Esrrb is a cell-cycle-dependent associated factor balancing pluripotency and XEN differentiation.

Cell cycle and differentiation decisions are linked; however, the underlying principles that drive these decisions are unclear. Here, we combined cell-cycle reporter system and single-cell RNA sequencing (scRNA-seq) profiling to study the transcriptomes of embryonic stem cells (ESCs) in the context of cell-cycle states and differentiation. By applying retinoic acid, to G1 and G2/M ESCs, we show that, while both populations can differentiate toward epiblast stem cells (EpiSCs), only G2/M ESCs could differentiate into extraembryonic endoderm cells. We identified Esrrb, a pluripotency factor that is upregulated during G2/M, as a driver of extraembryonic endoderm stem cell (XEN) differentiation. Furthermore, enhancer chromatin states based on wild-type (WT) and ESRRB knockout (KO) ESCs show association of ESRRB with XEN poised enhancers. G1 cells overexpressing Esrrb allow ESCs to produce XENs, while ESRRB-KO ESCs lost their potential to differentiate into XEN. Overall, this study reveals a vital link between Esrrb and cell-cycle states during the exit from pluripotency.

CloneSeq: A highly sensitive analysis platform for the characterization of 3D-cultured single-cell-derived clones.

Single-cell assays have revealed the importance of heterogeneity in many biological systems. However, limited sensitivity is a major hurdle for uncovering cellular variation. To overcome it, we developed CloneSeq, combining clonal expansion inside 3D hydrogel spheres and droplet-based RNA sequencing (RNA-seq). We show that clonal cells maintain similar transcriptional profiles and cell states. CloneSeq of lung cancer cells revealed cancer-specific subpopulations, including cancer stem-like cells, that were not revealed by scRNA-seq. Clonal expansion within 3D soft microenvironments supported cellular stemness of embryonic stem cells (ESCs) even without pluripotent media, and it improved epigenetic reprogramming efficiency of mouse embryonic fibroblasts. CloneSeq of ESCs revealed that the differentiation decision is made early during Oct4 downregulation and is maintained during early clonal expansion. Together, we show CloneSeq can be adapted to different biological systems to discover rare subpopulations by leveraging the enhanced sensitivity within clones.

DNA methylation patterns expose variations in enhancer-chromatin modifications during embryonic stem cell differentiation.

In mammals, cellular identity is defined through strict regulation of chromatin modifications and DNA methylation that control gene expression. Methylation of cytosines at CpG sites in the genome is mainly associated with suppression; however, the reason for enhancer-specific methylation is not fully understood. We used sequential ChIP-bisulfite-sequencing for H3K4me1 and H3K27ac histone marks. By collecting data from the same genomic region, we identified enhancers differentially methylated between these two marks. We observed a global gain of CpG methylation primarily in H3K4me1-marked nucleosomes during mouse embryonic stem cell differentiation. This gain occurred largely in enhancer regions that regulate genes critical for differentiation. The higher levels of DNA methylation in H3K4me1- versus H3K27ac-marked enhancers, despite it being the same genomic region, indicates cellular heterogeneity of enhancer states. Analysis of single-cell RNA-seq profiles demonstrated that this heterogeneity correlates with gene expression during differentiation. Furthermore, heterogeneity of enhancer methylation correlates with transcription start site methylation. Our results provide insights into enhancer-based functional variation in complex biological systems.

Fine-Resolution Mapping of TF Binding and Chromatin Interactions.

Transcription factor (TF) binding to DNA is crucial for transcriptional regulation. There are multiple methods for mapping such binding. These methods balance between input requirements, spatial resolution, and compatibility with high-throughput automation. Here, we describe SLIM-ChIP (short-fragment-enriched, low-input, indexed MNase ChIP), which combines enzymatic fragmentation of chromatin and on-bead indexing to address these desiderata. SLIM-ChIP reproduces a high-resolution binding map of yeast Reb1 comparable with existing methods, yet with less input material and full compatibility with high-throughput procedures. We demonstrate the robustness and flexibility of SLIM-ChIP by probing additional factors in yeast and mouse. Finally, we show that SLIM-ChIP provides information on the chromatin landscape surrounding the bound transcription factor. We identify a class of Reb1 sites where the proximal -1 nucleosome tightly interacts with Reb1 and maintains unidirectional transcription. SLIM-ChIP is an attractive solution for mapping DNA binding proteins and charting the surrounding chromatin occupancy landscape at a single-cell level.

SF3B1 association with chromatin determines splicing outcomes.

Much remains unknown concerning the mechanism by which the splicing machinery pinpoints short exons within intronic sequences and how splicing factors are directed to their pre-mRNA targets. One probable explanation lies in differences in chromatin organization between exons and introns. Proteomic, co-immunoprecipitation, and sedimentation analyses described here indicate that SF3B1, an essential splicing component of the U2 snRNP complex, is strongly associated with nucleosomes. ChIP-seq and RNA-seq analyses reveal that SF3B1 specifically binds nucleosomes located at exonic positions. SF3B1 binding is enriched at nucleosomes positioned over short exons flanked by long introns that are also characterized by differential GC content between exons and introns. Disruption of SF3B1 binding to such nucleosomes affects splicing of these exons similarly to SF3B1 knockdown. Our findings suggest that the association of SF3B1 with nucleosomes is functionally important for splice-site recognition and that SF3B1 conveys splicing-relevant information embedded in chromatin structure.

Differential association of chromatin proteins identifies BAF60a/SMARCD1 as a regulator of embryonic stem cell differentiation.

Embryonic stem cells (ESCs) possess a distinct chromatin conformation maintained by specialized chromatin proteins. To identify chromatin regulators in ESCs, we developed a simple biochemical assay named D-CAP (differential chromatin-associated proteins), using brief micrococcal nuclease digestion of chromatin, followed by liquid chromatography tandem mass spectrometry (LC-MS/MS). Using D-CAP, we identified several differentially chromatin-associated proteins between undifferentiated and differentiated ESCs, including the chromatin remodeling protein SMARCD1. SMARCD1 depletion in ESCs led to altered chromatin and enhanced endodermal differentiation. Gene expression and chromatin immunoprecipitation sequencing (ChIP-seq) analyses suggested that SMARCD1 is both an activator and a repressor and is enriched at developmental regulators and that its chromatin binding coincides with H3K27me3. SMARCD1 knockdown caused H3K27me3 redistribution and increased H3K4me3 around the transcription start site (TSS). One of the identified SMARCD1 targets was Klf4. In SMARCD1-knockdown clones, KLF4, as well as H3K4me3 at the Klf4 locus, remained high and H3K27me3 was abolished. These results propose a role for SMARCD1 in restricting pluripotency and activating lineage pathways by regulating H3K27 methylation.

Open chromatin in pluripotency and reprogramming.

Pluripotent stem cells can be derived from embryos or induced from adult cells by reprogramming. They are unique among stem cells in that they can give rise to all cell types of the body. Recent findings indicate that a particularly 'open' chromatin state contributes to maintenance of pluripotency. Two principles are emerging: specific factors maintain a globally open chromatin state that is accessible for transcriptional activation; and other chromatin regulators contribute locally to the silencing of lineage-specific genes until differentiation is triggered. These same principles may apply during reacquisition of an open chromatin state upon reprogramming to pluripotency, and during de-differentiation in cancer.

Chd1 regulates open chromatin and pluripotency of embryonic stem cells.

An open chromatin largely devoid of heterochromatin is a hallmark of stem cells. It remains unknown whether an open chromatin is necessary for the differentiation potential of stem cells, and which molecules are needed to maintain open chromatin. Here we show that the chromatin remodelling factor Chd1 is required to maintain the open chromatin of pluripotent mouse embryonic stem cells. Chd1 is a euchromatin protein that associates with the promoters of active genes, and downregulation of Chd1 leads to accumulation of heterochromatin. Chd1-deficient embryonic stem cells are no longer pluripotent, because they are incapable of giving rise to primitive endoderm and have a high propensity for neural differentiation. Furthermore, Chd1 is required for efficient reprogramming of fibroblasts to the pluripotent stem cell state. Our results indicate that Chd1 is essential for open chromatin and pluripotency of embryonic stem cells, and for somatic cell reprogramming to the pluripotent state.