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The First scMultiomic Atlas of Tissue Development – Transcriptomic and Epigenetic Profiling with CUT&RUN

By Stuart P. Atkinson, Ph.D.

March 17, 2025

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Introduction: Blurred Vision – A Lack of Single-cell Resolution in Lens Development Studies

Successful ocular lens development involves the differentiation of epithelial cells into fiber cells; however, this process can cause cataract formation when dysregulated. Genome-level approaches have evaluated lens tissues via microarrays and RNA sequencing, although we lack single-cell resolution studies of lens development. Advanced single-cell and single-nucleus RNA-sequencing techniques have supported the examination of gene expression profiles at high throughput and resolution; however, few studies have applied such technologies to developing lens tissue (Disatham et al.).

Researchers from the laboratory of Katia Del Rio-Tsonis (Miami University) sought to see lens development and disease more clearly by fashioning the first single-cell multiomic atlas of lens tissue development by applying single-nucleus RNA sequencing (snRNA-seq), single-nucleus assay for transposase-accessible chromatin with sequencing (snATAC-seq), and cleavage under targets and release using nuclease (CUT&RUN) assays in a chicken lens model system. The team's new study, published in Development, combines single-cell transcriptomic and epigenetic analyses of epithelial-to-fiber cell differentiation to describe those mechanisms controlling cell fate determination and explore cataract-linked regulatory networks (Tangeman et al.). Can single-cell multiomic analyses provide a clearer vision of lens development and disease?

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A Single-cell Multiomic Map of Epithelial-to-Fiber Cell Differentiation

Tangeman et al. analyzed whole embryonic chicken eyes via snRNA-seq and snATAC-seq and revealed three cell states associated with the epithelial cell-to-fiber cell transition – epithelial, intermediate, and fiber cells. Pseudotime analysis established a trajectory of epithelial-to-fiber cell differentiation that agreed with reported differentiation-associated gene expression profiles and supported the exploration of temporal gene regulation. The integration of transcriptomic and chromatin accessibility data identified nearly 1,000 regions exhibiting a significant change in chromatin accessibility during epithelial-to-fiber cell differentiation; notably, said regions generally mapped to cis-regulatory elements (CREs, such as enhancer elements) rather than promoters. The integrated dataset aided the prediction of interactions between differentially accessible chromatin regions and transcription start sites to identify CREs that controlled the expression pattern of specific genes: furthermore, this dataset served to reveal the dynamic expression of eighty transcription factor-encoding genes during lens development and to relate gene expression dynamics of transcription factors. to the chromatin signature at DNA-binding motifs. Overall, the integration of transcriptomic and epigenetic profiling permitted the creation of a single-cell multiomic map of epithelial-to-fiber cell differentiation that the authors hoped would prove valuable to future research.

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A Clear View of Transcription Factors Regulatory Networks Associated with Lens Pathology

The team next applied chromatin accessibility data to predict regulatory relationships associated with the MAF transcription factor in the lens, a known effector of fiber cell differentiation with reported links to cataract formation/ocular defects (Anand et al., Jamieson et al., and Niceta et al.). MAF expression increased during epithelial cell-to-fiber cell differentiation, correlating with increased accessibility at regions containing MAF-binding motifs, which associated with 114 genes that included cataract/lens defect-linked genes and other genes with essential roles in lens biology. Overall, these data demonstrate how multiomic mapping can predict transcription factor regulatory outputs associated with lens pathology.

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The Epigenetic Regulation of Epithelial-to-Fiber Cell Differentiation Revealed for All to See

When studying genes exhibiting significantly variable expression during epithelial-to-fiber cell differentiation, the authors identified components of the Polycomb repressive complex 2 (PRC2) complex - a H3K27me3 writer and critical regulator of gene silencing during development (Chammas et al.). Overall, these analyses suggested the loss of PRC2 and a gain in the expression of the PRC2-modifying gene JARID2 during epithelial cell-to-fiber cell differentiation. To validate the critical function of PRC2 in cell fate control during lens development, the team turned to CUT&RUN analysis of the embryonic chicken lens to identify EZH2 and JARID2 binding sites and evaluate correlative H3K27me3 levels. CUT&RUN analysis revealed extensive colocalization of signals for EZH2, JARID2, and H3K27me3 across regions devoid of H3K4me3 (which marks active transcription), which represented promoter regions of genes encoding transcription factors or DNA-binding factors involved in fate determination. These findings suggested that PRC2 controls differentiation and suppresses alternate cell fate emergence during lens development.

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Looking into FGF's Role of FGF in Lens Cell Fate and Exploring Cataract-associated Loci

Given the results achieved and the desire to gain in vivo mechanistic insights into signaling pathways at single-cell resolution, the team assessed the impact of fibroblast growth factors (FGF) signaling (Lovicu and McAvoy) on lens development by implanting beads delivering FGF2 to the developing lens before performing snRNA-seq. Results at the cell level suggested that FGF2 hyperstimulation increased intermediate cell frequency and drove fiber cell differentiation, while transcriptomics level data revealed the upregulation of genes related to FGF signaling (as expected) alongside the activation of fiber cell markers and the induction of MAF network genes. Finally, the team evaluated any overlap between genes identified in their study and those linked to cataract formation or lens biology/pathology to identify new proteins that may play a role in lens development, homeostasis, and pathology. Of the 72 identified genes linked to cataract formation and serve as cell population markers in the developing lens, 15 genes represented MAF targets and 80% were expressed by fiber cells.

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The Importance of Multiomic Analyses and CUT&RUN

The multiomic analyses carried out by the authors have provided us with a much clearer view of lens tissue development and defined those transcription factors and regulatory elements critical to the lens. Of significant note, the authors also highlighted the general importance of applying CUT&RUN analysis when profiling the complex features of DNA-binding proteins and transcription regulators in cells and tissues. What other model systems can multiomic analyses and CUT&RUN assays help to decipher?

For more on how multiomic analyses and CUT&RUN have helped us to see lens development and disease more clearly, see Development, January 2024.

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About the author

Stuart P. Atkinson, Ph.D.

Stuart was born and grew up in the idyllic town of Lanark (Scotland). He later studied biochemistry at the University of Strathclyde in Glasgow (Scotland) before gaining his Ph.D. in medical oncology; his thesis described the epigenetic regulation of the telomerase gene promoters in cancer cells. Following Post-doctoral stays in Newcastle (England) and Valencia (Spain) where his varied research aims included the exploration of epigenetics in embryonic and induced pluripotent stem cells, Stuart moved into project management and scientific writing/editing where his current interests include polymer chemistry, cancer research, regenerative medicine, and epigenetics. While not glued to his laptop, Stuart enjoys exploring the Spanish mountains and coastlines (and everywhere in between) and the food and drink that it provides!

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