Mono-allelic epigenetic regulation of bi-directional polycistronic transcription initiation by RNA Polymerase II in Trypanosoma brucei.

Unique for a eukaryote, protein-coding genes in trypanosomes are arranged in polycistronic units (PTUs). This genome arrangement has led to a model where Pol II transcription of PTUs is unregulated. The initial step in trypanosome lytic factor (TLF) mediated lysis of Trypanosoma brucei requires high affinity haptoglobin/hemoglobin receptor (HpHbR) binding. Here we demonstrate that by in vitro selection with TLF, resistance is obtained in a stepwise process correlating with loss of HpHbR expression at an allelic level. RNA-seq, Pol II ChIP and run-on analysis indicate HpHbR silencing is at the transcriptional level, where loss of Pol II binding at the promoter region specifically shuts down transcription of the HpHbR containing gene cluster and the adjacent opposing gene cluster. Reversible transcriptional silencing of the divergent PTUs correlates with DNA base J modification of the shared promoter region. Therefore, epigenetic mechanisms exist to regulate gene expression via Pol II transcription initiation of gene clusters in a mono-allelic fashion. These findings suggest epigenetic chromatin-based regulation of gene expression is deeply conserved among eukaryotes, including primitive eukaryotes that rely on polycistronic transcription.

Combined Genome-Wide Association Study and Transcriptome Analysis Reveal Candidate Genes for Resistance to Rust (Puccinia graminis) in Dactylis glomerata.

Rust disease is a common plant disease that can cause wilting, slow growth of plant leaves, and even affect the growth and development of plants. Orchardgrass (Dactylis glomerata L.) is native to temperate regions of Europe, which has been introduced as a superior forage grass in temperate regions worldwide. Orchardgrass has rich genetic diversity and is widely distributed in the world, which may contain rust resistance genes not found in other crops. Therefore, we collected a total of 333 orchardgrass accessions from different regions around the world. Through a genome-wide association study (GWAS) analysis conducted in four different environments, 91 genes that overlap or are adjacent to significant single nucleotide polymorphisms (SNPs) were identified as potential rust disease resistance genes. Combining transcriptome data from susceptible (PI292589) and resistant (PI251814) accessions, the GWAS candidate gene DG5C04160.1 encoding glutathione S-transferase (GST) was found to be important for orchardgrass rust (Puccinia graminis) resistance. Interestingly, by comparing the number of GST gene family members in seven species, it was found that orchardgrass has the most GST gene family members, containing 119 GST genes. Among them, 23 GST genes showed significant differential expression after inoculation with the rust pathogen in resistant and susceptible accessions; 82% of the genes still showed significantly increased expression 14 days after inoculation in resistant accessions, while the expression level significantly decreased in susceptible accessions. These results indicate that GST genes play an important role in orchardgrass resistance to rust (P. graminis) stress by encoding GST to reduce its oxidative stress response.

A spatially resolved multiomic single-cell atlas of soybean development.

Cis-regulatory elements (CREs) precisely control spatiotemporal gene expression in cells. Using a spatially resolved single-cell atlas of gene expression with chromatin accessibility across ten soybean tissues, we identified 103 distinct cell types and 303,199 accessible chromatin regions (ACRs). Nearly 40% of the ACRs showed cell-type-specific patterns and were enriched for transcription factor (TF) motifs defining diverse cell identities. We identified de novo enriched TF motifs and explored conservation of gene regulatory networks underpinning legume symbiotic nitrogen fixation. With comprehensive developmental trajectories for endosperm and embryo, we uncovered the functional transition of the three sub-cell types of endosperm, identified 13 sucrose transporters sharing the DOF11 motif that were co-up-regulated in late peripheral endosperm and identified key embryo cell-type specification regulators during embryogenesis, including a homeobox TF that promotes cotyledon parenchyma identity. This resource provides a valuable foundation for analyzing gene regulatory programs in soybean cell types across tissues and life stages.

Diagnosis and cardiac transplantation of a Carney syndrome-induced cardiac myxoma combined with dilated cardiomyopathy: a case report.

BACKGROUND: Carney syndrome is an uncommon autosomal disorder closely linked to mutations in the PRKAR1A gene. Skin lesions are the most pronounced feature of Carney syndrome, affecting over 80% of individuals with this condition. This syndrome is characterized by a triad of myxomas, skin pigmentation, and endocrine hyperfunction, featuring multiple endocrine neoplasms with skin and cardiac involvement. Dilated cardiomyopathy, a primary cardiomyopathy, is defined as the dilation and impaired systolic function of the left or both ventricles. Its clinical presentation varies from being asymptomatic to heart failure or sudden cardiac death, making it a leading global cause of heart failure. Currently, Dilated cardiomyopathy has an estimated prevalence of 1/2500-1/250 individuals, predominantly affecting those aged 30-40 years, with a male-to-female ratio of 3:1. This case report describes a heart failure patient with cardiac myxoma caused by Carney syndrome combined with dilated cardiomyopathy. The patient was successfully treated for heart failure by heart transplantation. CASE PRESENTATION: Herein, we report a case of heart failure due to Carney syndrome that resulted in cardiac myxoma combined with dilated cardiomyopathy. A 35-year-old male was admitted to the hospital three years ago because of sudden chest tightness and shortness of breath. Echocardiography indicated myxoma, and a combination of genetic screening and physical examination confirmed Carney syndrome with cardiac myxoma. Following symptomatic management, he was discharged. Surgical interventions were not considered at the time. However, the patient's chest tightness and shortness of breath symptoms worsened, and he returned to the hospital. A New York Heart Association grade IV heart function was confirmed, and echocardiography indicated the presence of dilated cardiomyopathy accompanied by cardiac myxoma. Ultimately, the patient's heart failure was successfully treated with heart transplantation. CONCLUSIONS: Cardiac myxoma caused by Carney syndrome combined with heart failure caused by dilated cardiomyopathy can be resolved by heart transplantation.

DNAJ protein gene expansion mechanism in Panicoideae and PgDNAJ functional identification in pearl millet.

KEY MESSAGE: Analyze the evolutionary pattern of DNAJ protein genes in the Panicoideae, including pearl millet, to identify and characterize the biological function of PgDNAJ genes in pearl millet. Global warming has become a major factor threatening food security and human development. It is urgent to analyze the heat-tolerant mechanism of plants and cultivate crops that are adapted to high temperature conditions. The Panicoideae are the second largest subfamily of the Poaceae, widely distributed in warm temperate and tropical regions. Many of these species have been reported to have strong adaptability to high temperature stress, such as pearl millet, foxtail millet and sorghum. The evolutionary differences in DNAJ protein genes among 12 Panicoideae species and 10 other species were identified and analyzed. Among them, 79% of Panicoideae DNAJ protein genes were associated with retrotransposon insertion. Analysis of the DNAJ protein pan-gene family in six pearl millet accessions revealed that the non-core genes contained significantly more TEs than the core genes. By identifying and analyzing the distribution and types of TEs near the DNAJ protein genes, it was found that the insertion of Copia and Gypsy retrotransposons provided the source of expansion for the DNAJ protein genes in the Panicoideae. Based on the analysis of the evolutionary pattern of DNAJ protein genes in Panicoideae, the PgDNAJ was obtained from pearl millet through identification. PgDNAJ reduces the accumulation of reactive oxygen species caused by high temperature by activating ascorbate peroxidase (APX), thereby improving the heat resistance of plants. In summary, these data provide new ideas for mining potential heat-tolerant genes in Panicoideae, and help to improve the heat tolerance of other crops.

Protein phosphatase PP1 regulation of RNA polymerase II transcription termination and allelic exclusion of VSG genes in trypanosomes.

The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.

scifi-ATAC-seq: massive-scale single-cell chromatin accessibility sequencing using combinatorial fluidic indexing.

Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called scifi-ATAC-seq, single-cell combinatorial fluidic indexing ATAC-sequencing, which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using the 10X Genomics platform. With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples can be indexed in a single emulsion reaction, representing an approximately 20-fold increase in throughput compared to the standard 10X Genomics workflow.

Investigating the cis-Regulatory Basis of C3 and C4 Photosynthesis in Grasses at Single-Cell Resolution.

While considerable knowledge exists about the enzymes pivotal for C4 photosynthesis, much less is known about the cis-regulation important for specifying their expression in distinct cell types. Here, we use single-cell-indexed ATAC-seq to identify cell-type-specific accessible chromatin regions (ACRs) associated with C4 enzymes for five different grass species. This study spans four C4 species, covering three distinct photosynthetic subtypes: Zea mays and Sorghum bicolor (NADP-ME), Panicum miliaceum (NAD-ME), Urochloa fusca (PEPCK), along with the C3 outgroup Oryza sativa. We studied the cis-regulatory landscape of enzymes essential across all C4 species and those unique to C4 subtypes, measuring cell-type-specific biases for C4 enzymes using chromatin accessibility data. Integrating these data with phylogenetics revealed diverse co-option of gene family members between species, showcasing the various paths of C4 evolution. Besides promoter proximal ACRs, we found that, on average, C4 genes have two to three distal cell-type-specific ACRs, highlighting the complexity and divergent nature of C4 evolution. Examining the evolutionary history of these cell-type-specific ACRs revealed a spectrum of conserved and novel ACRs, even among closely related species, indicating ongoing evolution of cis-regulation at these C4 loci. This study illuminates the dynamic and complex nature of CRE evolution in C4 photosynthesis, particularly highlighting the intricate cis-regulatory evolution of key loci. Our findings offer a valuable resource for future investigations, potentially aiding in the optimization of C3 crop performance under changing climatic conditions.

Evolution of plant cell-type-specific cis -regulatory elements.

Cis -regulatory elements (CREs) are critical in regulating gene expression, and yet our understanding of CRE evolution remains a challenge. Here, we constructed a comprehensive single-cell atlas of chromatin accessibility in Oryza sativa , integrating data from 104,029 nuclei representing 128 discrete cell states across nine distinct organs. We used comparative genomics to compare cell-type resolved chromatin accessibility between O. sativa and 57,552 nuclei from four additional grass species ( Zea mays, Sorghum bicolor, Panicum miliaceum , and Urochloa fusca ). Accessible chromatin regions (ACRs) had different levels of conservation depending on the degree of cell-type specificity. We found a complex relationship between ACRs with conserved noncoding sequences, cell-type specificity, conservation, and tissue-specific switching. Additionally, we found that epidermal ACRs were less conserved compared to other cell types, potentially indicating that more rapid regulatory evolution has occurred in the L1 epidermal layer of these species. Finally, we identified and characterized a conserved subset of ACRs that overlapped the repressive histone modification H3K27me3, implicating them as potentially critical silencer CREs maintained by evolution. Collectively, this comparative genomics approach highlights the dynamics of cell-type-specific CRE evolution in plants.

Massive-scale single-cell chromatin accessibility sequencing using combinatorial fluidic indexing.

Single-cell ATAC-seq has emerged as a powerful approach for revealing candidate cis-regulatory elements genome-wide at cell-type resolution. However, current single-cell methods suffer from limited throughput and high costs. Here, we present a novel technique called single-cell combinatorial fluidic indexing ATAC-sequencing ("scifi-ATAC-seq"), which combines a barcoded Tn5 pre-indexing step with droplet-based single-cell ATAC-seq using a widely commercialized microfluidics platform (10X Genomics). With scifi-ATAC-seq, up to 200,000 nuclei across multiple samples in a single emulsion reaction can be indexed, representing a ~20-fold increase in throughput compared to the standard 10X Genomics workflow.

Protein Phosphatase PP1 Regulation of Pol II Phosphorylation is Linked to Transcription Termination and Allelic Exclusion of VSG Genes and TERRA in Trypanosomes.

The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, suggesting a unique PP1-independent mechanism of termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei involving dephosphorylation of the C-terminal domain of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated large subunit of Pol II accompanied by readthrough transcription and pervasive transcription of the entire genome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation and production of TERRA RNA. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.

Novel discovery in roles of structural variations and RWP-RK transcription factors in heat tolerance for pearl millet.

Global warming adversely affects crop production worldwide. Massive efforts have been undertaken to study mechanisms regulating heat tolerance in plants. However, the roles of structural variations (SVs) in heat stress tolerance remain unclear. In a recent article, Yan et al. (Nat Genet 1-12, 2023) constructed the first pan-genome of pearl millet (Pennisetum glaucum) and identified key SVs linked to genes involved in regulating plant tolerance to heat stress for an important crop with a superior ability to thrive in extremely hot and arid climates. Through multi-omics analyses integrating by pan-genomics, comparative genomics, transcriptomics, population genetics and and molecular biological technologies, they found RWP-RK transcription factors cooperating with endoplasmic reticulum-related genes play key roles in heat tolerance in pearl millet. The results in this paper provided novel insights to advance the understanding of the genetic and genomic basis of heat tolerance and an exceptional resource for molecular breeding to improve heat tolerance in pearl millet and other crops.

Milletdb: a multi-omics database to accelerate the research of functional genomics and molecular breeding of millets.

Millets are a class of nutrient-rich coarse cereals with high resistance to abiotic stress; thus, they guarantee food security for people living in areas with extreme climatic conditions and provide stress-related genetic resources for other crops. However, no platform is available to provide a comprehensive and systematic multi-omics analysis for millets, which seriously hinders the mining of stress-related genes and the molecular breeding of millets. Here, a free, web-accessible, user-friendly millets multi-omics database platform (Milletdb, http://milletdb.novogene.com) has been developed. The Milletdb contains six millets and their one related species genomes, graph-based pan-genomics of pearl millet, and stress-related multi-omics data, which enable Milletdb to be the most complete millets multi-omics database available. We stored GWAS (genome-wide association study) results of 20 yield-related trait data obtained under three environmental conditions [field (no stress), early drought and late drought] for 2 years in the database, allowing users to identify stress-related genes that support yield improvement. Milletdb can simplify the functional genomics analysis of millets by providing users with 20 different tools (e.g., 'Gene mapping', 'Co-expression', 'KEGG/GO Enrichment' analysis, etc.). On the Milletdb platform, a gene PMA1G03779.1 was identified through 'GWAS', which has the potential to modulate yield and respond to different environmental stresses. Using the tools provided by Milletdb, we found that the stress-related PLATZs TFs (transcription factors) family expands in 87.5% of millet accessions and contributes to vegetative growth and abiotic stress responses. Milletdb can effectively serve researchers in the mining of key genes, genome editing and molecular breeding of millets.

Exploring plant cis-regulatory elements at single-cell resolution: overcoming biological and computational challenges to advance plant research.

Cis-regulatory elements (CREs) are important sequences for gene expression and for plant biological processes such as development, evolution, domestication, and stress response. However, studying CREs in plant genomes has been challenging. The totipotent nature of plant cells, coupled with the inability to maintain plant cell types in culture and the inherent technical challenges posed by the cell wall has limited our understanding of how plant cell types acquire and maintain their identities and respond to the environment via CRE usage. Advances in single-cell epigenomics have revolutionized the field of identifying cell-type-specific CREs. These new technologies have the potential to significantly advance our understanding of plant CRE biology, and shed light on how the regulatory genome gives rise to diverse plant phenomena. However, there are significant biological and computational challenges associated with analyzing single-cell epigenomic datasets. In this review, we discuss the historical and foundational underpinnings of plant single-cell research, challenges, and common pitfalls in the analysis of plant single-cell epigenomic data, and highlight biological challenges unique to plants. Additionally, we discuss how the application of single-cell epigenomic data in various contexts stands to transform our understanding of the importance of CREs in plant genomes.

Knockout of protein phosphatase 1 in Leishmania major reveals its role during RNA polymerase II transcription termination.

The genomes of kinetoplastids are organized into polycistronic transcription units that are flanked by a modified DNA base (base J, beta-D-glucosyl-hydroxymethyluracil). Previous work established a role of base J in promoting RNA polymerase II (Pol II) termination in Leishmania major and Trypanosoma brucei. We recently identified a PJW/PP1 complex in Leishmania containing a J-binding protein (JBP3), PP1 phosphatase 1, PP1 interactive-regulatory protein (PNUTS) and Wdr82. Analyses suggested the complex regulates transcription termination by recruitment to termination sites via JBP3-base J interactions and dephosphorylation of proteins, including Pol II, by PP1. However, we never addressed the role of PP1, the sole catalytic component, in Pol II transcription termination. We now demonstrate that deletion of the PP1 component of the PJW/PP1 complex in L. major, PP1-8e, leads to readthrough transcription at the 3'-end of polycistronic gene arrays. We show PP1-8e has in vitro phosphatase activity that is lost upon mutation of a key catalytic residue and associates with PNUTS via the conserved RVxF motif. Additionally, purified PJW complex with associated PP1-8e, but not complex lacking PP1-8e, led to dephosphorylation of Pol II, suggesting a direct role of PNUTS/PP1 holoenzymes in regulating transcription termination via dephosphorylating Pol II in the nucleus.

Carbon dots as specific fluorescent sensors for Hg2+ and glutathione imaging.

Nitrogen-doped carbon dots (NCDs) have been constructed in which coal washing wastewater is used as carbon precursor, tryptophan is added for nitrogen doping and surface functional together with polyethylene glycol. The nitrogen doping and surface functional with electron rich groups resulted in excellent fluorescent properties regarding stability, reversibility, printability with high quantum yield which not only enable the NCDs as fluorescent ink for advanced message encryption, but also realize specific on-off-on fluorescent sensing of Hg2+ and GSH as solution, hydrogel and filter paper sensors. The NCDs had a linear range of 0.01-100 µM and a detection limit of 6.27 nM (RSD 0.33%) for Hg2+ and the NCDs@Hg2+ had a linear range of 0.01-60 µM and a detection limit of 3.53 nM (RSD 1.53%) for GSH in sensing studies with aqueous solutions. In addition, with the low cytotoxicity and good biocompatibility NCDs have been successfully used for imaging Hg2+ and GSH in living MG-63 cells. The presented NCDs recycle waste coal washing water into worthwhile material which can be implemented as promising anti-counterfeiting and message encryption candidates as well as effective Hg2+ and GSH sensing, tracking and removing tools in complicated environmental and biological systems.

Pangenomic analysis identifies structural variation associated with heat tolerance in pearl millet.

Pearl millet is an important cereal crop worldwide and shows superior heat tolerance. Here, we developed a graph-based pan-genome by assembling ten chromosomal genomes with one existing assembly adapted to different climates worldwide and captured 424,085 genomic structural variations (SVs). Comparative genomics and transcriptomics analyses revealed the expansion of the RWP-RK transcription factor family and the involvement of endoplasmic reticulum (ER)-related genes in heat tolerance. The overexpression of one RWP-RK gene led to enhanced plant heat tolerance and transactivated ER-related genes quickly, supporting the important roles of RWP-RK transcription factors and ER system in heat tolerance. Furthermore, we found that some SVs affected the gene expression associated with heat tolerance and SVs surrounding ER-related genes shaped adaptation to heat tolerance during domestication in the population. Our study provides a comprehensive genomic resource revealing insights into heat tolerance and laying a foundation for generating more robust crops under the changing climate.

DNA hypermethylation promotes the flowering of orchardgrass during vernalization.

Vernalization, influenced by environmental factors, is an essential process associated with the productivity of temperate crops, during which epigenetic regulation of gene expression plays an important role. Although DNA methylation is one of the major epigenetic mechanisms associated with the control of gene expression, global changes in DNA methylation in the regulation of gene expression during vernalization-induced flowering of temperate plants remain largely undetermined. To characterize vernalization-associated DNA methylation dynamics, we performed whole-genome bisulfite-treated sequencing and transcriptome sequencing in orchardgrass (Dactylis glomerata) during vernalization. The results revealed that increased levels of genome DNA methylation during the early vernalization of orchardgrass were associated with transcriptional changes in DNA methyltransferase and demethylase genes. Upregulated expression of vernalization-related genes during early vernalization was attributable to an increase in mCHH in the promoter regions of these genes. Application of an exogenous DNA methylation accelerator or overexpression of orchardgrass NUCLEAR POLY(A) POLYMERASE (DgPAPS4) promoted earlier flowering, indicating that DNA hypermethylation plays an important role in vernalization-induced flowering. Collectively, our findings revealed that vernalization-induced hypermethylation is responsible for floral primordium initiation and development. These observations provide a theoretical foundation for further studies on the molecular mechanisms underlying the control of vernalization in temperate grasses.

Exploring transposable element-based markers to identify allelic variations underlying agronomic traits in rice.

Transposable elements (TEs) are a major force in the production of new alleles during domestication; nevertheless, their use in association studies has been limited because of their complexity. We have developed a TE genotyping pipeline (TEmarker) and applied it to whole-genome genome-wide association study (GWAS) data from 176 Oryza sativa subsp. japonica accessions to identify genetic elements associated with specific agronomic traits. TE markers recovered a large proportion (69%) of single-nucleotide polymorphism (SNP)-based GWAS peaks, and these TE peaks retained ca. 25% of the SNPs. The use of TEs in GWASs may reduce false positives associated with linkage disequilibrium (LD) among SNP markers. A genome scan revealed positive selection on TEs associated with agronomic traits. We found several cases of insertion and deletion variants that potentially resulted from the direct action of TEs, including an allele of LOC_Os11g08410 associated with plant height and panicle length traits. Together, these findings reveal the utility of TE markers for connecting genotype to phenotype and suggest a potential role for TEs in influencing phenotypic variations in rice that impact agronomic traits.

Molecular Dynamics Simulation of Sintering Densification of Multi-Scale Silver Layer.

Based on molecular dynamics (MD), in this study, a model was established to simulate the initial coating morphology of silver paste by using a random algorithm, and the effects of different sizes of particles on sintering porosity were also analyzed. The MD result reveals that compared with the sintering process using large-scale silver particles, the sintering process using multi-scale silver particles would enhance the densification under the same sintering conditions, which authenticates the feasibility of adding small silver particles to large-scale silver particles in theory. In addition, to further verify the feasibility of the multi-scale sintering, a semi in-situ observation was prepared for a sintering experiment using micro-nano multi-scale silver paste. The feasibility of multi-scale silver sintering is proved by theoretical and experimental means, which can provide a meaningful reference for optimizing the sintering process and the preparation of silver paste for die-attach in powering electronics industry. In addition, it is hoped that better progress can be made on this basis in the future.