SUMMARY Deciphering mechanisms in cell fate decisions requires single-cell holistic reconstructions of multi-dimensional epigenome in transcriptional regulation. Here we develop CoTECH, a combinatorial barcoding method allowing for high-throughput single-cell joint detection of chromatin occupancy and transcriptome. First, we used CoTECH to examine bivalent histone marks (H3K4me3 and H3K27me3) with transcription from naïve to primed mouse embryonic stem cells. Concurrent bivalent marks in pseudo-single cells linked via transcriptome were computationally derived, resolving pseudotemporal bivalency trajectories and disentangling a context-specific interplay between H3K4me3/H3K27me3 and transcription level. Next, CoTECH with H3K27ac, an active enhancer marker, 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 (HEC) emergence, indicating a unique epigenetic control of transcriptional regulation for hematopoietic stem cell priming. Together, CoTECH provides an efficient framework for single-cell co-assay of chromatin occupancy and transcription, thus, enabling higher-dimensional epigenomic reconstructions.
Abstract Rigorous exploration and dissection of potential actions and effects of long noncoding RNA (lncRNA) in animals remain challenging. Here using multiple knockout mouse models and single- cell RNA sequencing, we demonstrate that the divergent lncRNA Hand2as has a key, complex modulatory effect on the expression of its neighboring gene HAND2 and subsequently on heart development and function, largely independent of Hand2as transcription and transcripts. Full-length deletion of Hand2as in mouse causes moderate yet prevalent upregulation of HAND2 in hundreds of cardiac cells, resulting in profound biological consequences, including dysregulated cardiac gene programs, congenital heart defects and perinatal lethality. We propose a cis -functional role for the Hand2as locus in dampening HAND2 expression to restrain cardiomyocyte proliferation, thereby orchestrating a balanced development of cardiac cell lineages. This study highlights the need for complementary genetic and single-cell approaches to delineate the function and primary molecular effects of an lncRNA in animals. Impact statement The long noncoding RNA Hand2as critically controls the precise expression of its neighboring gene HAND2 , thereby balancing cardiac lineages and expression programs that are essential for heart development and function.
Abstract Background Cell type-specific transcriptional heterogeneity in embryonic mouse skin is well-documented, but few studies have investigated the regulatory mechanisms. Results Here, we present high throughput single-cell chromatin accessibility and transcriptome sequencing (HT-scCAT-seq), a method that simultaneously profiles transcriptome and chromatin accessibility. We utilized HT-scCAT-seq to dissect the gene regulatory mechanism governing epidermal stratification, periderm terminal differentiation, and fibroblast specification. Conclusions By linking chromatin accessibility to gene expression, we identified candidate cis- regulatory elements (cCREs) and target genes crucial for dermal and epidermal development. We described cells with similar gene expression profiles that exhibit distinct chromatin accessibility statuses during periderm terminal differentiation. Finally, we characterized the underlying lineage-determining transcription factors (TFs), and demonstrated that ALX4 and RUNX2 were candidate TF regulators of the dermal papilla lineage development through in silico perturbation analysis.
ABSTRACT Exploration and dissection of potential actions and effects of long noncoding RNA (lncRNA) in animals remain challenging. Here, using multiple knockout mouse models and single cell RNA sequencing, we demonstrate that the divergent lncRNA Hand2os1/Uph has a key complex modulatory effect on the expression of its neighboring gene HAND2 and subsequently on heart development and function. Short deletion of the Hand2os1 promoter in mouse diminishes Hand2os1 transcription to ∼8-32%, but fails to affect HAND2 expression and yields no discernable heart phenotypes. Interestingly, full-length deletion of Hand2os1 in mouse causes moderate yet prevalent upregulation of HAND2 in hundreds of cardiac cells, leading to profound biological consequences, including dysregulated cardiac gene programs, congenital heart defects and perinatal lethality. We propose that the Hand2os1 locus dampens HAND2 expression to restrain cardiomyocyte proliferation, thereby orchestrating a balanced development of cardiac cell lineages. This study highlights the regulatory complexity of the lncRNA Hand2os1 on HAND2 expression, emphasizing the need for complementary genetic and single cell approaches to delineate the function and primary molecular effects of an lncRNA in animals.
Abstract Here we present CoTECH, a high-throughput co-aasay that measures chromatin occupancy and transcriptome in single cells. The CoTECH method adopts a combinatorial indexing strategy to enrich chromatin fragments of interest as reported in CoBATCH in combination with a modified Smart-seq2 procedure to simultaneously capture the 3’ mRNA profiles in the same single cells. The whole experimental procedure can be handled within three days.The CoTECH acquires data quality of 1000-9000 unique mapped reads (DNA partition) and 1500-4000 expressed genes (RNA partition) per cell. Experimentally linking chromatin occupancy to transcriptional outputs and inferred molecular association between multimodal omics datasets made possible by CoTECH enables reconstructions of higher dimensional epigenomic landscape, providing new insights into epigenome-centric gene regulation and cellular heterogeneity in many biological processes. This step-by-step protocol is related to the publication “Single-cell joint detection of chromatin occupancy and transcriptome enables higher-dimensional epigenomic reconstructions” in Nature Methods.
Abstract Single-cell measurement of chromatin states, including histone modifications and non-histone protein binding, remains challenging. We present a low-cost, efficient ChIP-seq (simultaneous indexing and tagmentation-based ChIP-seq, itChIP), compatible to both low-input and single cells for profiling chromatin states. This single-cell itChIP approach combines chromatin opening, simultaneous cellular indexing and chromatin tagmentation in a single tube, processing samples with tens of single cells in rarity or with thousands of single cells per assay, and the entire procedure can be finished in two days. The sc-itChIP data acquire ~9,000 unique reads per cell, sufficiently capturing the earliest epigenetic priming along cell fate transition and the basis for cell-type specific enhancer usage. Our results demonstrate that itChIP is a generalizable technology for single-cell chromatin profiling of epigenetically heterogeneous cell populations in many biological processes. This step-by-step protocol is related to the publication “Profiling chromatin state by single-cell itChIP-seq” in Nature Cell Biology.
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 abstracteyJraWQiOiI4ZjUxYWNhY2IzYjhiNjNlNzFlYmIzYWFmYTU5NmZmYyIsImFsZyI6IlJTMjU2In0.eyJzdWIiOiI2NmZiMjUzMjAyNTE2ODVkNzI2MDhlZDQ1OGI5Nzk2YSIsImtpZCI6IjhmNTFhY2FjYjNiOGI2M2U3MWViYjNhYWZhNTk2ZmZjIiwiZXhwIjoxNjk0ODE2MjEzfQ.b_UfIUybk9KSGmNo6-yCZgCAm6M-CEAXVeh_LEqkvNEJnaT7QFHKDCM1Ilcpt82b9zLlmnrGErt1KV2O86OMYhOHkP7Us1oXe2h-uBGB9wagJn0s7izpUXd8togLxZLGLoi25E5_2S841hAugQ4NJ5pQfki5K4h0qCcADT8bDbAsQ510u38eNBxZ8uvcf4Rn7XiVXftgyXCiuOHT6mVGhf3XFaWYWkkgA6BO_9gAysezT2ocStEYfH6JXtW7XEDQ6r0Du2e4Wg3JS_Nt9SUWnmQoyjW7kaFOoqj6Qzoc4qIzx0uzCpdXt3MdwuYzoIh4_zZDClJBzT7fh0ueUJstHw(mp4, (80.85 MB) Download video
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