Lineage-Traced Single-Cell Analysis of Epigenetic Clocks

/ ABSTRACT It has long been known that DNA methylation profiles in the genome progressively change with increasing age. In the past decade, this knowledge has been leveraged to create epigenetic clock models calibrated to organismal chronological age or cellular replicative history. Environmental exposures, stress conditions, and disease states have been shown to accelerate estimated model age beyond actual chronological age. However, the mechanisms underlying this accelerated biological aging are not well understood. The discrete binary nature of DNA methylation at select CpG sites implies that quantitative shifts in DNA methylation levels reflect changes in underlying cellular heterogeneity in the sample. Bulk analysis of epigenetic clocks cannot distinguish between age-associated changes in cell populations, versus progressive DNA methylation changes within most cells in a population. Resolving this distinction, and understanding the biological mechanisms underlying epigenetic clock research would benefit from the ability to study epigenetic clocks at the single-cell level. However, existing technology for single-cell DNA methylation profiling provides too sparse genomic coverage to allow direct application of epigenetic clocks. We have developed advanced new single-cell whole- genome bisulfite sequencing technology that provides sufficiently deep genomic coverage to allow application of epigenetic clocks, and in-depth characterization of DNA methylation biology. Lineage tracing technology in mouse models has yielded new insights into stem-cell biology and the phylogenetic relationship among cells in vivo. Recently developed advanced lineage tracing technology employing CRISPR/Cas9-TdT cut and repair- generated unique barcodes has facilitated unprecedented high-resolution analysis of cell lineages. In the one Specific Aim for this project, we propose to use our advanced single-cell whole-genome bisulfite sequencing to characterize intercellular heterogeneity of epigenetic clocks in lineage-traced cells derived from the mouse colon and small intestine. We will use two pulsed inductions of barcode generation to both mark regional lineages in embryonic development and to secondarily mark postnatal intestinal crypt morphogenesis. We will collect and analyze single cells from the mouse colon and small intestine at four different ages to assess both within-mouse lineage relationships, as well as measure loss of barcode diversity as a measure of stem-cell exhaustion. Our high-coverage single-cell whole-genome bisulfite sequence analysis will allow us to both recover the unique lineage barcodes from each cell, as well as assess DNA methylation to apply available mouse epigenetic clocks, identify cell type by the analysis of hypomethylation of cell-type-specific enhancers, assess age-associated hypermethylation of polycomb repressive complex target genes, and measure replicative history by the degree of hypomethylation at late-replicating, lamina-attached regions of the genome. This study will provide a proof-of-principle for future studies that will explore the impacts of environmental interventions and genetic manipulation on biological aging and epigenetic clocks in various cell types. Project Number: 1R21AG087016-01A1 | Fiscal Year: 2025 | NIH Institute/Center: National Institute on Aging (NIA) | Principal Investigator: PETER LAIRD | Institution: VAN ANDEL RESEARCH INSTITUTE, GRAND RAPIDS, MI | Award Amount: $528,000 | Activity Code: R21 | Study Section: Genetic Variation and Evolution Study Section[GVE] View on NIH RePORTER: https://reporter.nih.gov/project-details/11233794