Single-cell EpiChem jointly measures drug-chromatin binding and multimodal epigenome.

Studies of molecular and cellular functions of small-molecule inhibitors in cancer treatment, eliciting effects by targeting genome and epigenome associated proteins, requires measurement of drug-target engagement in single-cell resolution. Here we present EpiChem for in situ single-cell joint mapping of small molecules and multimodal epigenomic landscape. We demonstrate single-cell co-assays of three small molecules together with histone modifications, chromatin accessibility or target proteins in human colorectal cancer (CRC) organoids.

Single-cell joint profiling of multiple epigenetic proteins and gene transcription.

Sculpting the epigenome with a combination of histone modifications and transcription factor occupancy determines gene transcription and cell fate specification. Here, we first develop uCoTarget, utilizing a split-pool barcoding strategy for realizing ultrahigh-throughput single-cell joint profiling of multiple epigenetic proteins. Through extensive optimization for sensitivity and multimodality resolution, we demonstrate that uCoTarget enables simultaneous detection of five histone modifications (H3K27ac, H3K4me3, H3K4me1, H3K36me3, and H3K27me3) in 19,860 single cells.

Novel enhancers conferring compensatory transcriptional regulation of Nkx2-5 in heart development.

Cell type-specific expression of the developmental gene is conferred by distinct enhancer elements. Current knowledge about mechanisms in transcriptional regulation and its specific roles in multistage heart morphogenesis is limited. We comprehensively interrogate enhancers U1 and U2 in controlling transcription during heart development.

Chlorination-induced p decrease: an improved strategy to design ratiometric hypochlorite fluorescent probes with ideally high selectivity.

Based on the selective ClO-triggered chlorination reaction and the subsequent p decrease of phenols, a new strategy was developed for rationally designing ratiometric ClO fluorescent probes with high selectivity. By investigating the fluorescence responses of 6-cyano-2-naphthol toward ClO and the p-dependent response mechanism, we developed a rapid, sensitive and selective two-photon ratiometric fluorescent probe, Naph-DFOB, to detect ClO. This probe displayed a ratiometric fluorescence change (from 509 nm to 628 nm) toward ClO and was successfully applied to image intracellular ClO in living cells with two-photon excitation.

Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions.

Deciphering mechanisms in cell-fate decisions requires single-cell holistic reconstructions of multidimensional epigenomic states in transcriptional regulation. Here we develop CoTECH, a combinatorial barcoding method allowing high-throughput single-cell joint detection of chromatin occupancy and transcriptome. We used CoTECH to examine bivalent histone marks (H3K4me3 and H3K27me3) with transcription from naive to primed mouse embryonic stem cells.

Generation and validation of versatile inducible CRISPRi embryonic stem cell and mouse model.

Clustered regularly interspaced short palindromic repeat (CRISPR)-CRISPR-associated (Cas) 9 has been widely used far beyond genome editing. Fusions of deactivated Cas9 (dCas9) to transcription effectors enable interrogation of the epigenome and controlling of gene expression. However, the large transgene size of dCas9-fusion hinders its applications especially in somatic tissues.

Rational Design of a Two-Photon Ratiometric Fluorescent Probe for Hypochlorous Acid with a Large Stokes Shift.

By introducing the tetrahydroquinoxaline group as the electron donor to enhance intramolecular charge transfer effect, we deliberately designed a coumarin derivative, TQC-HClO, to serve as a two-photon ratiometric fluorescent probe for imaging hypochlorous acid in cells and zebra fish with good sensitivity and high selectivity. TQC-HClO displayed an obvious two-photon absorbance cross-section (over 100 GM in 800-840 nm), large Stokes shifts (159 to 173 nm), and a ratiometric fluorescence change from orange (λ = 583 nm) to red (λ = 650 nm) in response to hypochlorous acid.

Single-Cell Transcriptomic Analysis of Cardiac Progenitor Differentiation.

Purpose Of Review: Emerging single-cell RNA sequencing technologies hold great promises to boost our understanding of the heterogeneity and molecular regulation of diverse cell phenotypes during organ development. In this review, we aimed at summarizing recent advances in employing single-cell transcriptomic analysis to depict the landscape of embryonic heart development, in particular, focusing on cardiac progenitor (CP) differentiation.

Recent Findings: Recent studies unbiasedly cataloged and characterized cardiac cell types in the spatial and temporal resolution during early heart development.

Investigation of the Relationship Between HO and HClO in Living Cells by a Bifunctional, Dual-ratiometric Responsive Fluorescent Probe.

As two important reactive oxygen species, hydrogen peroxide (HO) and hypochlorous acid (HClO) play vital roles in many physiological and pathological processes. However, the relationship between these two species is seldom investigated, in part, because of the lack of robust molecular tools that can simultaneously visualize HClO and HO in biosystems. In this work, we present a design strategy to construct a single fluorescent probe that can detect HO and HClO by simultaneously monitoring two distinct detection channels.

Profiling chromatin states using single-cell itChIP-seq.

Single-cell measurement of chromatin states, including histone modifications and non-histone protein binding, remains challenging. Here, we present a low-cost, efficient, simultaneous indexing and tagmentation-based ChIP-seq (itChIP-seq) method, compatible with both low cellular input and single cells for profiling chromatin states. itChIP combines chromatin opening, simultaneous cellular indexing and chromatin tagmentation within a single tube, enabling the processing of samples from tens of single cells to, more commonly, thousands of single cells per assay.

CoBATCH for High-Throughput Single-Cell Epigenomic Profiling.

An efficient, generalizable method for genome-wide mapping of single-cell histone modifications or chromatin-binding proteins is lacking. Here, we develop CoBATCH, combinatorial barcoding and targeted chromatin release, for single-cell profiling of genomic distribution of chromatin-binding proteins in cell culture and tissue. Protein A in fusion to Tn5 transposase is enriched through specific antibodies to genomic regions, and Tn5 generates indexed chromatin fragments ready for library preparation and sequencing.

Single-Cell Transcriptomics Reveals Chemotaxis-Mediated Intraorgan Crosstalk During Cardiogenesis.

Rationale: We hypothesized that the differentiation processes of cardiac progenitor cell (CP) from first and second heart fields (FHF and SHF) may undergo the unique instructive gene regulatory networks or signaling pathways, and the precise SHF progression is contingent on the FHF signaling developmental cues.

Objective: We investigated how the intraorgan communications control sequential building of discrete anatomic regions of the heart at single-cell resolution.

Methods And Results: By single-cell transcriptomic analysis of Nkx2-5 (NK2 homeobox 5) and Isl1 (ISL LIM homeobox 1) lineages at embryonic day 7.

An endoplasmic reticulum-targetable fluorescent probe for highly selective detection of hydrogen sulfide.

Hydrogen sulfide (H2S), a critical endogenous signaling molecule, is widely involved in many physiological processes. Endoplasmic reticulum, an important organelle with a sac-like structure, plays crucial roles in maintaining the normal function of cells. Accordingly, monitoring the H2S levels in endoplasmic reticulum is of great importance.

A red-emitting fluorescent probe for the detection of Hg in aqueous medium, living cells and organisms with a large Stokes shift.

A red-emitting fluorescent probe has been developed for the selective and sensitive detection of Hg2+. With the addition of Hg2+, the solution of probe 1 displayed a remarkable fluorescence enhancement (102 fold) with λemmax = 625 nm and a large Stokes shift (150 nm). The detection limit of this probe was as low as 7.