Multiome in the Same Cell Reveals the Impact of Osmotic Stress on Arabidopsis Root Tip Development at Single-Cell Level.

Cell-specific transcriptional regulatory networks (TRNs) play vital roles in plant development and response to environmental stresses. However, traditional single-cell mono-omics techniques are unable to directly capture the relationships and dynamics between different layers of molecular information within the same cells. While advanced algorithm facilitates merging scRNA-seq and scATAC-seq datasets, accurate data integration remains a challenge, particularly when investigating cell-type-specific TRNs. By examining gene expression and chromatin accessibility simultaneously in 16,670 Arabidopsis root tip nuclei, the TRNs are reconstructed that govern root tip development under osmotic stress. In contrast to commonly used computational integration at cell-type level, 12,968 peak-to-gene linkage is captured at the bona fide single-cell level and construct TRNs at an unprecedented resolution. Furthermore, the unprecedented datasets allow to more accurately reconstruct the coordinated changes of gene expression and chromatin states during cellular state transition. During root tip development, chromatin accessibility of initial cells precedes gene expression, suggesting that changes in chromatin accessibility may prime cells for subsequent differentiation steps. Pseudo-time trajectory analysis reveal that osmotic stress can shift the functional differentiation of trichoblast. Candidate stress-related gene-linked cis-regulatory elements (gl-cCREs) as well as potential target genes are also identified, and uncovered large cellular heterogeneity under osmotic stress.

Single-worm quantitative proteomics reveals aging heterogeneity in isogenic Caenorhabditis elegans.

The heterogeneity of aging has been investigated at cellular and organic levels in the mouse model and human, but the exploration of aging heterogeneity at whole-organism level is lacking. C. elegans is an ideal model organism for studying this question as they are self-fertilized and cultured in the same chamber. Despite the tremendous progress made in single-cell proteomic analysis, there is few single-worm proteomics studies about aging. Here, we apply single-worm quantitative mass spectrometry to quantify the heterogenous proteomic changes during aging across individuals, a total of 3524 proteins from 157 C. eleagns individuals were quantified. A reconstructed C. elegans aging trajectory and proteomic landscape of fast-aging individuals were used to analyze the heterogeneity of C. elegans aging. We characterized inter-individual proteomic variation during aging and revealed contributing factors that distinguish fast-aging individuals from their siblings.

Mapping protein direct interactome of oxidoreductases with small molecular chemical cross-linkers in live cells.

Identifying direct substrates of enzymes has been a long-term challenge. Here, we present a strategy using live cell chemical cross-linking and mass spectrometry to identify the putative substrates of enzymes for further biochemical validation. Compared with other methods, our strategy is based on the identification of cross-linked peptides supported by high-quality MS/MS spectra, which eliminates false-positive discoveries of indirect binders. Additionally, cross-linking sites allow the analysis of interaction interfaces, providing further information for substrate validation. We demonstrated this strategy by identifying direct substrates of thioredoxin in both E. coli and HEK293T cells using two bis-vinyl sulfone chemical cross-linkers BVSB and PDES. We confirmed that BVSB and PDES have high specificity in cross-linking the active site of thioredoxin with its substrates both in vitro and in live cells. Applying live cell cross-linking, we identified 212 putative substrates of thioredoxin in E. coli and 299 putative S-nitrosylation (SNO) substrates of thioredoxin in HEK293T cells. In addition to thioredoxin, we have shown that this strategy can be applied to other proteins in the thioredoxin superfamily. Based on these results, we believe future development of cross-linking techniques will further advance cross-linking mass spectrometry in identifying substrates of other classes of enzymes.

Somatic nuclear blebbing in Caenorhabditis elegans is not a feature of organismal aging but a potential indicator of germline proliferation in early adulthood.

Abnormal nuclear morphology is suggested to be a hallmark of aging and one such abnormality is nuclear blebbing. However, little is known about whether and how nuclear blebbing participates in animal aging, and what regulates it. In this study, we show that the frequency of nuclear blebbing in the hypodermis increases during aging in wild-type C. elegans. These nuclear blebs are enveloped by the nuclear lamina, the inner and the outer nuclear membrane, and 42% of them contain chromatin. Although nuclear blebbing could lead to DNA loss if chromatin-containing blebs detach and fuse with lysosomes, we find by time-lapse imaging that nuclear blebs rarely detach, and the estimated lifetime of a nuclear bleb is 772 h or 32 days. The amount of DNA lost through nuclear blebbing is estimated to be about 0.1% of the total DNA loss by adult Day 11. Furthermore, the frequency of nuclear blebbing does not correlate with the rate of aging in C. elegans. Old age does not necessarily induce nuclear blebbing, neither does starvation, heat stress, or oxidative stress. Intriguingly, we find that proliferation of germ cells promotes nuclear blebbing.

Detecting Active Deconjugating Enzymes with Genetically Encoded Activity-Based Ubiquitin and Ubiquitin-like Protein Probes.

Post-translational modification of proteins by Ubiquitin (Ub) and Ubiquitin-like proteins (Ubls) can be reversed by deconjugating enzymes, which have been implicated in different pathways and associated with various human diseases. To understand the activity and dynamics of deconjugating enzymes, multiple synthetic and semi-synthetic Ub/Ubl probes have been developed, and some of them have been applied to screen inhibitors of deconjugating enzymes. Since these Ub/Ubl probes are generally not cell-permeable, different strategies have been developed to deliver Ub/Ubl probes to live cells. However, till now, no Ub/Ubl probes can be expressed in live cells to directly report on the activities of deconjugating enzymes in the most relevant cellular environment. Here, we genetically encoded cross-linkable Ub/Ubl probes in live E. coli and HEK293T cells. These probes can cross-link with deconjugating enzymes in vitro and in vivo. Using these Ub probes combined with mass spectrometry, we have successfully identified endogenous deconjugating enzymes in live cells. We believe that these genetically encoded Ub/Ubl probes are valuable for investigating biological functions of deconjugating enzymes in physiological environments.

Uncover New Reactivity of Genetically Encoded Alkyl Bromide Non-Canonical Amino Acids.

Genetically encoded non-canonical amino acids (ncAAs) with electrophilic moieties are excellent tools to investigate protein-protein interactions (PPIs) both in vitro and in vivo. These ncAAs, including a series of alkyl bromide-based ncAAs, mainly target cysteine residues to form protein-protein cross-links. Although some reactivities towards lysine and tyrosine residues have been reported, a comprehensive understanding of their reactivity towards a broad range of nucleophilic amino acids is lacking. Here we used a recently developed OpenUaa search engine to perform an in-depth analysis of mass spec data generated for Thioredoxin and its direct binding proteins cross-linked with an alkyl bromide-based ncAA, BprY. The analysis showed that, besides cysteine residues, BprY also targeted a broad range of nucleophilic amino acids. We validated this broad reactivity of BprY with Affibody/Z protein complex. We then successfully applied BprY to map a binding interface between SUMO2 and SUMO-interacting motifs (SIMs). BprY was further applied to probe SUMO2 interaction partners. We identified 264 SUMO2 binders, including several validated SUMO2 binders and many new binders. Our data demonstrated that BprY can be effectively used to probe protein-protein interaction interfaces even without cysteine residues, which will greatly expand the power of BprY in studying PPIs.

The Meishan pig genome reveals structural variation-mediated gene expression and phenotypic divergence underlying Asian pig domestication.

There are wide genomic and phenotypic differences between Asian and European pig breeds, yet the current reference genome is the European Duroc pig genome. A high-quality pig genome is lacking for genetic analysis of agricultural traits in Asian pigs. Here, using a hybrid approach, a high-quality reference genome (MSCAAS v1) for the Asian Meishan breed is assembled with a contig N50 size of 48.05 Mb. MSCAAS v1 outperforms the Duroc genome as a reference genome for Asian breeds. Genomic comparison reveals 49,103 structural variations (SVs) between Meishan and Duroc, 4.02% of which are Asian-specific SVs (AP-SVs). Notably, a 30-Mb hotspot for AP-SVs on chromosome X enriched for genes associated with Asian-pig-specific phenotypes is present in Asian domestic pig breeds, but absent in Asian wild boars, suggesting that Asian domestic breeds share a common ancestor. Interbreed transcriptomics reveals transcriptional suppression roles of AP-SVs in multiple tissues. Finally, transcriptional regulation in the intron of IGF2R is reported, as genomic SV (274-bp deletion) in Tibetan pig limits its growth compared to domestic pig breeds. In summary, this study provides insights regarding the genetic changes underlying pig domestication and presents a benchmark-setting resource for the utilization of agricultural valuable loci in Asian pigs.

Identification of Protein Direct Interactome with Genetic Code Expansion and Search Engine OpenUaa.

Protein crosslinks occur endogenously such as modifications by ubiquitin-like proteins for signaling, or exogenously through genetically encoded chemical crosslinkers (GECX) for studying elusive protein-protein interactions. However, it remains challenging to identify these protein crosslinks efficiently at the proteomic scale. Herein, software OpenUaa is developed for identifying protein crosslinks generated by genetically encoded unnatural amino acids and endogenous protein conjugation. OpenUaa features inclusive and open search capability, dramatically improving identification sensitivity and coverage. Integrating GECX with OpenUaa, the direct interactome of thioredoxin is identified in Escherichia coli cells, yielding 289 crosslinked peptides and corresponding to 205 direct binding protein of thioredoxin. These identified direct binders provide evidence for thioredoxin's regulation of redox state and mitochondria energy metabolism. When identifying endogenous conjugation of small ubiquitin-like modifier (SUMO), OpenUaa also markedly improves coverage of SUMOylated peptides by ≈92%, revealing new SUMO targets. GECX-OpenUaa will enable efficient identification of direct interactomes of various proteins in live cells.

DAF-16 stabilizes the aging transcriptome and is activated in mid-aged Caenorhabditis elegans to cope with internal stress.

The roles and regulatory mechanisms of transcriptome changes during aging are unclear. It has been proposed that the transcriptome suffers decay during aging owing to age-associated down-regulation of transcription factors. In this study, we characterized the role of a transcription factor DAF-16, which is a highly conserved lifespan regulator, in the normal aging process of Caenorhabditis elegans. We found that DAF-16 translocates into the nucleus in aged wild-type worms and activates the expression of hundreds of genes in response to age-associated cellular stress. Most of the age-dependent DAF-16 targets are different from the canonical DAF-16 targets downstream of insulin signaling. This and other evidence suggest that activation of DAF-16 during aging is distinct from activation of DAF-16 due to reduced signaling from DAF-2. Further analysis showed that it is due in part to a loss of proteostasis during aging. We also found that without daf-16, dramatic gene expression changes occur as early as on adult day 2, indicating that DAF-16 acts to stabilize the transcriptome during normal aging. Our results thus reveal that normal aging is not simply a process in which the gene expression program descends into chaos due to loss of regulatory activities; rather, there is active transcriptional regulation during aging.

Spontaneous and specific chemical cross-linking in live cells to capture and identify protein interactions.

Covalently locking interacting proteins in situ is an attractive strategy for addressing the challenge of identifying weak and transient protein interactions, yet it is demanding to execute chemical reactions in live systems in a biocompatible, specific, and autonomous manner. Harnessing proximity-enabled reactivity of an unnatural amino acid incorporated in the bait toward a target residue of unknown proteins, here we genetically encode chemical cross-linkers (GECX) to cross-link interacting proteins spontaneously and selectively in live cells. Obviating an external trigger for reactivity and affording residue specificity, GECX enables the capture of low-affinity protein binding (affibody with Z protein), elusive enzyme-substrate interaction (ubiquitin-conjugating enzyme UBE2D3 with substrate PCNA), and endogenous proteins interacting with thioredoxin in E. coli cells, allowing for mass spectrometric identification of interacting proteins and crosslinking sites. This live cell chemistry-based approach should be valuable for investigating currently intangible protein interactions in vivo for better understanding of biology in physiological settings.

Segmentation and classification of two-channel C. elegans nucleus-labeled fluorescence images.

BACKGROUND: Aging is characterized by a gradual breakdown of cellular structures. Nuclear abnormality is a hallmark of progeria in human. Analysis of age-dependent nuclear morphological changes in Caenorhabditis elegans is of great value to aging research, and this calls for an automatic image processing method that is suitable for both normal and abnormal structures. RESULTS: Our image processing method consists of nuclear segmentation, feature extraction and classification. First, taking up the challenges of defining individual nuclei with fuzzy boundaries or in a clump, we developed an accurate nuclear segmentation method using fused two-channel images with seed-based cluster splitting and k-means algorithm, and achieved a high precision against the manual segmentation results. Next, we extracted three groups of nuclear features, among which five features were selected by minimum Redundancy Maximum Relevance (mRMR) for classifiers. After comparing the classification performances of several popular techniques, we identified that Random Forest, which achieved a mean class accuracy (MCA) of 98.69%, was the best classifier for our data set. Lastly, we demonstrated the method with two quantitative analyses of C. elegans nuclei, which led to the discovery of two possible longevity indicators. CONCLUSIONS: We produced an automatic image processing method for two-channel C. elegans nucleus-labeled fluorescence images. It frees biologists from segmenting and classifying the nuclei manually.

Microsatellite instability in chicken lymphoma induced by gallid herpesvirus 2.

Microsatellite instability (MSI) has been found in a range of human tumors, and little is known of the links between MSI and herpesvirus. In order to investigate the relationship between MSI and Gallid herpesvirus 2 (GaHV-2)-induced lymphoma, fifteen Marek's disease (MD) lymphomas were analyzed through using 46 microsatellite markers, which were amplified by PCR from DNA specimens of lymphoma and normal muscular tissues from the same chicken. PCR products were evaluated by denaturing polyacrylamide gel electrophoresis for MSI analysis. MSI was proved in all lymphomas, at least in one locus. Thirty of the 46 microsatellite markers had microsatellite alterations. These results suggested that GaHV-2-induced lymphoma in chickens is related to MSI, and this is the first report to demonstrate that MSI is associated with the GaHV-2 induced lymphoma in chicken.

CAMKII and calcineurin regulate the lifespan of Caenorhabditis elegans through the FOXO transcription factor DAF-16.

The insulin-like signaling pathway maintains a relatively short wild-type lifespan in Caenorhabditis elegans by phosphorylating and inactivating DAF-16, the ortholog of the FOXO transcription factors of mammalian cells. DAF-16 is phosphorylated by the AKT kinases, preventing its nuclear translocation. Calcineurin (PP2B phosphatase) also limits the lifespan of C. elegans, but the mechanism through which it does so is unknown. Herein, we show that TAX-6•CNB-1 and UNC-43, the C. elegans Calcineurin and Ca(2+)/calmodulin-dependent kinase type II (CAMKII) orthologs, respectively, also regulate lifespan through DAF-16. Moreover, UNC-43 regulates DAF-16 in response to various stress conditions, including starvation, heat or oxidative stress, and cooperatively contributes to lifespan regulation by insulin signaling. However, unlike insulin signaling, UNC-43 phosphorylates and activates DAF-16, thus promoting its nuclear localization. The phosphorylation of DAF-16 at S286 by UNC-43 is removed by TAX-6•CNB-1, leading to DAF-16 inactivation. Mammalian FOXO3 is also regulated by CAMKIIA and Calcineurin. DOI:http://dx.doi.org/10.7554/eLife.00518.001.