Single-cell RNA-seq reveals novel regulators of human embryonic stem cell differentiation to definitive endoderm

Background: Human pluripotent stem cells offer the best available model to study the underlying cellular and molecular mechanisms of human embryonic lineage specification. However, it is not fully understood how individual stem cells exit the pluripotent state and transition towards their respective progenitor states. Results: Here, we analyze the transcriptomes of human embryonic stem cell-derived lineage-specific progenitors by single-cell RNA-sequencing (scRNA-seq). We identify a definitive endoderm (DE) transcriptomic signature that leads us to pinpoint a critical time window when DE differentiation is enhanced by hypoxia. The molecular mechanisms governing the emergence of DE are further examined by time course scRNA-seq experiments, employing two new statistical tools to identify stage-specific genes over time (SCPattern) and to reconstruct the differentiation trajectory from the pluripotent state through mesendoderm to DE (Wave-Crest). Importantly, presumptive DE cells can be detected during the transitory phase from Brachyury (T)+ mesendoderm toward a CXCR4+ DE state. Novel regulators are identified within this time window and are functionally validated on a screening platform with a T-2A-EGFP knock-in reporter engineered by CRISPR/Cas9. Through loss-of-function and gain-of-function experiments, we demonstrate that KLF8 plays a pivotal role modulating mesendoderm to DE differentiation. Conclusions: We report the analysis of 1776 cells by scRNA-seq covering distinct human embryonic stem cell-derived progenitor states. By reconstructing a differentiation trajectory at single-cell resolution, novel regulators of the mesendoderm transition to DE are elucidated and validated. Our strategy of combining single-cell analysis and genetic approaches can be applied to uncover novel regulators governing cell fate decisions in a variety of systems. Electronic supplementary material The online version of this article (doi:10.1186/s13059-016-1033-x) contains supplementary material, which is available to authorized users

The Origin Recognition Complex Interacts with a Subset of Metabolic Genes Tightly Linked to Origins of Replication

The origin recognition complex (ORC) marks chromosomal sites as replication origins and is essential for replication initiation. In yeast, ORC also binds to DNA elements called silencers, where its primary function is to recruit silent information regulator (SIR) proteins to establish transcriptional silencing. Indeed, silencers function poorly as chromosomal origins. Several genetic, molecular, and biochemical studies of HMR-E have led to a model proposing that when ORC becomes limiting in the cell (such as in the orc2-1 mutant) only sites that bind ORC tightly (such as HMR-E) remain fully occupied by ORC, while lower affinity sites, including many origins, lose ORC occupancy. Since HMR-E possessed a unique non-replication function, we reasoned that other tight sites might reveal novel functions for ORC on chromosomes. Therefore, we comprehensively determined ORC “affinity” genome-wide by performing an ORC ChIP–on–chip in ORC2 and orc2-1 strains. Here we describe a novel group of orc2-1–resistant ORC–interacting chromosomal sites (ORF–ORC sites) that did not function as replication origins or silencers. Instead, ORF–ORC sites were comprised of protein-coding regions of highly transcribed metabolic genes. In contrast to the ORC–silencer paradigm, transcriptional activation promoted ORC association with these genes. Remarkably, ORF–ORC genes were enriched in proximity to origins of replication and, in several instances, were transcriptionally regulated by these origins. Taken together, these results suggest a surprising connection among ORC, replication origins, and cellular metabolism

Introducing Large Genomic Deletions in Human Pluripotent Stem Cells Using CRISPR‐Cas3

CRISPR‐Cas systems provide researchers with eukaryotic genome editing tools and therapeutic platforms that make it possible to target disease mutations in somatic organs. Most of these tools employ Type II (e.g., Cas9) or Type V (e.g., Cas12a) CRISPR enzymes to create RNA‐guided precise double‐strand breaks in the genome. However, such technologies are limited in their capacity to make targeted large deletions. Recently, the Type I CRISPR system, which is prevalent in microbes and displays unique enzymatic features, has been harnessed to effectively create large chromosomal deletions in human cells. Type I CRISPR first uses a multisubunit ribonucleoprotein (RNP) complex called Cascade to find its guide‐complementary target site, and then recruits a helicase‐nuclease enzyme, Cas3, to travel along and shred the target DNA over a long distance with high processivity. When introduced into human cells as purified RNPs, the CRISPR‐Cas3 complex can efficiently induce large genomic deletions of varying lengths (1‐100 kb) from the CRISPR‐targeted site. Because of this unique editing outcome, CRISPR‐Cas3 holds great promise for tasks such as the removal of integrated viral genomes and the interrogation of structural variants affecting gene function and human disease. Here, we provide detailed protocols for introducing large deletions using CRISPR‐Cas3. We describe step‐by‐step procedures for purifying the Type I‐E CRISPR proteins Cascade and Cas3 from Thermobifida fusca, electroporating RNPs into human cells, and characterizing DNA deletions using PCR and sequencing. We focus here on human pluripotent stem cells due to their clinical potential, but these protocols will be broadly useful for other cell lines and model organisms for applications including large genomic deletion, full‐gene or ‐chromosome removal, and CRISPR screening for noncoding elements, among others. © 2022 Wiley Periodicals LLC.Basic Protocol 1: Expression and purification of Tfu Cascade RNPSupport Protocol 1: Expression and purification of TfuCas3 proteinSupport Protocol 2: Culture of human pluripotent stem cellsBasic Protocol 2: Introduction of Tfu Cascade RNP and Cas3 protein into hPSCs via electroporationBasic Protocol 3: Characterization of genomic DNA lesions using long‐range PCR, TOPO cloning, and Sanger sequencingAlternate Protocol: Comprehensive analysis of genomic lesions by Tn5‐based next‐generation sequencingSupport Protocol 3: Single‐cell clonal isolationPeer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/171838/1/cpz1361.pdfhttp://deepblue.lib.umich.edu/bitstream/2027.42/171838/2/cpz1361_am.pd

Interplay of transcription factors and signaling proteins in specifying the regulatory programs of modules.

<p><b>A.</b> Shown are the fraction of modules that are regulated by TFs alone, signaling proteins alone or both <b>B.</b> Shown are the co-regulatory, genetic and protein-protein interactions between regulators associated with HOG1 associated modules. HOG1 is a protein kinase involved in osmotic stress and cell wall organization. HOG1 is predicted to be a regulator for Modules 2 and 37, and is known to be directly upstream of SKO1 which is predicted to regulate genes in Module 19. Co-regulatory relations are inferred between two regulators if they share common targets. Genetic and protein-protein interactions are obtained from BioGRID <a href="http://www.ploscompbiol.org/article/info:doi/10.1371/journal.pcbi.1003252#pcbi.1003252-Chatraryamontri1" target="_blank">[66]</a>.</p