Profiling of pluripotency factors in single cells and early embryos
Ontology highlight
ABSTRACT: Cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small populations of cells within developing embryos. To understand their functions in vivo, it is important to identify TF binding sites in these cells. However, current methods cannot profile TFs genome-wide at or near the single cell level. Here we adapt the CUT&RUN method to profile TFs in low cell numbers, including single cells and individual pre-implantation embryos. Single-cell experiments demonstrate that only a fraction of TF binding sites appear to be occupied in most cells, in a manner broadly consistent with measurements of peak intensity from multi-cell studies. We further show that chromatin binding by the pluripotency TF NANOG is highly dependent on the SWI/SNF chromatin remodeling complex in individual blastocysts but not in cultured cells. Ultra-low input CUT&RUN (uliCUT&RUN) therefore enables interrogation of TF binding from rare cell populations of particular importance in development or disease.
Project description:Though the in vitro structural and in vivo spatial characteristics of transcription factor (TF) binding are well defined, TF interactions with chromatin and other companion TFs during development are poorly understood. To analyze such interactions in vivo, we profiled several TFs across a time course of human embryonic stem cell differentiation via CUT&RUN epigenome profiling, and studied their interactions with nucleosomes and co-occurring TFs by Enhanced Chromatin Occupancy (EChO), a computational strategy for classifying TF binding characteristics across time and space. EChO shows that at different enhancer targets, the same TF can employ either direct DNA binding, or “pioneer” nucleosome binding to access them. Pioneer binding is correlated with local binding of other TFs and enhancer motif character, including degeneracy at key bases in the pioneer factor target motif. Our strategy reveals a dynamic exchange of TFs at enhancers across developmental time that is aided by pioneer nucleosome binding.
Project description:Enhancers harbor binding motifs that recruit transcription factors (TFs) for gene activation. While cooperative binding of TFs at enhancers is known to be critical for transcriptional activation of a handful of developmental enhancers, the extent TF cooperativity genome-wide is unknown. Here, we couple high-resolution nuclease footprinting with single-molecule methylation profiling to characterize TF cooperativity at active enhancers in the Drosophila genome. Enrichment of short MNase-protected DNA segments indicates that the majority of enhancers harbor two or more TF binding sites, and we uncover protected fragments that correspond to co-bound sites in thousands of enhancers. We integrate MNase-seq, methylation accessibility profiling, and CUT&RUN chromatin profiling as a comprehensive strategy to characterize co-binding of the Trithorax-like (TRL) DNA binding protein and multiple other TFs. We identify states where an enhancer is bound by no TF, by either single factor, by multiple factors, or where binding sites are occluded by nucleosomes. From the analysis of co-binding, we find that cooperativity dominates TF binding in vivo at a majority of active enhancers. Factor cooperativity occurs predominantly between sites spaced 50 bp apart in active enhancers, indicating that most TF cooperativity in cells occurs without apparent protein-protein interactions. Our findings suggest a mechanism for nucleosomes to promote cooperativity by requiring co-binding to effectively clear nucleosomes and promote enhancer function.
Project description:We performed genome-wide analysis of protein-DNA binding using data obtained from CUT&RUN of BOS patient and control individual fibroblast samples to dissect the effects of truncating ASXL1 mutations. We performed genome-wide analysis of protein-DNA binding using data obtained from CUT&RUN of BOS patient and control individual fibroblast samples.
Project description:Cell fate transitions are accompanied by global transcriptional, epigenetic and topological changes driven by transcription factors (TFs), as is strikingly exemplified by reprogramming somatic cells to pluripotent stem cells (PSCs) via expression of OCT4, KLF4, SOX2 and cMYC. How TFs orchestrate the complex molecular changes around their target gene loci in a temporal manner remains incompletely understood. Here, using KLF4 as a paradigm, we provide the first TF-centric view of chromatin reorganization and its association to 3D enhancer rewiring and transcriptional changes of linked genes during reprogramming of mouse embryonic fibroblasts (MEFs) to PSCs. Inducible depletion of KLF factors in PSCs caused a genome-wide decrease in the connectivity of enhancers, while disruption of individual KLF4 binding sites from PSC-specific enhancers was sufficient to impair enhancer-promoter contacts and reduce expression of associated genes. Our study provides an integrative view of the complex activities of a lineage-specifying TF during a controlled cell fate transition and offers novel insights into the order and nature of molecular events that follow TF binding.
Project description:DNA sequence-specific transcription factors (TFs) modulate transcription and chromatin architecture, acting from regulatory sites in enhancers and promoters of eukaryotic genes. How multiple TFs cooperate to regulate individual genes is still unclear. Most yeast TFs are thought to regulate transcription via binding to Upstream Activating Sequences, situated within a few hundred base pairs upstream of the regulated gene1. While this model has been validated for individual TFs and specific genes, it has not been tested in a systematic way. We integrated information on the binding and expression targets for the near-complete set of yeast TFs. Here we show that, contrary to expectations, there are few TFs with dedicated activator or repressor functions with most TFs having a dual role. While nearly all protein coding genes are regulated by one or more TFs, our analysis revealed limited overlap between TF binding and gene regulation. Rapid depletion of many TFs also revealed numerous regulatory targets distant from detectable TF binding sites, suggesting unexpected regulatory mechanisms. Our study provides a comprehensive survey of TF functions, offering insights into interactions between the set of TFs expressed in a single cell type and how they contribute to the complex program of gene regulation.
Project description:DNA sequence-specific transcription factors (TFs) modulate transcription and chromatin architecture, acting from regulatory sites in enhancers and promoters of eukaryotic genes. How multiple TFs cooperate to regulate individual genes is still unclear. Most yeast TFs are thought to regulate transcription via binding to Upstream Activating Sequences, situated within a few hundred base pairs upstream of the regulated gene1. While this model has been validated for individual TFs and specific genes, it has not been tested in a systematic way. We integrated information on the binding and expression targets for the near-complete set of yeast TFs. Here we show that, contrary to expectations, there are few TFs with dedicated activator or repressor functions with most TFs having a dual role. While nearly all protein coding genes are regulated by one or more TFs, our analysis revealed limited overlap between TF binding and gene regulation. Rapid depletion of many TFs also revealed numerous regulatory targets distant from detectable TF binding sites, suggesting unexpected regulatory mechanisms. Our study provides a comprehensive survey of TF functions, offering insights into interactions between the set of TFs expressed in a single cell type and how they contribute to the complex program of gene regulation.
Project description:DNA sequence-specific transcription factors (TFs) modulate transcription and chromatin architecture, acting from regulatory sites in enhancers and promoters of eukaryotic genes. How multiple TFs cooperate to regulate individual genes is still unclear. Most yeast TFs are thought to regulate transcription via binding to Upstream Activating Sequences, situated within a few hundred base pairs upstream of the regulated gene1. While this model has been validated for individual TFs and specific genes, it has not been tested in a systematic way. We integrated information on the binding and expression targets for the near-complete set of yeast TFs. Here we show that, contrary to expectations, there are few TFs with dedicated activator or repressor functions with most TFs having a dual role. While nearly all protein coding genes are regulated by one or more TFs, our analysis revealed limited overlap between TF binding and gene regulation. Rapid depletion of many TFs also revealed numerous regulatory targets distant from detectable TF binding sites, suggesting unexpected regulatory mechanisms. Our study provides a comprehensive survey of TF functions, offering insights into interactions between the set of TFs expressed in a single cell type and how they contribute to the complex program of gene regulation.
Project description:Transcription factors (TFs) play crucial roles in kidney development and disease by recognizing specific DNA sequences to control gene expression programs. The kidney’s cellular heterogeneity poses substantial challenges to identifying the genomic binding sites and direct target genes of TFs in vivo. We apply the CUT&RUN (cleavage under targets and release using nuclease) technique, together with transcriptomic analysis, to identify cAMP-response element-binding protein (CREB) target genes in cystic epithelial cells of autosomal dominant polycystic kidney disease (ADPKD). Our results reveal that CREB binds to and activates ribosomal biogenesis genes, and that inhibition of CREB retards cyst growth in ADPKD mouse models. Our findings demonstrate that CUT&RUN is a powerful method for genome-scale profiling and identifying direct targets of TFs from small numbers of specific kidney cells.
Project description:Transcription factors (TFs) play crucial roles in kidney development and disease by recognizing specific DNA sequences to control gene expression programs. The kidney’s cellular heterogeneity poses substantial challenges to identifying the genomic binding sites and direct target genes of TFs in vivo. We apply the CUT&RUN (cleavage under targets and release using nuclease) technique, together with transcriptomic analysis, to identify cAMP-response element-binding protein (CREB) target genes in cystic epithelial cells of autosomal dominant polycystic kidney disease (ADPKD). Our results reveal that CREB binds to and activates ribosomal biogenesis genes, and that inhibition of CREB retards cyst growth in ADPKD mouse models. Our findings demonstrate that CUT&RUN is a powerful method for genome-scale profiling and identifying direct targets of TFs from small numbers of specific kidney cells.