Joint single-cell multiomic analysis in Wnt3a induced asymmetric stem cell division.

Wnt signaling usually functions through a spatial gradient. Localized Wnt3a signaling can induce the asymmetric division of mouse embryonic stem cells, where proximal daughter cells maintain self-renewal and distal daughter cells acquire hallmarks of differentiation. Here, we develop an approach, same cell epigenome and transcriptome sequencing, to jointly profile the epigenome and transcriptome in the same single cell. Utilizing this method, we profiled H3K27me3 and H3K4me3 levels along with gene expression in mouse embryonic stem cells with localized Wnt3a signaling, revealing the cell type-specific maps of the epigenome and transcriptome in divided daughter cells. H3K27me3, but not H3K4me3, is correlated with gene expression changes during asymmetric cell division. Furthermore, cell clusters identified by H3K27me3 recapitulate the corresponding clusters defined by gene expression. Our study provides a convenient method to jointly profile the epigenome and transcriptome in the same cell and reveals mechanistic insights into the gene regulatory programs that maintain and reset stem cell fate during differentiation.

The H3K36me2 methyltransferase NSD1 modulates H3K27ac at active enhancers to safeguard gene expression.

Epigenetics, especially histone marks, functions beyond the DNA sequences to regulate gene expression. Depletion of NSD1, which catalyzes H3K36me2, leads to both up- and down-regulation of gene expression, indicating NSD1 is associated with both active and repressed gene expression. It's known that NSD1 regulates the deposition and expansion of H3K27me3, a repressive mark for gene expression, to keep active gene transcription. However, how NSD1 functions to repress gene expression is largely unknown. Here, we find that, when NSD1 is knocked out in mouse embryonic stem cells (mESCs), H3K27ac increases correlatively with the decrease of H3K36me2 at active enhancers, which is associated with mesoderm differentiation genes, leading to elevated gene expression. Mechanistically, NSD1 recruits HDAC1, the deacetylase of H3K27ac, to chromatin. Moreover, HDAC1 knockout (KO) recapitulates the increase of H3K27ac at active enhancers as the NSD1 depletion. Together, we propose that NSD1 deposits H3K36me2 and recruits HDAC1 at active enhancers to serve as a 'safeguard', preventing further activation of active enhancer-associated genes.

Correction: Small-molecule-based human genome G4 profiling reveals potential gene regulation activity.

Correction for 'Small-molecule-based human genome G4 profiling reveals potential gene regulation activity' by Weiwu Zeng et al., Chem. Commun., 2019, 55, 2269-2272, DOI: 10.1039/C8CC10052G.

Facile cell patterning induced by combined surface topography and chemistry on polydopamine-defined nanosubstrates.

Cell patterning holds significant implications for cell-based analysis and high-throughput screening. The challenge and key factor for formation of cell patterns is to precisely modulate the interaction between cells and substrate surfaces. Many nanosubstrates have been developed to control cell adhesion and patterning, however, requirements of complicated fabrication procedures, harsh reaction conditions, and delicate manipulation are not routinely feasible. Here, we developed a hierarchical polydimethylsiloxane nanosubstrate (HPNS) coated with mussel-inspired polydopamine (PDA) micropatterns for effective cell patterning, depending on both surface topography and chemistry. HPNSs obtained by facile template-assisted replication brought enhanced topographic interaction between cells and substrates, but they were innately hydrophobic and cell-repellent. The hydrophobic nanosubstrates were converted to be hydrophilic after PDA coatings formed via spontaneous self-polymerization, which greatly facilitated cell adhesion. As such, without resorting to any external forces or physical constraints, cells selectively adhered and spread on spatially defined PDA regions with high efficiency, and well-defined cell microarrays could be formed within 20 min. Therefore, this easy-to-fabricate nanosubstrate with no complex chemical modification will afford a facile yet effective platform for rapid cell patterning.

Acrylonitrile-Mediated Nascent RNA Sequencing for Transcriptome-Wide Profiling of Cellular RNA Dynamics.

RNA sequencing has greatly facilitated gene expression studies but is weak in studying temporal RNA dynamics; this issue can be addressed by analyzing nascent RNAs. A famous method for nascent RNA analysis is metabolic labeling with noncanonic nucleoside followed by affinity purification, however, purification processes can always introduce biases into data analysis. Here, a chemical method for nascent RNA sequencing that avoids affinity purification based on acrylonitrile-mediated uridine-to-cytidine (U-to-C) conversion (AMUC-seq) via 4-thiouridine (s4U) cyanoethylation is presented. This method converts s4U base-pairing with guanine through the nucleophilic addition of s4U to acrylonitrile. The high reaction efficiency permits AMUC-seq directly and efficiently to recover nascent RNA information from total RNAs. AMUC-seq is validated by being used to detect mRNA half-lives and investigating the direct gene targets of a G-quadruplex stabilizer, which can be regarded as potential anticancer drug, in human cells. Thousands of direct gene targets of this drug are verified (these genes are significantly enriched in cancer such as SRC and HRAS). AMUC-seq also confirms G-quadruplex stabilization that impacts RNA polyadenylation. These results show AMUC-seq is qualified for the study of temporal RNA dynamics, and it can be a promising strategy to study the therapeutic mechanism of transcription-modulating drugs.

Biotinylation and isolation of an RNA G-quadruplex based on its peroxidase-mimicking activity.

RNA G-quadruplexes (rG4s) are important RNA secondary structures considering their significance in regulating numerous cellular processes. Described herein is an rG4 detecting and isolation method, which exploits the complex of rG4 and hemin to mimic peroxidase. In the presence of biotin tyramide and hydrogen peroxide, rG4s can be selectively self-biotinylated and easily isolated from a complex RNA mixture using streptavidin magnetic beads.

Small-molecule-based human genome G4 profiling reveals potential gene regulation activity.

Small-molecule-based G4 isolation from genomic DNA has enabled the identification of a total of 51 446 PQSs (potential G-quadruplex sites). Incubating cells with PDP for 3 h to 72 h resulted in the differential expression of a variety of genes, indicating a potential function of G4s in gene regulation.

Highly Selective 5-Formyluracil Labeling and Genome-wide Mapping Using (2-Benzimidazolyl)Acetonitrile Probe.

Chemical modifications to nucleobases have a great influence on various cellular processes, by making gene regulation more complex, thus indicating their profound impact on aspects of heredity, growth, and disease. Here, we provide the first genome-wide map of 5-formyluracil (5fU) in living tissues and evaluate the potential roles for 5fU in genomics. We show that an azido derivative of (2-benzimidazolyl)acetonitrile has high selectivity for enriching 5fU-containing genomic DNA. The results have demonstrated the feasibility of using this method to determine the genome-wide distribution of 5fU. Intriguingly, most 5fU sites were found in intergenic regions and introns. Also, distribution of 5fU in human thyroid carcinoma tissues is positively correlated with binding sites of POLR2A protein, which indicates that 5fU may distributed around POLR2A-binding sites.

Fluorogenic labeling and single-base resolution analysis of 5-formylcytosine in DNA.

5-Formylcytosine (5fC), which plays an important role in epigenetic functions, has received widespread attention in many related fields. Here, we demonstrate a new design for both the fluorogenic switch-on detection and single-base resolution analysis of 5fC through selectively reacting a reagent with 5fC to yield an intramolecular cyclization nucleobase. The generated product, bearing a similar benzothiazole-iminocoumarin scaffold, is highly fluorescent and enables us to qualitatively and quantitatively detect 5fC moieties in γ-irradiated calf thymus DNA. Additionally, losing the exocyclic 4-amino group in 5fC causes the incorporation of dATP through base pairing with the generated nucleobase during polymerase extension, which helped us to analyze the 5fC sites in both single- and double-stranded oligonucleotides. Our Sanger and Illumina sequencing results show great potential in single-base resolution analysis of 5fC. It is hopeful that a similar design may be used for more detection targets.

Obtaining More Accurate Signals: Spatiotemporal Imaging of Cancer Sites Enabled by a Photoactivatable Aptamer-Based Strategy.

Early cancer diagnosis is of great significance to relative cancer prevention and clinical therapy, and it is crucial to efficiently recognize cancerous tumor sites at the molecular level. Herein, we proposed a versatile and efficient strategy based on aptamer recognition and photoactivation imaging for cancer diagnosis. This is the first time that a visible light-controlled photoactivatable aptamer-based platform has been applied for cancer diagnosis. The photoactivatable aptamer-based strategy can accurately detect nucleolin-overexpressed tumor cells and can be used for highly selective cancer cell screening and tissue imaging. This strategy is available for both formalin-fixed paraffin-embedded tissue specimens and frozen sections. Moreover, the photoactivation techniques showed great progress in more accurate and persistent imaging to the use of traditional fluorophores. Significantly, the application of this strategy can produce the same accurate results in tissue specimen analysis as with classical hematoxylin-eosin staining and immunohistochemical technology.