Effective methods for bulk RNA-seq deconvolution using scnRNA-seq transcriptomes.

Background: RNA profiling technologies at single-cell resolutions, including single-cell and single-nuclei RNA sequencing (scRNA-seq and snRNA-seq, scnRNA-seq for short), can help characterize the composition of tissues and reveal cells that influence key functions in both healthy and disease tissues. However, the use of these technologies is operationally challenging because of high costs and stringent sample-collection requirements. Computational deconvolution methods that infer the composition of bulk-profiled samples using scnRNA-seq-characterized cell types can broaden scnRNA-seq applications, but their effectiveness remains controversial.

Single cell Hi-C identifies plastic chromosome conformations underlying the gastrulation enhancer landscape.

Embryonic development involves massive proliferation and differentiation of cell lineages. This must be supported by chromosome replication and epigenetic reprogramming, but how proliferation and cell fate acquisition are balanced in this process is not well understood. Here we use single cell Hi-C to map chromosomal conformations in post-gastrulation mouse embryo cells and study their distributions and correlations with matching embryonic transcriptional atlases.

TATTOO-seq delineates spatial and cell type-specific regulatory programs in the developing limb.

The coordinated differentiation of progenitor cells into specialized cell types and their spatial organization into distinct domains is central to embryogenesis. Here, we developed and applied an unbiased spatially resolved single-cell transcriptomics method to identify the genetic programs underlying the emergence of specialized cell types during mouse limb development and their spatial integration. We identify multiple transcription factors whose expression patterns are predominantly associated with cell type specification or spatial position, suggesting two parallel yet highly interconnected regulatory systems.

DNA methyltransferases 3A and 3B target specific sequences during mouse gastrulation.

In mammalian embryos, DNA methylation is initialized to maximum levels in the epiblast by the de novo DNA methyltransferases DNMT3A and DNMT3B before gastrulation diversifies it across regulatory regions. Here we show that DNMT3A and DNMT3B are differentially regulated during endoderm and mesoderm bifurcation and study the implications in vivo and in meso-endoderm embryoid bodies. Loss of both Dnmt3a and Dnmt3b impairs exit from the epiblast state.

The intrinsic and extrinsic effects of TET proteins during gastrulation.

Mice deficient for all ten-eleven translocation (TET) genes exhibit early gastrulation lethality. However, separating cause and effect in such embryonic failure is challenging. To isolate cell-autonomous effects of TET loss, we used temporal single-cell atlases from embryos with partial or complete mutant contributions.

A single-embryo, single-cell time-resolved model for mouse gastrulation.

Mouse embryonic development is a canonical model system for studying mammalian cell fate acquisition. Recently, single-cell atlases comprehensively charted embryonic transcriptional landscapes, yet inference of the coordinated dynamics of cells over such atlases remains challenging. Here, we introduce a temporal model for mouse gastrulation, consisting of data from 153 individually sampled embryos spanning 36 h of molecular diversification.

Single-cell transcriptomic analyses provide insights into the developmental origins of neuroblastoma.

Neuroblastoma is a pediatric tumor of the developing sympathetic nervous system. However, the cellular origin of neuroblastoma has yet to be defined. Here we studied the single-cell transcriptomes of neuroblastomas and normal human developing adrenal glands at various stages of embryonic and fetal development.

Multi-clonal SARS-CoV-2 neutralization by antibodies isolated from severe COVID-19 convalescent donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild versus severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection.

Multi-Clonal Live SARS-CoV-2 In Vitro Neutralization by Antibodies Isolated from Severe COVID-19 Convalescent Donors.

The interactions between antibodies, SARS-CoV-2 and immune cells contribute to the pathogenesis of COVID-19 and protective immunity. To understand the differences between antibody responses in mild severe cases of COVID-19, we analyzed the B cell responses in patients 1.5 months post SARS-CoV-2 infection.

Single-cell analysis of clonal maintenance of transcriptional and epigenetic states in cancer cells.

Propagation of clonal regulatory programs contributes to cancer development. It is poorly understood how epigenetic mechanisms interact with genetic drivers to shape this process. Here, we combine single-cell analysis of transcription and DNA methylation with a Luria-Delbrück experimental design to demonstrate the existence of clonally stable epigenetic memory in multiple types of cancer cells.

MetaCell: analysis of single-cell RNA-seq data using K-nn graph partitions.

scRNA-seq profiles each represent a highly partial sample of mRNA molecules from a unique cell that can never be resampled, and robust analysis must separate the sampling effect from biological variance. We describe a methodology for partitioning scRNA-seq datasets into metacells: disjoint and homogenous groups of profiles that could have been resampled from the same cell. Unlike clustering analysis, our algorithm specializes at obtaining granular as opposed to maximal groups.

Deterministic Somatic Cell Reprogramming Involves Continuous Transcriptional Changes Governed by Myc and Epigenetic-Driven Modules.

The epigenetic dynamics of induced pluripotent stem cell (iPSC) reprogramming in correctly reprogrammed cells at high resolution and throughout the entire process remain largely undefined. Here, we characterize conversion of mouse fibroblasts into iPSCs using Gatad2a-Mbd3/NuRD-depleted and highly efficient reprogramming systems. Unbiased high-resolution profiling of dynamic changes in levels of gene expression, chromatin engagement, DNA accessibility, and DNA methylation were obtained.

Neutralizing Gatad2a-Chd4-Mbd3/NuRD Complex Facilitates Deterministic Induction of Naive Pluripotency.

Mbd3, a member of nucleosome remodeling and deacetylase (NuRD) co-repressor complex, was previously identified as an inhibitor for deterministic induced pluripotent stem cell (iPSC) reprogramming, where up to 100% of donor cells successfully complete the process. NuRD can assume multiple mutually exclusive conformations, and it remains unclear whether this deterministic phenotype can be attributed to a specific Mbd3/NuRD subcomplex. Moreover, since complete ablation of Mbd3 blocks somatic cell proliferation, we aimed to explore functionally relevant alternative ways to neutralize Mbd3-dependent NuRD activity.

Early metazoan cell type diversity and the evolution of multicellular gene regulation.

A hallmark of metazoan evolution is the emergence of genomic mechanisms that implement cell-type-specific functions. However, the evolution of metazoan cell types and their underlying gene regulatory programmes remains largely uncharacterized. Here, we use whole-organism single-cell RNA sequencing to map cell-type-specific transcription in Porifera (sponges), Ctenophora (comb jellies) and Placozoa species.

Cnidarian Cell Type Diversity and Regulation Revealed by Whole-Organism Single-Cell RNA-Seq.

The emergence and diversification of cell types is a leading factor in animal evolution. So far, systematic characterization of the gene regulatory programs associated with cell type specificity was limited to few cell types and few species. Here, we perform whole-organism single-cell transcriptomics to map adult and larval cell types in the cnidarian Nematostella vectensis, a non-bilaterian animal with complex tissue-level body-plan organization.

Transient acquisition of pluripotency during somatic cell transdifferentiation with iPSC reprogramming factors.

Somatic cells can be transdifferentiated to other cell types without passing through a pluripotent state by ectopic expression of appropriate transcription factors. Recent reports have proposed an alternative transdifferentiation method in which fibroblasts are directly converted to various mature somatic cell types by brief expression of the induced pluripotent stem cell (iPSC) reprogramming factors Oct4, Sox2, Klf4 and c-Myc (OSKM) followed by cell expansion in media that promote lineage differentiation. Here we test this method using genetic lineage tracing for expression of endogenous Nanog and Oct4 and for X chromosome reactivation, as these events mark acquisition of pluripotency.

Stem cells. m6A mRNA methylation facilitates resolution of naïve pluripotency toward differentiation.

Naïve and primed pluripotent states retain distinct molecular properties, yet limited knowledge exists on how their state transitions are regulated. Here, we identify Mettl3, an N(6)-methyladenosine (m(6)A) transferase, as a regulator for terminating murine naïve pluripotency. Mettl3 knockout preimplantation epiblasts and naïve embryonic stem cells are depleted for m(6)A in mRNAs, yet are viable.

Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells.

Stable maintenance of gene regulatory programs is essential for normal function in multicellular organisms. Epigenetic mechanisms, and DNA methylation in particular, are hypothesized to facilitate such maintenance by creating cellular memory that can be written during embryonic development and then guide cell-type-specific gene expression. Here we develop new methods for quantitative inference of DNA methylation turnover rates, and show that human embryonic stem cells preserve their epigenetic state by balancing antagonistic processes that add and remove methylation marks rather than by copying epigenetic information from mother to daughter cells.

Deterministic direct reprogramming of somatic cells to pluripotency.

Somatic cells can be inefficiently and stochastically reprogrammed into induced pluripotent stem (iPS) cells by exogenous expression of Oct4 (also called Pou5f1), Sox2, Klf4 and Myc (hereafter referred to as OSKM). The nature of the predominant rate-limiting barrier(s) preventing the majority of cells to successfully and synchronously reprogram remains to be defined. Here we show that depleting Mbd3, a core member of the Mbd3/NuRD (nucleosome remodelling and deacetylation) repressor complex, together with OSKM transduction and reprogramming in naive pluripotency promoting conditions, result in deterministic and synchronized iPS cell reprogramming (near 100% efficiency within seven days from mouse and human cells).