A new study of significant importance combines single-cell transcriptomic and epigenetic analyses in organoids to generate the first single-cell epigenomic atlas of human central nervous system development.
Embracing the Potential of Integrated Single-cell Epigenetic and Transcriptomic Analyses
Due to the importance of histone modifications in regulating the tightly controlled programs of gene expression required for normal cell and tissue function, we currently possess a vast catalog of genome-wide profiles from studies employing techniques such as chromatin immunoprecipitation (ChIP)-seq, cleavage under targets & release using nuclease (CUT&RUN), and Cleavage Under Targets and Tagmentation (CUT&Tag). These techniques generally employ millions of cells as input, with the outputs representing the average characteristics of such “bulk” samples. This represents a problem when studying tissues composed of multiple distinct cell types/subtypes or developmental processes involving the intricate transitioning/branching of cell identities. The ongoing development of single-cell/single-nucleus epigenetic and transcriptomic techniques may represent a solution to such problems.
Developments in single-cell histone modification profiling techniques include Paired-Tag technology from Epigenome Technologies, which enables antibody-targeted profiling of histones or transcription factors and RNA and facilitates precise annotation of cis-regulatory elements, linking their states directly to gene activity. While the application of single-cell epigenetic techniques such as these remains few and far between, we highlight a study from Zenk, Fleck, and Colleagues published in Nature Neuroscience, which represents a robust example of the power of single-cell analyses (Zenk and Fleck et al.).
The team behind this study understood that dynamic and complex epigenetic alterations underpinned the in vivo differentiation of cells during development (Millan-Zambrano et al.) and that any epigenetic dysregulation could prompt the onset of various diseases/disorders (Paulsen et al. and Li et al.). Unfortunately, bulk studies of histone modifications in developing tissues fail to support the analysis of single-cell trajectories as they pass through multiple fates (Preissl, Gaulton, and Ren). In their recent study, the team applied single-cell epigenetic and transcriptomic analyses to brain and retina organoids – in vitro stem cell-derived “mini-organs” – to fully appreciate the tight control over gene expression required for normal neural development.
Histone Modification Profiling Describes a Single-Cell Epigenomic Atlas of Organoid Development
Researchers from the laboratories of Fides Zenk (ETH Zürich), J. Gray Camp (Roche Innovation Center Basel), and Barbara Treutlein (ETH Zürich) profiled three histone modifications at the single-cell level in brain and retina organoids by performing single-cell cleavage under targets and tagmentation (scCUT&Tag) during organoid development. The histone modifications chosen included H3K27me3, which marks repressed developmental genes; H3K27ac, which marks active gene enhancers and promoters; and H3K4me3, which marks active or bivalent promoters of developmental genes. The time points employed covered the pluripotent state of induced pluripotent stem cells and differentiation through a neuroepithelium stage to the final stage of retinal and brain region/cell-type specification (e.g., neurons and glia), which would support the reconstruction of the epigenomic trajectories regulating cell fate. In addition, they performed single-cell RNA-sequencing (scRNA-seq) to generate transcriptomic profiles that would match the epigenetic profiles.
Of note, this approach fails to support epigenetic and transcriptomic profiling in the same cell as these monomodal techniques do not support simultaneous analysis, which can prompt problems when integrating datasets at later stages. Paired-Tag technology from Epigenome Technologies supports the simultaneous profiling of one histone modification and RNA in the same cell, thereby generating more data with greater surety over the results.
In their study, Zenk and Fleck et al. clustered cells for each modality separately and then matched clusters since increased levels of H3K27ac and H3K4me3 tend to associate with active transcription while increased levels of H3K27me3 tended to associate with transcriptional silencing. The annotation of cell types in clusters based on gene expression supported the definition of histone modification profiles in distinct cell types during organoid development; overall, the accumulated data provided evidence that specific chromatin modification profiles associated with specific cell populations during differentiation and supported the development of a single-cell epigenomic atlas of human central nervous system development in organoids comprising activating and repressive histone modifications and RNA expression. This resource is free to explore and can be viewed at https://episcape.ethz.ch.
Understanding Histone Modification Dynamics During Organoid Development
This single-cell epigenomic atlas developed by the team permitted a view of histone modification dynamics as pluripotent stem cells differentiated through a neuroepithelial phase into distinct neuronal cell types. Observing levels of all three histone modifications at gene regulatory elements (promoters and enhancers) during this time supported the identification of region-specific regulatory elements near cell fate determinants that may play critical roles during organoid development. Interestingly, the authors discovered that H3K27ac and H3K4me3 accumulation (and H3K27me3 loss) at such regulatory elements preceded the onset of gene expression, in agreement with activating histone marks functioning to prime chromatin for gene expression. Further exploration of this concept employed a single-cell multiome assay to profile chromatin accessibility and RNA expression simultaneously; this approach provided evidence for the establishment of H3K27ac and H3K4me3 profiles before an increase in chromatin accessibility and subsequent gene expression. Analysis of transcription factor binding (or binding motifs) at identified region-specific regulatory elements also allowed the inference of the transcription factors and gene regulatory network driving organoid development.
Finally, the team evaluated how the loss of one histone modification may affect differentiation/organoid development; treatment with an inhibitor of a non-redundant member of the Polycomb Repressive Complex 2 (PRC2) complex - embryonic ectoderm development (EED) – prompted H3K27me3 loss at the early neuroepithelium stage, induced the activation of repressed genes, disrupted fate restriction decisions, and resulted in the emergence of aberrant cell identities. Overall, these findings suggested a need for tight control over chromatin modifiers for appropriate regional diversification and cell fate stabilization.
Paired-Tag Technology: Towards Improved Single-cell Epigenomic Analyses
In summary, the authors performed scCUT&Tag and scRNA-seq to separately histone modification patterns and RNA expression at the single-cell level to generate what they report as the first single-cell epigenomic atlas of brain/retina organoid development, which simulates critical aspects of early human regionalization and cell type formation in vitro. The immense effort revealed the dynamic nature of gene regulatory elements and cell identity alterations during organoid development and underscored the importance of histone modification profiles in regulating cell fate determination and brain regionalization.
While the current approach has provided invaluable insight, the potential of Paired-Tag technology from Epigenome Technologies to support the simultaneous profiling of histone modification and RNA expression in the same cell remains a promising approach, which could significantly enhance the robustness and depth of future single-cell epigenomic analyses.
Authors: Haruhiko Ishii, Stuart P. Atkinson
Comments