that rescue is very slow compared to both elongation and termination. We propose that the very slow clearance of stalled ribosomes provides the basis for the cell to distinguish between transient and deleterious stalls, and that the human quality control apparatus predominantly targets the nascent protein rather than the mRNA.

biochemistry↗

bioRxiv2020

Cryo-EM Structure of Native Human Uromodulin, a Zona Pellucida Module Polymer

Assembly of extracellular filaments and matrices mediating fundamental biological processes such as morphogenesis, hearing, fertilization and antibacterial defense is driven by a ubiquitous polymerization module known as zona pellucida (ZP) "domain". Despite the conservation of this element from hydra to human, no information is available on the filamentous conformation of any ZP module protein. Here we report the cryo-electron microscopy structure of uromodulin (UMOD)/Tamm-Horsfall protein, the most abundant protein in human urine and an archetypal ZP module-containing molecule, in its mature homopolymeric state. UMOD forms a one-start helix with an unprecedented 180-degree twist between subunits enfolded by interdomain linkers that have completely reorganized as a result of propeptide dissociation. Lateral interaction between filaments in the urine generates sheets exposing a checkerboard of binding sites to capture uropathogenic bacteria, and UMOD-based models of mammalian and avian heteromeric egg coat filaments identify a common sperm-binding region at the interface between subunits.

biophysics↗

bioRxiv2020

Dual located WHIRLY1 affects salicylic acid homeostasis via coordination of ICS1, 1 PAL1 and BSMT1 during Arabidopsis plant aging

Salicylic acid (SA) homeostasis determines also developmental senescence and is spatiotemporally controlled by various mechanisms, including biosynthesis, transport and conjugate formation. The alteration of WHIRLY1 (WHY1), a repressor of leaf natural senescence, with respect to allocation in the nucleus or chloroplast causes a perturbation in SA homeostasis, resulting in adverse plant senescence phenotypes. Loss of WHY1 resulted in a 5 days earlier SA peak compared to wild type plants which accumulated SA at 42 days after germination. SA accumulation coincided with an early leaf senescence phenotype, which could be prevented by ectopic expression of the nuclear WHY1 isoform (nWHY1). However, expressing the plastid WHY1 isoform (pWHY1) greatly enhanced cellular SA levels. A global transcriptional analysis in WHY1 loss-of-function background by expressing either pWHY1 or nWHY1 indicated that hormone metabolism related genes were most significantly altered. The pWHY1 isoform predominantly affected stress related gene expression, while the nWHY1 controlled rather developmental gene expression. Chromatin immunoprecipitation-qPCR (ChIP-qPCR) assays indicated that nWHY1 directly binds to the promoter region of isochorismate synthase (ICS1) to activate its expression at later stage, but indirectly activated S-adenosyl-L-methionine-dependent methyltransferase (BSMT1) gene expression via ethylene response factor 109 (ERF109), while repressing phenylalanine ammonia lyase (PAL1) expression via R2R3-MYB member 15 (MYB15) at the early stage of development. Interestingly, rising SA levels exerted a feedback effect by inducing nWHY1 modification and pWHY1 accumulation. Thus, the alteration of WHY1 organelle isoforms and the feedback of SA intervened in a circularly integrated regulatory network during developmental or stress-induced senescence in Arabidopsis.

plant biology↗

bioRxiv2020

DNA methylation is indispensable for leukemia inhibitory factor dependent embryonic stem cells reprogramming

Naive pluripotency can be maintained by the 2i/LIF supplements (CHIR99021, PD0325901 and LIF), which primarily affect canonical WNT, FGF/ERK, and JAK/STAT3 signaling. However, whether one of these tripartite supplements alone is sufficient to maintain naive self-renewal remain unclear. Here we show that LIF alone is sufficient to induce reprogramming of 2i/LIF cultured ESCs (2i/L-ESCs) to ESCs with hypermethylated state (L-ESCs). In vitro, upon withdrawal of 2i, 2i/L-ESCs overcome the epigenetic barrier and DNA hypermethylated, which accompanies transcriptional changes and subsequent establishment of epigenetic memory. Global transcriptome features also show that L-ESCs are close to 2i/L-ESCs and in a stable state between naive and primed pluripotency. Notably, our results demonstrate that DNA methylation was indispensable for LIF-dependent mouse ESCs reprogramming and self-renew. LIF-dependent ESCs reprogramming efficiency is significantly increased in serum treatment and reduced in Dnmt3a or Dnmt3l knockout ESCs. Importantly, unlike epiblast and EpiSCs, L-ESCs contribute to somatic tissues and germ cells in chimaeras. Such simple culture system of ESCs is more conducive to clarify the molecular mechanism of ESCs in vitro culture. SignificanceEmbryonic stem cell (ESCs) exhibit naive pluripotency which reflects their ability to contribute to all embryonic lineages upon injection into blastocyst. ESCs were originally derived by co-culture with feeder cells and fetal calf serum. In this manuscript, we took a detailed approach to dissect the roles of LIF alone in ESC reprogramming of 2i/LIF cultured ESCs (2i/L-ESCs). Here, for the first time, we derived stable hypermethylated pluripotent ESCs under culture of LIF alone (L-ESCs). We further assessed L-ESCs properties both in vitro and in vivo, and provide molecular insights to the mechanism which allows LIF alone to maintain pluripotency and a hypermethylated state. We believe these findings are novel and valuable for future ESCs study.

cell biology↗