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.

Effects of source-sink regulation and nodal position of the main crop on the sprouting of regenerated buds and grain yield of ratoon rice.

Ratoon rice (Oryza sativa L.) is the production of a second season rice that utilizes the dormant buds surviving on the stubble left behind after the harvest of the main crop. However, the sprouting mechanism of regenerated buds at separate nodes is rarely reported. Field experiments were conducted to examine the effects of leaf-cutting and spikelet thinning on the sprouting of regenerated buds at the separate node, the contributions of regenerated panicles at the separate node to the total grain yield in the ratoon crop, and the associated mechanism. The results showed that the contribution of separate node yields to the total grain yield in the ratoon crop was D2 (panicles regenerated from the 2nd node from the top) >D3 (panicles regenerated from the 3rd node from the top) >D4 (panicles regenerated from the lower nodes below the 3rd node), and the contribution of D2 and D3 made up approximately 80% of the total yield in the ratoon crop. In addition, the effect of leaf-cutting treatment and spikelet-thinning treatment on the grain yield of ratoon season was mainly realized by regulating the relative contribution rate of D2 and D4 grain yield to the total yield of ratoon season. Further analysis indicated that the sprouting of regenerated buds at the D2 node was mainly affected by the content of CTK, while D3 was mainly regulated by GAs and CTK, and D4 was mainly regulated by ABA and CTK. However, only the CTK content in stems and buds was positively correlated with single bud length and bud number at each nodes. These results indicated that CTK might be the main signal regulating the sprouting of regenerated buds and the grain yield at separate nodes, which might change the transport of assimilates to stems and buds.

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.

GATA4 Regulates Developing Endocardium Through Interaction With ETS1.

BACKGROUND: The pioneer transcription factor (TF) GATA4 (GATA Binding Protein 4) is expressed in multiple cardiovascular lineages and is essential for heart development. GATA4 lineage-specific occupancy in the developing heart underlies its lineage specific activities. Here, we characterized GATA4 chromatin occupancy in cardiomyocyte and endocardial lineages, dissected mechanisms that control lineage specific occupancy, and analyzed GATA4 regulation of endocardial gene expression. METHODS: We mapped GATA4 chromatin occupancy in cardiomyocyte and endocardial cells of embryonic day 12.5 (E12.5) mouse heart using lineage specific, Cre-activated biotinylation of GATA4. Regulation of GATA4 pioneering activity was studied in cell lines stably overexpressing GATA4. GATA4 regulation of endocardial gene expression was analyzed using single cell RNA sequencing and luciferase reporter assays. RESULTS: Cardiomyocyte-selective and endothelial-selective GATA4 occupied genomic regions had features of lineage specific enhancers. Footprints within cardiomyocyte- and endothelial-selective GATA4 regions were enriched for NKX2-5 (NK2 homeobox 5) and ETS1 (ETS Proto-Oncogene 1) motifs, respectively, and both of these TFs interacted with GATA4 in co-immunoprecipitation assays. In stable NIH3T3 cell lines expressing GATA4 with or without NKX2-5 or ETS1, the partner TFs re-directed GATA4 pioneer binding and augmented its ability to open previously inaccessible regions, with ETS1 displaying greater potency as a pioneer partner than NKX2-5. Single-cell RNA sequencing of embryonic hearts with endothelial cell-specific Gata4 inactivation identified Gata4-regulated endocardial genes, which were adjacent to GATA4-bound, endothelial regions enriched for both GATA4 and ETS1 motifs. In reporter assays, GATA4 and ETS1 cooperatively stimulated endothelial cell enhancer activity. CONCLUSIONS: Lineage selective non-pioneer TFs NKX2-5 and ETS1 guide the activity of pioneer TF GATA4 to bind and open chromatin and create active enhancers and mechanistically link ETS1 interaction to GATA4 regulation of endocardial development.

Loss of sphingosine kinase 2 promotes the expansion of hematopoietic stem cells by improving their metabolic fitness.

Hematopoietic stem cells (HSCs) have reduced capacities to properly maintain and replenish the hematopoietic system during myelosuppressive injury or aging. Expanding and rejuvenating HSCs for therapeutic purposes has been a long-sought goal with limited progress. Here, we show that the enzyme Sphk2 (sphingosine kinase 2), which generates the lipid metabolite sphingosine-1-phosphate, is highly expressed in HSCs. The deletion of Sphk2 markedly promotes self-renewal and increases the regenerative potential of HSCs. More importantly, Sphk2 deletion globally preserves the young HSC gene expression pattern, improves the function, and sustains the multilineage potential of HSCs during aging. Mechanistically, Sphk2 interacts with prolyl hydroxylase 2 and the Von Hippel-Lindau protein to facilitate HIF1α ubiquitination in the nucleus independent of the Sphk2 catalytic activity. Deletion of Sphk2 increases hypoxic responses by stabilizing the HIF1α protein to upregulate PDK3, a glycolysis checkpoint protein for HSC quiescence, which subsequently enhances the function of HSCs by improving their metabolic fitness; specifically, it enhances anaerobic glycolysis but suppresses mitochondrial oxidative phosphorylation and generation of reactive oxygen species. Overall, targeting Sphk2 to enhance the metabolic fitness of HSCs is a promising strategy to expand and rejuvenate functional HSCs.

Mitochondrial base editor induces substantial nuclear off-target mutations.

DddA-derived cytosine base editors (DdCBEs)-which are fusions of split DddA halves and transcription activator-like effector (TALE) array proteins from bacteria-enable targeted C•G-to-T•A conversions in mitochondrial DNA1. However, their genome-wide specificity is poorly understood. Here we show that the mitochondrial base editor induces extensive off-target editing in the nuclear genome. Genome-wide, unbiased analysis of its editome reveals hundreds of off-target sites that are TALE array sequence (TAS)-dependent or TAS-independent. TAS-dependent off-target sites in the nuclear DNA are often specified by only one of the two TALE repeats, challenging the principle that DdCBEs are guided by paired TALE proteins positioned in close proximity. TAS-independent off-target sites on nuclear DNA are frequently shared among DdCBEs with distinct TALE arrays. Notably, they co-localize strongly with binding sites for the transcription factor CTCF and are enriched in topologically associating domain boundaries. We engineered DdCBE to alleviate such off-target effects. Collectively, our results have implications for the use of DdCBEs in basic research and therapeutic applications, and suggest the need to thoroughly define and evaluate the off-target effects of base-editing tools.

Enhancement in Seed Priming-Induced Starch Degradation of Rice Seed Under Chilling Stress via GA-Mediated α-Amylase Expression.

Chilling stress is the major abiotic stress that severely limited the seedling establishment of direct-seeded rice in temperate and sub-tropical rice production regions. While seed priming is an efficient pre-sowing seed treatment in enhancing crop establishment under abiotic stress. Our previous research has identified two seed priming treatments, selenium priming (Se) and salicylic priming (SA) that effectively improved the seed germination and seedling growth of rice under chilling stress. To further explore how seed priming enhance the starch degradation of rice seeds under chilling stress, the present study evaluated the effects of Se and SA priming on germination and seedling growth, α-amylase activity, total soluble sugar content, hormone content and associated gene relative expression under chilling stress. The results showed that both Se and SA priming significantly increased the seed germination and seedling growth attributes, and enhanced the starch degradation ability by increasing α-amylase activity and total soluble sugar content under chilling stress. Meanwhile, seed priming increased the transcription level of OsRamy1A, OsRamy3B that regulated by GA, and increased the transcription level of OsRamy3E that regulated by sugar signals. Furthermore, seed priming significantly improved the GA3 contents in rice seeds by up-regulating the expression of OsGA3ox1 and OsGA20ox1, and decreased the ABA content and the expression of OsNCED1, indicating that the improved starch degradation ability in primed rice seeds under chilling stress might be attributed to the increased GA3 and decreased ABA levels in primed rice seeds, which induced the expression of GA-mediated α-amylase. However, studies to explore how seed priming mediate hormonal metabolism and the expression of OsRamy3E are desperately needed.

Pre-configuring chromatin architecture with histone modifications guides hematopoietic stem cell formation in mouse embryos.

The gene activity underlying cell differentiation is regulated by a diverse set of transcription factors (TFs), histone modifications, chromatin structures and more. Although definitive hematopoietic stem cells (HSCs) are known to emerge via endothelial-to-hematopoietic transition (EHT), how the multi-layered epigenome is sequentially unfolded in a small portion of endothelial cells (ECs) transitioning into the hematopoietic fate remains elusive. With optimized low-input itChIP-seq and Hi-C assays, we performed multi-omics dissection of the HSC ontogeny trajectory across early arterial ECs (eAECs), hemogenic endothelial cells (HECs), pre-HSCs and long-term HSCs (LT-HSCs) in mouse embryos. Interestingly, HSC regulatory regions are already pre-configurated with active histone modifications as early as eAECs, preceding chromatin looping dynamics within topologically associating domains. Chromatin looping structures between enhancers and promoters only become gradually strengthened over time. Notably, RUNX1, a master TF for hematopoiesis, enriched at half of these loops is observed early from eAECs through pre-HSCs but its enrichment further increases in HSCs. RUNX1 and co-TFs together constitute a central, progressively intensified enhancer-promoter interactions. Thus, our study provides a framework to decipher how temporal epigenomic configurations fulfill cell lineage specification during development.

Nuclear peripheral chromatin-lamin B1 interaction is required for global integrity of chromatin architecture and dynamics in human cells.

The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.

Continuous live imaging reveals a subtle pathological alteration with cell behaviors in congenital heart malformation.

To form fully functional four-chambered structure, mammalian heart development undergoes a transient finger-shaped trabeculae, crucial for efficient contraction and exchange for gas and nutrient. Although its developmental origin and direct relevance to congenital heart disease has been studied extensively, the time-resolved cellular mechanism underlying hypotrabeculation remains elusive. Here, we employed in toto live imaging and reconstructed the holistic cell lineages and cellular behavior landscape of control and hypotrabeculed hearts of mouse embryos from E9.5 for up to 24 h. Compared to control, hypotrabeculation in ErbB2 mutants arose mainly through dual mechanisms: both reduced proliferation of trabecular cardiomyocytes from early cell fate segregation and markedly impaired oriented cell division and migration. Further examination of mosaic mutant hearts confirmed alterations in cellular behaviors in a cell autonomous manner. Thus, our work offers a framework for continuous live imaging and digital cell lineage analysis to better understand subtle pathological alterations in congenital heart disease.

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.

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.

Long-term, in toto live imaging of cardiomyocyte behaviour during mouse ventricle chamber formation at single-cell resolution.

Mapping of the holistic cell behaviours sculpting the four-chambered mammalian heart has been a goal or previous studies, but so far only success in transparent invertebrates and lower vertebrates with two-chambered hearts has been achieved. Using a live-imaging system comprising a customized vertical light-sheet microscope equipped with a mouse embryo culture module, a heartbeat-gated imaging strategy and a digital image processing framework, we realized volumetric imaging of developing mouse hearts at single-cell resolution and with uninterrupted cell lineages for up to 1.5 d. Four-dimensional landscapes of Nppa+ cardiomyocyte cell behaviours revealed a blueprint for ventricle chamber formation by which biased outward migration of the outermost cardiomyocytes is coupled with cell intercalation and horizontal division. The inner-muscle architecture of trabeculae was developed through dual mechanisms: early fate segregation and transmural cell arrangement involving both oriented cell division and directional migration. Thus, live-imaging reconstruction of uninterrupted cell lineages affords a transformative means for deciphering mammalian organogenesis.

Meningeal lymphatic vessels regulate brain tumor drainage and immunity.

Recent studies have shown that meningeal lymphatic vessels (MLVs), which are located both dorsally and basally beneath the skull, provide a route for draining macromolecules and trafficking immune cells from the central nervous system (CNS) into cervical lymph nodes (CLNs), and thus represent a potential therapeutic target for treating neurodegenerative and neuroinflammatory diseases. However, the roles of MLVs in brain tumor drainage and immunity remain unexplored. Here we show that dorsal MLVs undergo extensive remodeling in mice with intracranial gliomas or metastatic melanomas. RNA-seq analysis of MLV endothelial cells revealed changes in the gene sets involved in lymphatic remodeling, fluid drainage, as well as inflammatory and immunological responses. Disruption of dorsal MLVs alone impaired intratumor fluid drainage and the dissemination of brain tumor cells to deep CLNs (dCLNs). Notably, the dendritic cell (DC) trafficking from intracranial tumor tissues to dCLNs decreased in mice with defective dorsal MLVs, and increased in mice with enhanced dorsal meningeal lymphangiogenesis. Strikingly, disruption of dorsal MLVs alone, without affecting basal MLVs or nasal LVs, significantly reduced the efficacy of combined anti-PD-1/CTLA-4 checkpoint therapy in striatal tumor models. Furthermore, mice bearing tumors overexpressing VEGF-C displayed a better response to anti-PD-1/CTLA-4 combination therapy, and this was abolished by CCL21/CCR7 blockade, suggesting that VEGF-C potentiates checkpoint therapy via the CCL21/CCR7 pathway. Together, the results of our study not only demonstrate the functional aspects of MLVs as classic lymphatic vasculature, but also highlight that they are essential in generating an efficient immune response against brain tumors.

Polycomb Group Proteins Regulate Chromatin Architecture in Mouse Oocytes and Early Embryos.

In mammals, chromatin organization undergoes drastic reorganization during oocyte development. However, the dynamics of three-dimensional chromatin structure in this process is poorly characterized. Using low-input Hi-C (genome-wide chromatin conformation capture), we found that a unique chromatin organization gradually appears during mouse oocyte growth. Oocytes at late stages show self-interacting, cohesin-independent compartmental domains marked by H3K27me3, therefore termed Polycomb-associating domains (PADs). PADs and inter-PAD (iPAD) regions form compartment-like structures with strong inter-domain interactions among nearby PADs. PADs disassemble upon meiotic resumption from diplotene arrest but briefly reappear on the maternal genome after fertilization. Upon maternal depletion of Eed, PADs are largely intact in oocytes, but their reestablishment after fertilization is compromised. By contrast, depletion of Polycomb repressive complex 1 (PRC1) proteins attenuates PADs in oocytes, which is associated with substantial gene de-repression in PADs. These data reveal a critical role of Polycomb in regulating chromatin architecture during mammalian oocyte growth and early development.

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.

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.