Modeling post-implantation stages of human development into early organogenesis with stem-cell-derived peri-gastruloids.

In vitro stem cell models that replicate human gastrulation have been generated, but they lack the essential extraembryonic cells needed for embryonic development, morphogenesis, and patterning. Here, we describe a robust and efficient method that prompts human extended pluripotent stem cells to self-organize into embryo-like structures, termed peri-gastruloids, which encompass both embryonic (epiblast) and extraembryonic (hypoblast) tissues. Although peri-gastruloids are not viable due to the exclusion of trophoblasts, they recapitulate critical stages of human peri-gastrulation development, such as forming amniotic and yolk sac cavities, developing bilaminar and trilaminar embryonic discs, specifying primordial germ cells, initiating gastrulation, and undergoing early neurulation and organogenesis. Single-cell RNA-sequencing unveiled transcriptomic similarities between advanced human peri-gastruloids and primary peri-gastrulation cell types found in humans and non-human primates. This peri-gastruloid platform allows for further exploration beyond gastrulation and may potentially aid in the development of human fetal tissues for use in regenerative medicine.

In Situ Capture of Chromatin Interactions by Biotinylated dCas9.

Cis-regulatory elements (CREs) are commonly recognized by correlative chromatin features, yet the molecular composition of the vast majority of CREs in chromatin remains unknown. Here, we describe a CRISPR affinity purification in situ of regulatory elements (CAPTURE) approach to unbiasedly identify locus-specific chromatin-regulating protein complexes and long-range DNA interactions. Using an in vivo biotinylated nuclease-deficient Cas9 protein and sequence-specific guide RNAs, we show high-resolution and selective isolation of chromatin interactions at a single-copy genomic locus. Purification of human telomeres using CAPTURE identifies known and new telomeric factors. In situ capture of individual constituents of the enhancer cluster controlling human ß-globin genes establishes evidence for composition-based hierarchical organization. Furthermore, unbiased analysis of chromatin interactions at disease-associated cis-elements and developmentally regulated super-enhancers reveals spatial features that causally control gene transcription. Thus, comprehensive and unbiased analysis of locus-specific regulatory composition provides mechanistic insight into genome structure and function in development and disease.

Base-resolution analysis of 5-hydroxymethylcytosine in the mammalian genome.

The study of 5-hydroxylmethylcytosines (5hmC) has been hampered by the lack of a method to map it at single-base resolution on a genome-wide scale. Affinity purification-based methods cannot precisely locate 5hmC nor accurately determine its relative abundance at each modified site. We here present a genome-wide approach, Tet-assisted bisulfite sequencing (TAB-Seq), that when combined with traditional bisulfite sequencing can be used for mapping 5hmC at base resolution and quantifying the relative abundance of 5hmC as well as 5mC. Application of this method to embryonic stem cells not only confirms widespread distribution of 5hmC in the mammalian genome but also reveals sequence bias and strand asymmetry at 5hmC sites. We observe high levels of 5hmC and reciprocally low levels of 5mC near but not on transcription factor-binding sites. Additionally, the relative abundance of 5hmC varies significantly among distinct functional sequence elements, suggesting different mechanisms for 5hmC deposition and maintenance.

Blastocyst-like structures generated from human pluripotent stem cells.

Limited access to embryos has hampered the study of human embryogenesis and disorders that occur during early pregnancy. Human pluripotent stem cells provide an alternative means to study human development in a dish1-7. Recent advances in partial embryo models derived from human pluripotent stem cells have enabled human development to be examined at early post-implantation stages8-14. However, models of the pre-implantation human blastocyst are lacking. Starting from naive human pluripotent stem cells, here we developed an effective three-dimensional culture strategy with successive lineage differentiation and self-organization to generate blastocyst-like structures in vitro. These structures-which we term 'human blastoids'-resemble human blastocysts in terms of their morphology, size, cell number, and composition and allocation of different cell lineages. Single-cell RNA-sequencing analyses also reveal the transcriptomic similarity of blastoids to blastocysts. Human blastoids are amenable to embryonic and extra-embryonic stem cell derivation and can further develop into peri-implantation embryo-like structures in vitro. Using chemical perturbations, we show that specific isozymes of protein kinase C have a critical function in the formation of the blastoid cavity. Human blastoids provide a readily accessible, scalable, versatile and perturbable alternative to blastocysts for studying early human development, understanding early pregnancy loss and gaining insights into early developmental defects.

Multiplexed Engineering and Analysis of Combinatorial Enhancer Activity in Single Cells.

The study of enhancers has been hampered by the scarcity of methods to systematically quantify their endogenous activity. We develop Mosaic-seq to systematically perturb enhancers and measure their endogenous activities at single-cell resolution. Mosaic-seq uses a CRISPR barcoding system to jointly measure a cell's transcriptome and its sgRNA modulators, thus quantifying the effects of dCas9-KRAB-mediated enhancer repression in single cells. Applying Mosaic-seq to 71 constituent enhancers from 15 super-enhancers, our analysis of 51,448 sgRNA-induced transcriptomes finds that only a small number of constituents are major effectors of target gene expression. Binding of p300 and RNAPII are key features of these constituents. We determine two key parameters of enhancer activity in single cells: their penetrance in a population and their contribution to expression in these cells. Through combinatorial interrogation, we find that simultaneous repression of multiple weak constituents can alter super-enhancer activity in a manner greatly exceeding repression of individual constituents.

A single factor elicits multilineage reprogramming of astrocytes in the adult mouse striatum.

SignificanceOutside the neurogenic niches, the adult brain lacks multipotent progenitor cells. In this study, we performed a series of in vivo screens and reveal that a single factor can induce resident brain astrocytes to become induced neural progenitor cells (iNPCs), which then generate neurons, astrocytes, and oligodendrocytes. Such a conclusion is supported by single-cell RNA sequencing and multiple lineage-tracing experiments. Our discovery of iNPCs is fundamentally important for regenerative medicine since neural injuries or degeneration often lead to loss/dysfunction of all three neural lineages. Our findings also provide insights into cell plasticity in the adult mammalian brain, which has largely lost the regenerative capacity.

Identification and characterization of cellular heterogeneity within the developing renal interstitium.

Kidney formation requires the coordinated growth of multiple cell types including the collecting ducts, nephrons, vasculature and interstitium. There is a long-held belief that interactions between progenitors of the collecting ducts and nephrons are primarily responsible for kidney development. However, over the last several years, it has become increasingly clear that multiple aspects of kidney development require signaling from the interstitium. How the interstitium orchestrates these various roles is poorly understood. Here, we show that during development the interstitium is a highly heterogeneous patterned population of cells that occupies distinct positions correlated to the adjacent parenchyma. Our analysis indicates that the heterogeneity is not a mere reflection of different stages in a linear developmental trajectory but instead represents several novel differentiated cell states. Further, we find that ß-catenin has a cell autonomous role in the development of a medullary subset of the interstitium and that this non-autonomously affects the development of the adjacent epithelia. These findings suggest the intriguing possibility that the different interstitial subtypes may create microenvironments that play unique roles in development of the adjacent epithelia and endothelia.

EGFR Mutation Promotes Glioblastoma through Epigenome and Transcription Factor Network Remodeling.

Epidermal growth factor receptor (EGFR) gene amplification and mutations are the most common oncogenic events in glioblastoma (GBM), but the mechanisms by which they promote aggressive tumor growth are not well understood. Here, through integrated epigenome and transcriptome analyses of cell lines, genotyped clinical samples, and TCGA data, we show that EGFR mutations remodel the activated enhancer landscape of GBM, promoting tumorigenesis through a SOX9 and FOXG1-dependent transcriptional regulatory network in vitro and in vivo. The most common EGFR mutation, EGFRvIII, sensitizes GBM cells to the BET-bromodomain inhibitor JQ1 in a SOX9, FOXG1-dependent manner. These results identify the role of transcriptional/epigenetic remodeling in EGFR-dependent pathogenesis and suggest a mechanistic basis for epigenetic therapy.

Computational identification of clonal cells in single-cell CRISPR screens.

BACKGROUND: Single-cell CRISPR screens are powerful tools to understand genome function by linking genetic perturbations to transcriptome-wide phenotypes. However, since few cells can be affordably sequenced in these screens, biased sampling of cells could affect data interpretation. One potential source of biased sampling is clonal cell expansion. RESULTS: Here, we identify clonal cells in single cell screens using multiplexed sgRNAs as barcodes. We find that the cells in each clone share transcriptional similarities and bear segmental copy number changes. These analyses suggest that clones are genetically distinct. Finally, we show that the transcriptional similarities of clonally expanded cells contribute to false positives in single-cell CRISPR screens. CONCLUSIONS: Experimental conditions that reduce clonal expansion or computational filtering of clonal cells will improve the reliability of single-cell CRISPR screens.

FBA: feature barcoding analysis for single cell RNA-Seq.

MOTIVATION: Single cell RNA-Seq (scRNA-Seq) has broadened our understanding of cellular heterogeneity and provided valuable insights into cellular functions. Recent experimental strategies extend scRNA-Seq readouts to include additional features, including cell surface proteins and genomic perturbations. These 'feature barcoding' strategies rely on converting molecular and cellular features to unique sequence barcodes, which are then detected with the transcriptome. RESULTS: Here, we introduce FBA, a flexible and streamlined package to perform quality control, quantification, demultiplexing, multiplet detection, clustering and visualization of feature barcoding assays. AVAILABILITYAND IMPLEMENTATION: FBA is available on PyPi at https://pypi.org/project/fba and on GitHub at https://github.com/jlduan/fba. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

5mC oxidation by Tet2 modulates enhancer activity and timing of transcriptome reprogramming during differentiation.

In mammals, cytosine methylation (5mC) is widely distributed throughout the genome but is notably depleted from active promoters and enhancers. While the role of DNA methylation in promoter silencing has been well documented, the function of this epigenetic mark at enhancers remains unclear. Recent experiments have demonstrated that enhancers are enriched for 5-hydroxymethylcytosine (5hmC), an oxidization product of the Tet family of 5mC dioxygenases and an intermediate of DNA demethylation. These results support the involvement of Tet proteins in the regulation of dynamic DNA methylation at enhancers. By mapping DNA methylation and hydroxymethylation at base resolution, we find that deletion of Tet2 causes extensive loss of 5hmC at enhancers, accompanied by enhancer hypermethylation, reduction of enhancer activity, and delayed gene induction in the early steps of differentiation. Our results reveal that DNA demethylation modulates enhancer activity, and its disruption influences the timing of transcriptome reprogramming during cellular differentiation.

N6-methyladenosine-dependent regulation of messenger RNA stability.

N(6)-methyladenosine (m(6)A) is the most prevalent internal (non-cap) modification present in the messenger RNA of all higher eukaryotes. Although essential to cell viability and development, the exact role of m(6)A modification remains to be determined. The recent discovery of two m(6)A demethylases in mammalian cells highlighted the importance of m(6)A in basic biological functions and disease. Here we show that m(6)A is selectively recognized by the human YTH domain family 2 (YTHDF2) 'reader' protein to regulate mRNA degradation. We identified over 3,000 cellular RNA targets of YTHDF2, most of which are mRNAs, but which also include non-coding RNAs, with a conserved core motif of G(m(6)A)C. We further establish the role of YTHDF2 in RNA metabolism, showing that binding of YTHDF2 results in the localization of bound mRNA from the translatable pool to mRNA decay sites, such as processing bodies. The carboxy-terminal domain of YTHDF2 selectively binds to m(6)A-containing mRNA, whereas the amino-terminal domain is responsible for the localization of the YTHDF2-mRNA complex to cellular RNA decay sites. Our results indicate that the dynamic m(6)A modification is recognized by selectively binding proteins to affect the translation status and lifetime of mRNA.

Next-generation genomics: an integrative approach.

Integrating results from diverse experiments is an essential process in our effort to understand the logic of complex systems, such as development, homeostasis and responses to the environment. With the advent of high-throughput methods--including genome-wide association (GWA) studies, chromatin immunoprecipitation followed by sequencing (ChIP-seq) and RNA sequencing (RNA-seq)--acquisition of genome-scale data has never been easier. Epigenomics, transcriptomics, proteomics and genomics each provide an insightful, and yet one-dimensional, view of genome function; integrative analysis promises a unified, global view. However, the large amount of information and diverse technology platforms pose multiple challenges for data access and processing. This Review discusses emerging issues and strategies related to data integration in the era of next-generation genomics.

CBX3 regulates efficient RNA processing genome-wide.

CBX5, CBX1, and CBX3 (HP1α, ß, and γ, respectively) play an evolutionarily conserved role in the formation and maintenance of heterochromatin. In addition, CBX5, CBX1, and CBX3 may also participate in transcriptional regulation of genes. Recently, CBX3 binding to the bodies of a subset of genes has been observed in human and murine cells. However, the generality of this phenomenon and the role CBX3 may play in this context are unknown. Genome-wide localization analysis reveals CBX3 binding at genic regions, which strongly correlates with gene activity across multiple cell types. Depletion of CBX3 resulted in down-regulation of a subset of target genes. Loss of CBX3 binding leads to a more dramatic accumulation of unspliced nascent transcripts. In addition, we observed defective recruitment of splicing factors, including SNRNP70, to CBX3 target genes. Collectively, our data suggest a role for CBX3 in aiding in efficient cotranscriptional RNA processing.

Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer.

While genetic mutation is a hallmark of cancer, many cancers also acquire epigenetic alterations during tumorigenesis including aberrant DNA hypermethylation of tumor suppressors, as well as changes in chromatin modifications as caused by genetic mutations of the chromatin-modifying machinery. However, the extent of epigenetic alterations in cancer cells has not been fully characterized. Here, we describe complete methylome maps at single nucleotide resolution of a low-passage breast cancer cell line and primary human mammary epithelial cells. We find widespread DNA hypomethylation in the cancer cell, primarily at partially methylated domains (PMDs) in normal breast cells. Unexpectedly, genes within these regions are largely silenced in cancer cells. The loss of DNA methylation in these regions is accompanied by formation of repressive chromatin, with a significant fraction displaying allelic DNA methylation where one allele is DNA methylated while the other allele is occupied by histone modifications H3K9me3 or H3K27me3. Our results show a mutually exclusive relationship between DNA methylation and H3K9me3 or H3K27me3. These results suggest that global DNA hypomethylation in breast cancer is tightly linked to the formation of repressive chromatin domains and gene silencing, thus identifying a potential epigenetic pathway for gene regulation in cancer cells.

Histone modifications at human enhancers reflect global cell-type-specific gene expression.

The human body is composed of diverse cell types with distinct functions. Although it is known that lineage specification depends on cell-specific gene expression, which in turn is driven by promoters, enhancers, insulators and other cis-regulatory DNA sequences for each gene, the relative roles of these regulatory elements in this process are not clear. We have previously developed a chromatin-immunoprecipitation-based microarray method (ChIP-chip) to locate promoters, enhancers and insulators in the human genome. Here we use the same approach to identify these elements in multiple cell types and investigate their roles in cell-type-specific gene expression. We observed that the chromatin state at promoters and CTCF-binding at insulators is largely invariant across diverse cell types. In contrast, enhancers are marked with highly cell-type-specific histone modification patterns, strongly correlate to cell-type-specific gene expression programs on a global scale, and are functionally active in a cell-type-specific manner. Our results define over 55,000 potential transcriptional enhancers in the human genome, significantly expanding the current catalogue of human enhancers and highlighting the role of these elements in cell-type-specific gene expression.

Breaking enhancers to gain insights into developmental defects.

Despite ground-breaking genetic studies that have identified thousands of risk variants for developmental diseases, how these variants lead to molecular and cellular phenotypes remains a gap in knowledge. Many of these variants are non-coding and occur at enhancers, which orchestrate key regulatory programs during development. The prevailing paradigm is that non-coding variants alter the activity of enhancers, impacting gene expression programs, and ultimately contributing to disease risk. A key obstacle to progress is the systematic functional characterization of non-coding variants at scale, especially since enhancer activity is highly specific to cell type and developmental stage. Here, we review the foundational studies of enhancers in developmental disease and current genomic approaches to functionally characterize developmental enhancers and their variants at scale. In the coming decade, we anticipate systematic enhancer perturbation studies to link non-coding variants to molecular mechanisms, changes in cell state, and disease phenotypes.

Protocol to dissociate epithelia from non-pregnant and pregnant mouse cervical tissue for single-cell RNA-sequencing.

A challenge in studying cervical epithelial cell biology at the single-cell level is that differentiated subtypes, in particular mucus-secreting goblet cells, are sensitive to disassociating enzymes making isolation of all epithelial subpopulations difficult. Here we present a protocol to dissociate epithelia from non-pregnant and pregnant mouse cervical tissue for single-cell RNA-sequencing. We describe steps for harvesting cervices, preparing cervical tissue, dissociation of cervical cells, and viability checks. We then detail library preparation, sequencing, and procedure for data analysis. For complete details on the use and execution of this protocol, please refer to Cooley et al. (2023).1.