Single-cell lineage tracing with endogenous markers.

Resolving lineage relationships between cells in an organism provides key insights into the fate of individual cells and drives a fundamental understanding of the process of development and disease. A recent rapid increase in experimental and computational advances for detecting naturally occurring somatic nuclear and mitochondrial mutation at single-cell resolution has expanded lineage tracing from model organisms to humans. This review discusses the advantages and challenges of experimental and computational techniques for cell lineage tracing using somatic mutation as endogenous DNA barcodes to decipher the relationships between cells during development and tumour evolution. We outlook the advantages of spatial clonal evolution analysis and single-cell lineage tracing using endogenous genetic markers.

A single-cell atlas of conventional central chondrosarcoma reveals the role of endoplasmic reticulum stress in malignant transformation.

The transformation of benign lesions to malignant tumours is a crucial aspect of understanding chondrosarcomas, which are malignant cartilage tumours that could develop from benign chondroid lesions. However, the process of malignant transformation for chondroid lesions remains poorly understood, and no reliable markers are available to aid clinical decision-making. To address this issue, we conducted a study analysing 11 primary cartilage tumours and controls using single-cell RNA sequencing. By creating a single-cell atlas, we were able to identify the role of endoplasmic reticulum (ER) stress in the malignant transformation of conventional central chondrosarcomas (CCCS). Our research revealed that lower levels of ER stress promote chondrosarcoma growth in a patient-derived xenograft mouse model, while intensive ER stress reduces primary chondrosarcoma cell viability. Furthermore, we discovered that the NF-κB pathway alleviates ER stress-induced apoptosis during chondrosarcoma progression. Our single-cell signatures and large public data support the use of key ER stress regulators, such as DNA Damage Inducible Transcript 3 (DDIT3; also known as CHOP), as malignant markers for overall patient survival. Ultimately, our study highlights the significant role that ER stress plays in the malignant transformation of cartilaginous tumours and provides a valuable resource for future diagnostic markers and therapeutic strategies.

MOF-mediated acetylation of SIRT6 disrupts SIRT6-FOXA2 interaction and represses SIRT6 tumor-suppressive function by upregulating ZEB2 in NSCLC.

Mammalian sirtuin 6 (SIRT6) regulates a spectrum of vital biological processes and has long been implicated in the progression of cancer. However, the mechanisms underlying the regulation of SIRT6 in tumorigenesis remain elusive. Here, we report that the tumor-suppressive function of SIRT6 in non-small cell lung cancer (NSCLC) is regulated by acetylation. Specifically, males absent on the first (MOF) acetylates SIRT6 at K128, K160, and K267, resulting in a decreased deacetylase activity of SIRT6 and attenuated SIRT6 tumor-suppressive function in NSCLC. Mechanistically, MOF-mediated SIRT6 acetylation hinders the interaction between SIRT6 and transcriptional factor FOXA2, which in turn leads to the transcriptional activation of ZEB2, thus promoting NSCLC progression. Collectively, these data indicate an acetylation-dependent mechanism that modulates SIRT6 tumor-suppressive function in NSCLC. Our findings suggest that the MOF-SIRT6-ZEB2 axis may represent a promising therapeutic target for the management of NSCLC.

Inferring cell diversity in single cell data using consortium-scale epigenetic data as a biological anchor for cell identity.

Methods for cell clustering and gene expression from single-cell RNA sequencing (scRNA-seq) data are essential for biological interpretation of cell processes. Here, we present TRIAGE-Cluster which uses genome-wide epigenetic data from diverse bio-samples to identify genes demarcating cell diversity in scRNA-seq data. By integrating patterns of repressive chromatin deposited across diverse cell types with weighted density estimation, TRIAGE-Cluster determines cell type clusters in a 2D UMAP space. We then present TRIAGE-ParseR, a machine learning method which evaluates gene expression rank lists to define gene groups governing the identity and function of cell types. We demonstrate the utility of this two-step approach using atlases of in vivo and in vitro cell diversification and organogenesis. We also provide a web accessible dashboard for analysis and download of data and software. Collectively, genome-wide epigenetic repression provides a versatile strategy to define cell diversity and study gene regulation of scRNA-seq data.

Molecular determinants of intrinsic cellular stiffness in health and disease.

In recent years, the role of intrinsic biophysical features, especially cellular stiffness, in diverse cellular and disease processes is being increasingly recognized. New high throughput techniques for the quantification of cellular stiffness facilitate the study of their roles in health and diseases. In this review, we summarized recent discovery about how cellular stiffness is involved in cell stemness, tumorigenesis, and blood diseases. In addition, we review the molecular mechanisms underlying the gene regulation of cellular stiffness in health and disease progression. Finally, we discussed the current understanding on how the cytoskeleton structure and the regulation of these genes contribute to cellular stiffness, highlighting where the field of cellular stiffness is headed.

Overexpression of the Arabidopsis MACPF Protein AtMACP2 Promotes Pathogen Resistance by Activating SA Signaling.

Immune response in plants is tightly regulated by the coordination of the cell surface and intracellular receptors. In animals, the membrane attack complex/perforin-like (MACPF) protein superfamily creates oligomeric pore structures on the cell surface during pathogen infection. However, the function and molecular mechanism of MACPF proteins in plant pathogen responses remain largely unclear. In this study, we identified an Arabidopsis MACP2 and investigated the responsiveness of this protein during both bacterial and fungal pathogens. We suggest that MACP2 induces programmed cell death, bacterial pathogen resistance, and necrotrophic fungal pathogen sensitivity by activating the biosynthesis of tryptophan-derived indole glucosinolates and the salicylic acid signaling pathway dependent on the activity of enhanced disease susceptibility 1 (EDS1). Moreover, the response of MACP2 mRNA isoforms upon pathogen attack is differentially regulated by a posttranscriptional mechanism: alternative splicing. In comparison to previously reported MACPFs in Arabidopsis, MACP2 shares a redundant but nonoverlapping role in plant immunity. Thus, our findings provide novel insights and genetic tools for the MACPF family in maintaining SA accumulation in response to pathogens in Arabidopsis.

Alternative splicing and translation play important roles in hypoxic germination in rice.

Post-transcriptional mechanisms (PTMs), including alternative splicing (AS) and alternative translation initiation (ATI), may explain the diversity of proteins involved in plant development and stress responses. Transcriptional regulation is important during the hypoxic germination of rice seeds, but the potential roles of PTMs in this process have not been characterized. We used a combination of proteomics and RNA sequencing to discover how AS and ATI contribute to plant responses to hypoxia. In total, 10 253 intron-containing genes were identified. Of these, ~1741 differentially expressed AS (DAS) events from 811 genes were identified in hypoxia-treated seeds compared with controls. Over 95% of these were not present in the list of differentially expressed genes. In particular, regulatory pathways such as the spliceosome, ribosome, endoplasmic reticulum protein processing and export, proteasome, phagosome, oxidative phosphorylation, and mRNA surveillance showed substantial AS changes under hypoxia, suggesting that AS responses are largely independent of transcriptional regulation. Considerable AS changes were identified, including the preferential usage of some non-conventional splice sites and enrichment of splicing factors in the DAS data sets. Taken together, these results not only demonstrate that AS and ATI function during hypoxic germination but they have also allowed the identification of numerous novel proteins/peptides produced via ATI.

Comparative performance of the BGISEQ-500 and Illumina HiSeq4000 sequencing platforms for transcriptome analysis in plants.

BACKGROUND: The next-generation sequencing (NGS) technology has greatly facilitated genomic and transcriptomic studies, contributing significantly in expanding the current knowledge on genome and transcriptome. However, the continually evolving variety of sequencing platforms, protocols and analytical pipelines has led the research community to focus on cross-platform evaluation and standardization. As a NGS pioneer in China, the Beijing Genomics Institute (BGI) has announced its own NGS platform designated as BGISEQ-500, since 2016. The capability of this platform in large-scale DNA sequencing and small RNA analysis has been already evaluated. However, the comparative performance of BGISEQ-500 platform in transcriptome analysis remains yet to be elucidated. The Illumina series, a leading sequencing platform in China's sequencing market, would be a preferable reference to evaluate new platforms. METHODS: To this end, we describe a cross-platform comparative study between BGISEQ-500 and Illumina HiSeq4000 for analysis of Arabidopsis thaliana WT (Col 0) transcriptome. The key parameters in RNA sequencing and transcriptomic data processing were assessed in biological replicate experiments, using aforesaid platforms. RESULTS: The results from the two platforms BGISEQ-500 and Illumina HiSeq4000 shared high concordance in both inter- (correlation, 0.88-0.93) and intra-platform (correlation, 0.95-0.98) comparison for gene quantification, identification of differentially expressed genes and alternative splicing events. However, the two platforms yielded highly variable interpretation results for single nucleotide polymorphism and insertion-deletion analysis. CONCLUSION: The present case study provides a comprehensive reference dataset to validate the capability of BGISEQ-500 enabling it to be established as a competitive and reliable platform in plant transcriptome analysis.

Natural variation in the promoter of rice calcineurin B-like protein10 (OsCBL10) affects flooding tolerance during seed germination among rice subspecies.

Rice (Oryza sativa L.) has two ecotypes, upland and lowland rice, that have been observed to show different tolerance levels under flooding stress. In this study, two rice cultivars, upland (Up221, flooding-intolerant) and lowland (Low88, flooding-tolerant), were initially used to study their molecular mechanisms in response to flooding germination. We observed that variations in the OsCBL10 promoter sequences in these two cultivars might contribute to this divergence in flooding tolerance. Further analysis using another eight rice cultivars revealed that the OsCBL10 promoter could be classified as either a flooding-tolerant type (T-type) or a flooding-intolerant type (I-type). The OsCBL10 T-type promoter only existed in japonica lowland cultivars, whereas the OsCBL10 I-type promoter existed in japonica upland, indica upland and indica lowland cultivars. Flooding-tolerant rice cultivars containing the OsCBL10 T-type promoter have shown lower Ca2+ flow and higher α-amylase activities in comparison to those in flooding-intolerant cultivars. Furthermore, the OsCBL10 overexpression lines were sensitive to both flooding and hypoxic treatments during rice germination with enhanced Ca2+ flow in comparison to wild-type. Subsequent findings also indicate that OsCBL10 may affect OsCIPK15 protein abundance and its downstream pathways. In summary, our results suggest that the adaptation to flooding stress during rice germination is associated with two different OsCBL10 promoters, which in turn affect OsCBL10 expression in different cultivars and negatively affect OsCIPK15 protein accumulation and its downstream cascade.

The AMP-Activated Protein Kinase KIN10 Is Involved in the Regulation of Autophagy in Arabidopsis.

Autophagy is a highly conserved system in eukaryotes for the bulk degradation and recycling of intracellular components. Autophagy is involved in many physiological processes including development, senescence, and responses to abiotic and biotic stress. The adenosine 5'-monophosphate (AMP)-activated protein kinase AMPK positively regulates autophagy in mammals; however, the potential function of AMPK in plant autophagy remains largely unknown. Here, we identified KIN10, a plant ortholog of the mammalian AMPK, as a positive regulator of plant autophagy and showed that it acts by affecting the phosphorylation of ATG1 (AUTOPHAGY-RELATED GENE 1) proteins in Arabidopsis. Transgenic Arabidopsis lines overexpressing KIN10 (KIN10-OE) showed delays in leaf senescence, and increased tolerance to nutrient starvation, these phenotypes required a functional autophagy pathway. Consistent with KIN10 having a potential role in autophagy, the nutrient starvation-induced formation of autophagosomes and cleavage of GFP-ATG8e were accelerated in the KIN10-OE lines compared to the wild type. Moreover, the KIN10-OE lines were less sensitive to drought and hypoxia treatments, compared with wild type. Carbon starvation enhanced the level of phosphorylated YFP-ATG1a in the KIN10-OE lines compared to that of wild type. Together, these findings suggest that KIN10 is involved in positive regulation of autophagy, possibly by affecting the phosphorylation of ATG1s in Arabidopsis.