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. Importantly, this strategy enables not only low-input epigenomic profiling in intact tissues but also measures scalable up to tens of thousands of single cells per experiment under both native and cross-linked conditions. CoBATCH produces ∼12,000 reads/cell with extremely low background. Mapping of endothelial cell lineages from ten embryonic mouse organs through CoBATCH allows for efficient deciphering of epigenetic heterogeneity of cell populations and cis-regulatory mechanisms. Thus, obviating specialized devices, CoBATCH is broadly applicable and easily deployable for single-cell profiling of protein-DNA interactions.

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. We also derived concurrent bivalent marks in pseudosingle cells using transcriptome as an anchor for resolving pseudotemporal bivalency trajectories and disentangling a context-specific interplay between H3K4me3/H3K27me3 and transcription level. Next, we revealed the regulatory basis of endothelial-to-hematopoietic transition in two waves of hematopoietic cells and distinctive enhancer-gene-linking schemes guiding hemogenic endothelial cell emergence, indicating a unique epigenetic control of transcriptional regulation for hematopoietic stem cell priming. CoTECH provides an efficient framework for single-cell coassay of chromatin occupancy and transcription, thus enabling higher-dimensional epigenomic reconstructions.

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. Here, we develop a robust CRISPR interference (CRISPRi) system by transgenic expression of doxycycline (Dox) inducible dCas9-KRAB in mouse embryonic stem cells (iKRAB ESC). After introduction of specific single-guide RNAs (sgRNAs), the induced dCas9-KRAB efficiently maintains gene inactivation, although it modestly down-regulates the expression of active genes. The proper timing of Dox addition during cell differentiation or reprogramming allows us to study or screen spatiotemporally activated promoters or enhancers and thereby the gene functions. Furthermore, taking the ESC for blastocyst injection, we generate an iKRAB knock-in (KI) mouse model that enables the shutdown of gene expression and loss-of-function (LOF) studies ex vivo and in vivo by a simple transduction of gRNAs. Thus, our inducible CRISPRi ESC line and KI mouse provide versatile and convenient platforms for functional interrogation and high-throughput screens of specific genes and potential regulatory elements in the setting of development or diseases.

Chlorination-induced pKa 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 pKa 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 pKa-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. Using Naph-DFOB as a useful tool, the investigation of lipopolysaccharide (LPS)-induced acute lung injury in a mouse model was effectively performed.

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 7.75, embryonic day 8.25, embryonic day 8.75, and embryonic day 9.25, we present a panoramic view of distinct CP differentiation hierarchies. Computational identifications of FHF- and SHF-CP descendants revealed that SHF differentiation toward cardiomyocytes underwent numerous step-like transitions, whereas earlier FHF progressed toward cardiomyocytes in a wave-like manner. Importantly, single-cell pairing analysis demonstrated that SHF-CPs were attracted to and expanded FHF-populated heart tube region through interlineage communications mediated by the chemotactic guidance (MIF [macrophage migration inhibitory factor]-CXCR2 [C-X-C motif chemokine receptor 2]). This finding was verified by pharmacological blockade of this chemotaxis in embryos manifesting limited SHF cell migration and contribution to the growth of the outflow tract and right ventricle but undetectable effects on the left ventricle or heart tube initiation. Genetic loss-of-function assay of Cxcr2 showed that the expression domain of CXCR4 was expanded predominantly at SHF. Furthermore, double knockout of Cxcr2/Cxcr4 exhibited defective SHF development, corroborating the redundant function. Mechanistically, NKX2-5 directly bound the Cxcr2 and Cxcr4 genomic loci and activated their transcription in SHF. CONCLUSIONS: Collectively, we propose a model in which the chemotaxis-mediated intraorgan crosstalk spatiotemporally guides the successive process of positioning SHF-CP and promoting primary heart expansion and patterning upon FHF-derived heart tube initiation.

Investigation of the Relationship Between H2O2 and HClO in Living Cells by a Bifunctional, Dual-ratiometric Responsive Fluorescent Probe.

As two important reactive oxygen species, hydrogen peroxide (H2O2) 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 H2O2 in biosystems. In this work, we present a design strategy to construct a single fluorescent probe that can detect H2O2 and HClO by simultaneously monitoring two distinct detection channels. In the design, one phenothiazine-based coumarin serves as a chromophore and sensor for HClO, while a second coumarin precursor containing a boronate ester acts as a sensor for H2O2. After a head-to-head screening of three candidates differing in their coumarin precursor moieties, probe CSU1 was found to have the optimal characteristics. As shown experimentally, it is able to detect them selectively and sensitively to generate distinct fluorescence signals and patterns in living cells. Furthermore, the endogenous generation of HClO from H2O2 and Cl- catalyzed by myeloperoxidase enzyme in living cells can be clearly monitored by the probe. These studies demonstrate the potential of the probe as a powerful tool to investigate the interplay of HClO and H2O2 in oxidative stress.

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 (λmaxem = 583 nm) to red (λmaxem = 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. Pseudo-time analysis revealed a temporal continuum of the differentiation progress from embryonic day (E) 6.5 to E9.5, implicating early cardiac lineage restriction during mouse gastrulation. First and second heart field (FHF and SHF) CPs adopted different differentiation strategies and underwent distinct transcriptional regulation. Collectively, the comprehensive molecular atlases yield a rich resource for identification of the key cardiac regulators and signaling molecules within the key cardiac gene regulatory network (GRN) governing cardiac cell fate determinations. This review offers insights into the exquisite process and its regulation of CP differentiation at single-cell resolution. As single-cell technologies continuously grow and evolve, computational integration of multimodal single-cell data with well-designed experimental validation promises to further delineate molecular basis in deploying cardiac progenitors of distinct sources with anatomical information.

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. Herein, we have developed an endoplasmic reticulum-targetable fluorescent probe, ER-CN, for H2S detection. ER-CN features excellent sensing properties, such as high sensitivity and selectivity. In addition, ER-CN exhibits low cytotoxicity and a fine endoplasmic reticulum targeting property (with a Pearson's colocalization coefficient of 0.95). Significantly, visualization of H2S in the endoplasmic reticulum of living HeLa cells by using ER-CN was successfully realized.

A red-emitting fluorescent probe for the detection of Hg2+ 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 7.1 nM based on S/N = 3. This probe exhibited a good performance in detecting Hg2+ in real water samples, living cells and organisms.

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. We applied uCoTarget to the in vitro generation of hematopoietic stem/progenitor cells (HSPCs) from human embryonic stem cells, presenting multimodal epigenomic profiles in 26,418 single cells. uCoTarget reveals establishment of pairing of HSPC enhancers (H3K27ac) and promoters (H3K4me3) and RUNX1 engagement priming for H3K27ac activation along the HSPC path. We then develop uCoTargetX, an expansion of uCoTarget to simultaneously measure transcriptome and multiple epigenome targets. Together, our methods enable generalizable, versatile multimodal profiles for reconstructing comprehensive epigenome and transcriptome landscapes and analyzing the regulatory interplay at single-cell level.

Genetic reporter for live tracing fluid flow forces during cell fate segregation in mouse blastocyst development.

Mechanical forces are known to be important in mammalian blastocyst formation; however, due to limited tools, specific force inputs and how they relay to first cell fate control of inner cell mass (ICM) and/or trophectoderm (TE) remain elusive. Combining in toto live imaging and various perturbation experiments, we demonstrate and measure fluid flow forces existing in the mouse blastocyst cavity and identify Klf2(Krüppel-like factor 2) as a fluid force reporter with force-responsive enhancers. Long-term live imaging and lineage reconstructions reveal that blastomeres subject to higher fluid flow forces adopt ICM cell fates. These are reinforced by internal ferrofluid-induced flow force assays. We also utilize ex vivo fluid flow force mimicking and pharmacological perturbations to confirm mechanosensing specificity. Together, we report a genetically encoded reporter for continuously monitoring fluid flow forces and cell fate decisions and provide a live imaging framework to infer force information enriched lineage landscape during development. VIDEO ABSTRACT.

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 Nkx2-5 transcriptional regulation and its specific roles in multistage heart morphogenesis is limited. We comprehensively interrogate enhancers U1 and U2 in controlling Nkx2-5 transcription during heart development. Serial genomic deletions in mice reveal U1 and U2 function redundantly to confer Nkx2-5 expression at early stages, but U2 instead of U1 supports its expression at later stages. Combined deletions markedly reduce Nkx2-5 dosage as early as E7.5, despite being largely reinstated two days later, displaying heart malformations with precocious differentiation of cardiac progenitors. Cutting-edge low-input chromatin immunoprecipitation sequencing (ChIP-seq) confirmed that not only genomic NKX2-5 occupancy but also its regulated enhancer landscape is mostly disturbed in the double-deletion mouse hearts. Together, we propose a model that the temporal and partially compensatory regulatory function of two enhancers dictates a transcription factor (TF)'s dosage and specificity during development.

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. We demonstrate that single-cell itChIP-seq (sc-itChIP-seq) yields ~9,000 unique reads per cell. Using sc-itChIP-seq to profile H3K27ac, we sufficiently capture the earliest epigenetic priming event during the cell fate transition from naive to primed pluripotency, and reveal the basis for cell-type specific enhancer usage during the differentiation of bipotent cardiac progenitor cells into endothelial cells and cardiomyocytes. Our results demonstrate that itChIP is a widely applicable technology for single-cell chromatin profiling of epigenetically heterogeneous cell populations in many biological processes.