CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo

Aughey, Gabriel; Estacio-Gómez, Alicia; Thomson, Jamie; Yin, Hang; Southall, Tony D.

doi:10.7554/elife.32341

Cited by 72 publications

(80 citation statements)

References 65 publications

Supporting

Mentioning

Contrasting

Order By: Relevance

“…Although previous studies have demonstrated a global reduction in chromatin accessibility and/or plasticity during lineage commitment in specific developmental settings (e.g. embryonic stem cells, intestinal stem cells, and neural stem cells 35,36,38,64 ), our quantitative findings extend the scope of this result. Moreover, as has been previously shown 65 , our data indicate that variability in gene counts between phenotypically identical single cells is not exclusively due to drop-out events, but also due to differential sampling of the transcriptome (Fig.…”

Section: Discussionsupporting

confidence: 63%

“…Since the transcriptional output of a cell is associated with its genome-wide chromatin profile, we hypothesized that single-cell gene counts might ultimately be a surrogate for global chromatin accessibility, which has been previously shown to decrease with differentiation [35][36][37][38] . To test this, we compared single-cell gene counts derived from scRNA-seq data with paired bulk ATAC-seq (assay for transposase-accessible chromatin sequencing) profiles obtained from a recent study of in vitro mesodermal differentiation from human embryonic stem cells (hESCs) 32 ( Fig.…”

Section: Robustness and Biological Basis Of Gene Countsmentioning

confidence: 99%

See 1 more Smart Citation

Single-cell transcriptional diversity is a hallmark of developmental potential

Gulati

Sikandar

Wesche

et al. 2019

Preprint

133

245

View full text Add to dashboard Cite

Single-cell RNA-sequencing (scRNA-seq) is a powerful approach for reconstructing cellular differentiation trajectories. However, inferring both the state and direction of differentiation without prior knowledge has remained challenging. Here we describe a simple yet robust determinant of developmental potential-the number of detectably expressed genes per celland leverage this measure of transcriptional diversity to develop a new framework for predicting ordered differentiation states from scRNA-seq data. When evaluated on ~150,000 single-cell transcriptomes spanning 53 lineages and five species, our approach, called CytoTRACE, outperformed previous methods and ~19,000 molecular signatures for resolving experimentallyconfirmed developmental trajectories. In addition, it enabled unbiased identification of tissueresident stem cells, including cells with long-term regenerative potential. When used to analyze human breast tumors, we discovered candidate genes associated with less-differentiated luminal progenitor cells and validated GULP1 as a novel gene involved in tumorigenesis. Our study establishes a key RNA-based correlate of developmental potential and provides a new platform for robust delineation of cellular hierarchies (https://cytotrace.stanford.edu).

show abstract

Section: Discussionsupporting

confidence: 63%

Section: Robustness and Biological Basis Of Gene Countsmentioning

confidence: 99%

Single-cell transcriptional diversity is a hallmark of developmental potential

Gulati

Sikandar

Wesche

et al. 2019

Preprint

133

245

View full text Add to dashboard Cite

show abstract

“…We designed MaTaDa to take advantage of the large collection of Cre-expressing constructs, cell lines and model organisms for targeted expression in vivo. As has been demonstrated in Drosophila (Cheetham and Brand, 2018;Marshall and Brand, 2017;Southall et al, 2013;Aughey et al, 2018), the generation of MaTaDa transgenic animals for key transcription factors, lncRNAs, chromatin complexes and RNA polymerase II will permit the characterisation of the molecular landscape of gene regulation in almost any cell type. Analysing the interactions between transcription factors and enhancers in small and pure populations of stem cells in vivo will be of vital importance for an increased understanding of the transcriptional control of development.…”

Section: Discussionmentioning

confidence: 99%

Targeted DamID reveals differential binding of mammalian pluripotency factors

et al. 2018

View full text Add to dashboard Cite

The precise control of gene expression by transcription factor networks is crucial to organismal development. The predominant approach for mapping transcription factor-chromatin interactions has been chromatin immunoprecipitation (ChIP). However, ChIP requires a large number of homogeneous cells and antisera with high specificity. A second approach, DamID, has the drawback that high levels of Dam methylase are toxic. Here, we modify our targeted DamID approach (TaDa) to enable cell type-specific expression in mammalian systems, generating an inducible system (mammalian TaDa or MaTaDa) to identify genome-wide protein/DNA interactions in 100 to 1000 times fewer cells than ChIP-based approaches. We mapped the binding sites of two key pluripotency factors, OCT4 and PRDM14, in mouse embryonic stem cells, epiblast-like cells and primordial germ cell-like cells (PGCLCs). PGCLCs are an important system for elucidating primordial germ cell development in mice. We monitored PRDM14 binding during the specification of PGCLCs, identifying direct targets of PRDM14 that are key to understanding its crucial role in PGCLC development. We show that MaTaDa is a sensitive and accurate method for assessing cell type-specific transcription factor binding in limited numbers of cells.

show abstract

“…Because of this range of methylation, DamID is unlikely to reach the resolution offered by ChIP-based techniques; DamID is not, however, constrained by the same input limitations, and has been used to profile transcription factor binding from 1000 ES cells (Tosti et al 2018) and even single cells (Lai et al 2019). Although ChIP-seq (and more recently, CUT&RUN) has largely superseded DamID for factor localization, DamID is becoming more popular in studying broader chromatin features; for instance, Chromatin Accessibility Targeted DamID (CATaDA) has been developed to assess open chromatin (Aughey et al 2018). CATaDa utilizes an untethered Dam protein to methylate regions of open chromatin, leaving nucleosome-bound DNA unmethylated (Aughey et al 2018).…”

Section: Damidmentioning

confidence: 99%

“…Although ChIP-seq (and more recently, CUT&RUN) has largely superseded DamID for factor localization, DamID is becoming more popular in studying broader chromatin features; for instance, Chromatin Accessibility Targeted DamID (CATaDA) has been developed to assess open chromatin (Aughey et al 2018). CATaDa utilizes an untethered Dam protein to methylate regions of open chromatin, leaving nucleosome-bound DNA unmethylated (Aughey et al 2018). Split DamID has also been used to profile cooccupancy of two proteins at genomic loci, acting in a similar manner to a yeast two-hybrid screen (Hass et al 2015), and a catalytically inactive DpnI-GFP fusion construct has been used to examine Dam-driven GATC methylation in real-time using microscopy (Kind et al 2015).…”

Section: Damidmentioning

confidence: 99%

Genomic methods in profiling DNA accessibility and factor localization

Klein

Hainer

2019

Chromosome Res

View full text Add to dashboard Cite

Recent advancements in next-generation sequencing technologies and accompanying reductions in cost have led to an explosion of techniques to examine DNA accessibility and protein localization on chromatin genome-wide. Generally, accessible regions of chromatin are permissive for factor binding and are therefore hotspots for regulation of gene expression; conversely, genomic regions that are highly occupied by histone proteins are not permissive for factor binding and are less likely to be active regulatory regions. Identifying regions of differential accessibility can be useful to uncover putative gene regulatory regions, such as enhancers, promoters, and insulators. In addition, DNA-binding proteins, such as transcription factors that preferentially bind certain DNA sequences and histone proteins that form the core of the nucleosome, play essential roles in all DNA-templated processes. Determining the genomic localization of chromatin-bound proteins is therefore e s s e n t i a l i n d e t e r m i n i n g f u n c t i o n a l r o l e s , sequence motifs important for factor binding, and regulatory networks controlling gene expression. In this review, we discuss techniques for determining DNA accessibility and nucleosome positioning (DNase-seq, FAIRE-seq, MNase-seq, and ATACseq) and techniques for detecting and functionally characterizing chromatin-bound proteins (ChIP-seq, DamID, and CUT&RUN). These methods have been optimized to varying degrees of resolution, specificity, and ease of use. Here, we outline some advantages and disadvantages of these techniques, their general protocols, and a brief discussion of their development. Together, these complimentary approaches have provided an unparalleled view of chromatin architecture and functional gene regulation. Keywords Chromatin. DNase. MNase. ATAC. ChIP. CUT&RUN. nucleosome occupancy. transcription factors. genomics Abbreviations DHS DNase I hypersensitive site DNase-seq DNase I coupled with deep sequencing XL-DNaseseq Crosslinking DNase I coupled with deep sequencing scDNaseseq Single-cell DNase I coupled with deep sequencing FAIRE-seq Formaldehyde-assisted isolation of regulatory elements MNase-seq Micrococcal nuclease digestion coupled with deep sequencing MPE-seq Methidiumpropyl-EDTA cleavage coupled with deep sequencing ATAC-seq An assay for transposase accessibility

show abstract

CATaDa reveals global remodelling of chromatin accessibility during stem cell differentiation in vivo

Cited by 72 publications

References 65 publications

Single-cell transcriptional diversity is a hallmark of developmental potential

Single-cell transcriptional diversity is a hallmark of developmental potential

Targeted DamID reveals differential binding of mammalian pluripotency factors

Genomic methods in profiling DNA accessibility and factor localization

Contact Info

Product

Resources

About