Differential Transcriptomic Landscapes of SARS-CoV-2 Variants in Multiple Organs from Infected Rhesus Macaques

Severe acute respiratory syndrome coronavirus 2(SARS-CoV-2)caused the persistent coronavirus disease 2019(COVID-19)pandemic,which has resulted in millions of deaths worldwide and brought an enormous public health and global economic burden.The recurring global wave of infections has been exacerbated by growing variants of SARS-CoV-2.In this study,the virological characteristics of the original SARS-CoV-2 strain and its variants of concern(VOCs;including Alpha,Beta,and Delta)in vitro,as well as differential transcriptomic landscapes in multiple organs(lung,right ventricle,blood,cerebral cortex,and cerebellum)from the infected rhesus macaques,were elucidated.The original strain of SARS-CoV-2 caused a stronger innate immune response in host cells,and its VOCs markedly increased the levels of subgenomic RNAs,such as N,Orf9b,Orf6,and Orf7ab,which are known as the innate immune antagonists and the inhibitors of antiviral factors.Intriguingly,the original SARS-CoV-2 strain and Alpha variant induced larger alteration of RNA abundance in tissues of rhesus monkeys than Beta and Delta variants did.Moreover,a hyperinflammatory state and active immune response were shown in the right ventricles of rhesus monkeys by the up-regulation of inflammation-and immune-related RNAs.Furthermore,peripheral blood may mediate signaling transmission among tissues to coordinate the molecular changes in the infected individuals.Collectively,these data provide insights into the pathogenesis of COVID-19 at the early stage of infection by the original SARS-CoV-2 strain and its VOCs.

CTCF acetylation at lysine 20 is required for the early cardiac mesoderm differentiation of embryonic stem cells

The CCCTC-binding factor(CTCF)protein and its modified forms regulate gene expression and genome organization.However,information on CTCF acetylation and its biological function is still lacking.Here,we show that CTCF can be acetylated at lysine 20(CTCF-K20)by CREB-binding protein(CBP)and deacetylated by histone deacetylase 6(HDAC6).CTCF-K20 is required for the CTCF interaction with CBP.A CTCF point mutation at lysine 20 had no effect on self-renewal but blocked the mesoderm differentiation of mouse embryonic stem cells(mESCs).The CTCF-K20 mutation reduced CTCF binding to the promoters and enhancers of genes associated with early cardiac mesoderm differentia-tion,resulting in diminished chromatin accessibility and decreased enhancer-promoter interactions,impairing gene expression.In summary,this study reveals the important roles of CTCF-K20 in regulating CTCF genomic functions and mESC differentiation into mesoderm.

CRISPR/Cas9-mediated gene knockout reveals a guardian role of NF-κB/RelA in maintaining the homeostasis of human vascular cells

Vascular cell functionality is critical to blood vessel homeostasis. Constitutive NF-κB activation in vascular cells results in chronic vascular inflammation, leading to various cardiovascular diseases. However, how NF-κB regulates human blood vessel homeostasis remains largely elusive. Here, using CRISPR/Cas9-mediated gene editing, we generated RelA knockout human embryonic stem cells （hESCs） and differentiated them into various vascular cell derivatives to study how NF- KS modulates human vascular cells under basal and inflammatory conditions. Multi-dimensional phenotypic assessments and transcriptomic analyses revealed that RelA deficiency affected vascular cells via modulatinginflammation, survival, vasculogenesis, cell differentia- tion and extracellular matrix organization in a cell type- specific manner under basal condition, and that RelA protected vascular cells against apoptosis and modu- lated vascular inflammatory response upon tumor necrosis factor a （TNFa） stimulation. Lastly, further evaluation of gene expression patterns in IKBo knockout vascular cells demonstrated that IKBa acted largely independent of RelA signaling. Taken together, our data reveal a protective role of NF-κB/ReiA in modulating human blood vessel homeostasis and map the human vascular transcriptomic landscapes for the discovery of novel therapeutic targets.

Differential transcriptomic landscapes of multiple organs from SARS-CoV-2 early infected rhesus macaques

SARS-CoV-2 infection causes complicated clinical manifestations with variable multi-organ injuries,how-ever,the underlying mechanism,in particular immune responses in different organs,remains elusive.In this study,comprehensive transcriptomic alterations of 14 tissues from rhesus macaque infected with SARS-CoV-2 were analyzed.Compared to normal controls,SARS-CoV-2 infection resulted in dysregulation of genes involving diverse functions in various examined tissues/organs,with drastic transcriptomic changes in cerebral cortex and right ventricle.Intriguingly,cerebral cortex exhibited a hyperinflammatory state evidenced by sig-nificant upregulation of inflammation response-related genes.Meanwhile,expressions of coagulation,angio-genesis and fibrosis factors were also up-regulated in cerebral cortex.Based on our findings,neuropilin 1(NRP1),a receptor of SARS-CoV-2,was significantly elevated in cerebral cortex post infection,accompanied by active immune response releasing inflammatory factors and signal transmission among tissues,which enhanced infection of the central nervous system(CNS)in a positive feedback way,leading to viral encephalitis.Overall,our study depicts a multi-tissue/organ tran-scriptomic landscapes of rhesus macaque with early infection of SARS-CoV-2,and provides important insights into the mechanistic basis for COVID-19-asso-ciated clinical complications.

Characteristics of N^(6)-methyladenosine Modification During Sexual Reproduction of Chlamydomonas reinhardtii

The unicellular green alga Chlamydomonas reinhardtii(hereafter Chlamydomonas)possesses both plant and animal attributes,and it is an ideal model organism for studying fundamental processes such as photosynthesis,sexual reproduction,and life cycle.N^(6)-methyladenosine(m^(6)A)is the most prevalent mRNA modification,and it plays important roles during sexual reproduction in animals and plants.However,the pattern and function of m^(6)A modification during the sexual reproduction of Chlamydomonas remain unknown.Here,we performed transcriptome and methylated RNA immunoprecipitation sequencing(MeRIP-seq)analyses on six samples from different stages during sexual reproduction of the Chlamydomonas life cycle.The results show that m^(6)A modification frequently occurs at the main motif of DRAC(D=G/A/U,R=A/G)in Chlamydomonas mRNAs.Moreover,m^(6)A peaks in Chlamydomonas mRNAs are mainly enriched in the 30 untranslated regions(30 UTRs)and negatively correlated with the abundance of transcripts at each stage.In particular,there is a significant negative correlation between the expression levels and the m^(6)A levels of genes involved in the microtubule-associated pathway,indicating that m^(6)A modification influences the sexual reproduction and the life cycle of Chlamydomonas by regulating microtubule-based movement.In summary,our findings are the first to demonstrate the distribution and the functions of m^(6)A modification in Chlamydomonas mRNAs and provide new evolutionary insights into m^(6)A modification in the process of sexual reproduction in other plant organisms.

Dynamic DNA 5-hydroxylmethylcytosine and RNA 5-methycytosine Reprogramming During Early Human Development

After implantation,complex and highly specialized molecular events render functionally distinct organ formation,whereas how the epigenome shapes organ-specific development remains to be fully elucidated.Here,nano-hmC-Seal,RNA bisulfite sequencing(RNA-BisSeq),and RNA sequencing(RNA-Seq)were performed,and the first multilayer landscapes of DNA 5-hydroxymethylcytosine(5hmC)and RNA 5-methylcytosine(m^(5)C)epigenomes were obtained in the heart,kidney,liver,and lung of the human foetuses at 13-28 weeks with 123 samples in total.We identified 70,091 and 503 organ-and stage-specific differentially hydroxymethylated regions(DhMRs)and m^(5)C-modified mRNAs,respectively.The key transcription factors(TFs),T-box transcription factor 20(TBX20),paired box 8(PAX8),krueppel-like factor 1(KLF1),transcription factor 21(TCF21),and CCAAT enhancer binding protein beta(CEBPB),specifically contribute to the formation of distinct organs at different stages.Additionally,5hmC-enriched Alu elements may participate in the regulation of expression of TF-targeted genes.Our integrated studies reveal a putative essential link between DNA modification and RNA methylation,and illustrate the epigenetic maps during human foetal organogenesis,which provide a foundation for an in-depth understanding of the epigenetic mechanisms underlying early development and birth defects.

Omics Views of Mechanisms for Cell Fate Determination in Early Mammalian Development

During mammalian preimplantation development,a totipotent zygote undergoes several cell cleavages and two rounds of cell fate determination,ultimately forming a mature blastocyst.Along with compaction,the establishment of apicobasal cell polarity breaks the symmetry of an embryo and guides subsequent cell fate choice.Although the lineage segregation of the inner cell mass(ICM)and trophectoderm(TE)is the first symbol of cell differentiation,several molecules have been shown to bias the early cell fate through their inter-cellular variations at much earlier stages,including the 2-and 4-cell stages.The underlying mechanisms of early cell fate determination have long been an important research topic.In this review,we summarize the molecular events that occur during early embryogenesis,as well as the current understanding of their regulatory roles in cell fate decisions.Moreover,as powerful tools for early embryogenesis research,single-cell omics techniques have been applied to both mouse and human preimplantation embryos and have contributed to the discovery of cell fate regulators.Here,we summarize their applications in the research of preimplantation embryos,and provide new insights and perspectives on cell fate regulation.

神经退行性疾病研究及治疗新方向:核糖体相关蛋白质量控制

Ribosome-associated protein quality control (RQC) is a surveillance system that detects translation stalling events and degrades incomplete or defective polypeptides. This process maintains protein homeostasis and protects cells from proteotoxic stress. Evidence of compromised RQC functionality has been observed in the aging process and in several age-related neurodegenerative afflictions, including amyotrophic lateral sclerosis, Parkinson’s disease (PD), Huntington’s disease (HD), and Alzheimer’s disease (AD)。

m^6A Regulates Neurogenesis and Neuronal Development by Modulating Histone Methyltransferase Ezh2

N6-methyladenosine (m6A),catalyzed by the methyltransferase complex consisting of Mettl3 and Mettl14,is the most abundant RNA modification in mRNAs and participates in diverse biological processes. However,the roles and precise mechanisms of m6A modification in regulating neuronal development and adult neurogenesis remain unclear. Here,we examined the function of Mettl3,the key component of the complex,in neuronal development and adult neurogenesis of mice. We found that the depletion of Mettl3 significantly reduced m6A levels in adult neural stem cells (aNSCs) and inhibited the proliferation of aNSCs. Mettl3 depletion not only inhibited neu-ronal development and skewed the differentiation of aNSCs more toward glial lineage,but also affected the morphological maturation of newborn neurons in the adult brain. m6A immunoprecip-itation combined with deep sequencing (MeRIP-seq) revealed that m6A was predominantly enriched in transcripts related to neurogenesis and neuronal development. Mechanistically,m6A was present on the transcripts of histone methyltransferase Ezh2,and its reduction upon Mettl3 knockdown decreased both Ezh2 protein expression and consequent H3K27me3 levels. The defects of neurogenesis and neuronal development induced by Mettl3 depletion could be rescued by Ezh2 overexpression. Collectively,our results uncover a crosstalk between RNA and histone modifica-tions and indicate that Mettl3-mediated m6A modification plays an important role in regulating neurogenesis and neuronal development through modulating Ezh2.

Chemical screen identifies a geroprotective role of quercetin in premature aging

Aging increases the risk of various diseases. The main goal of aging research is to find therapies that attenuate aging and alleviate aging-related diseases. In this study, we screened a natural product library for geroprotective compounds using Werner syndrome (WS) human mesenchymal stem cells (hMSCs), a premature aging model that we recently established. Ten candidate compounds were identified and quercetin was investigated in detail due to its leading effects. Mechanistic studies revealed that quercetin alleviated senescence via the enhancement of cell proliferation and restoration of heterochromatin architecture in WS hMSCs. RNA-sequencing analysis revealed the transcriptional commonalities and differences in the geroprotective effects by quercetin and Vitamin C. Besides WS hMSCs, quercetin also attenuated cellular senescence in Hutchinson-Gilford progeria syndrome (HGPS) and physiological-aging hMSCs. Taken together, our study identifies quercetin as a geroprotective agent against accelerated and natural aging in hMSCs, providing a potential therapeutic intervention for treating age-associated disorders.

FOXO3-engineered human mesenchymal progenitor cells efficiently promote cardiac repair after myocardial infarction

Dear Editor,Myocardial infarction(MI)is the irreversible cardiomyocyte death resulting from prolonged oxygen deprivation due to obstructed blood supply(ischemia),leading to contractile dysfunction and cardiac remodeling.In recent decades,stem cell transplantation has been extensively investigated for the repair of injured heart in animal studies and clinical trials(Kanelidis et al.,2017;Gyongyosi et al.,2018).

Epitranscriptomic 5-Methylcytosine Profile in PM_(2.5)-induced Mouse Pulmonary Fibrosis

Exposure of airborne particulate matter(PM)with an aerodynamic diameter less than 2.5μm(PM2.5)is epidemiologically associated with lung dysfunction and respiratory symptoms,including pulmonary fibrosis.However,whether epigenetic mechanisms are involved in PM2.5-induced pulmonary fibrosis is currently poorly understood.Herein,using a PM2.5-induced pulmonary fibrosis mouse model,we found that PM2.5 exposure leads to aberrant mRNA5-methylcytosine(m5C)gain and loss in fibrotic lung tissues.Moreover,we showed the m5C-mediated regulatory map of gene functions in pulmonary fibrosis after PM2.5 exposure.Several genes act as m5C gain-upregulated factors,probably critical for the development of PM2.5-induced fibrosis in mouse lungs.These genes,including Lcn2,Mmp9,Chi3l1,Adipoq,Atp5j2,Atp5l,Atpif1,Ndufb6,Fgr,Slc11 a1,and Tyrobp,are highly related to oxidative stress response,inflammatory responses,and immune system processes.Our study illustrates the first epitranscriptomic RNA m5C profile in PM2.5-induced pulmonary fibrosis and will be valuable in identifying biomarkers for PM2.5 exposure-related lung pathogenesis with translational potential.

Global Profiling of the Lysine Crotonylome in Different Pluripotent States

Pluripotent stem cells(PSCs)can be expanded in vitro in different culture conditions,resulting in a spectrum of cell states with distinct properties.Understanding how PSCs transition from one state to another,ultimately leading to lineage-specific differentiation,is important for developmental biology and regenerative medicine.Although there is significant information regarding gene expression changes controlling these transitions,less is known about post-translational modifications of proteins.Protein crotonylation is a newly discovered post-translational modification where lysine residues are modified with a crotonyl group.Here,we employed affinity purification of crotonylated(LC–MS/MS)to systematically profile protein crotonylation in mouse PSCs in different states including ground,metastable,and primed states,as well as metastable PSCs undergoing early pluripotency exit.We successfully identified 3628 high-confidence crotonylated sites in 1426 proteins.These crotonylated proteins are enriched for factors involved in functions/processes related to pluripotency such as RNA biogenesis,central carbon metabolism,and proteasome function.Moreover,we found that increasing the cellular levels of crotonyl-coenzyme A(crotonyl-CoA)through crotonic acid treatment promotes proteasome activity in metastable PSCs and delays their differentiation,consistent with previous observations showing that enhanced proteasome activity helps to sustain pluripotency.Our atlas of protein crotonylation will be valuable for further studies of pluripotency regulation and may also provide insights into the role of metabolism in other cell fate transitions.

5-Hydroxymethylome in Circulating Cell-free DNA as A Potential Biomarker for Non-small-cell Lung Cancer

Non-small-cell lung cancer （NSCLC）, the most common type of lung cancer accounting for 85% of the cases, is often diagnosed at advanced stages owing to the lack of efficient early diagnostic tools. 5-Hydroxymetbylcytosine （ShmC） signatures in circulating cell-free DNA （cfDNA） that carries the cancer-specific epigenetic patterns may represent the valuable biomarkers for discriminat- ing tumor and healthy individuals, and thus could be potentially useful for NSCLC diagnosis. Here, we employed a sensitive and reliable method to map genome-wide 5hmC in the cfDNA of Chinese NSCLC patients and detected a significant 5hmC gain in both the gene bodies and promoter regions in the blood samples from tumor patients compared with healthy controls. Specifically, we identi- fied six potential biomarkers from 66 patients and 67 healthy controls （mean decrease accuracy 〉 3.2, P 〈 3.68E-19） using machine-learning-based tumor classifiers with high accuracy. Thus, the unique signature of 5hmC in tumor patient＇s cfDNA identified in our study may provide valuable information in facilitating the development of new diagnostic and therapeutic modalities for NSCLC.

Adenine base editing disease mutations in to mimic or correcl rodents

Cytidine base editors （CBEs, rAPOBECI-nCas9-UGI） and adenine base editors （ABEs, TadA-TadA＊-nCas9） are newly developed genome-editing tools that have enabled highly efficient base conversions （CoG to T·A or A·T to G·C） at designated target sites （Komor et al., 2016; Gaudelli et al., 2017）. Both types of base editors enzymatically catalyze deamination of target bases and respectively replace C with U or A with I.