Our epigenomics research uses novel tools to understand genome regulation in a cell type specific fashion within the CNS.
DNA modifications in specific CNS cell types
Cellular identity is determined partly by cell type-specific epigenomic profiles that regulate gene expression. In neuroscience, there is a pressing need to isolate and characterize the epigenomes of specific CNS cell types in health and disease. We have developed a variety of in vivo tagging mouse models for paired isolation of cell type-specific DNA and RNA without cell sorting. Comparing data on DNA modifications from neurons, microglia and astrocyte models, each cell type demonstrates different genome-wide levels of mCG, hmCG, and mCH that are reproducible across analytical methods. However, modification-gene expression relationships are conserved across cell types. Enrichment of differential modifications across cell types in gene bodies and distal regulatory elements, but not proximal promoters, highlights epigenomic patterning in these regions as potentially greater determinants of cell identity. These findings also demonstrate the importance of differentiating between mC and hmC in neuroepigenomic analyses, as up to 30% of what is conventionally interpreted as mCG can be hmCG, which often has a different relationship to gene expression than mCG.
Native detection of DNA modifications with nanopore long-read sequencing. Nanopore long-read sequencing was performed on INTACT-isolated high molecular weight gDNA from neurons, astrocytes, and microglia (n = 2/group). Native mCG and hmCG calling was performed to obtain total whole genome %mCG A and %hmCG B. %modC (%mCG or %hmCG) was plotted across chromosome 15 C to demonstrate modification differences between cell types and reproducibility across biological replicates. Modification values were smoothed in CpG-only coordinate space (One-way ANOVA with Tukey’s multiple comparisons test, *p < 0.05, **p < 0.01)
From Differential usage of DNA modifications in neurons, astrocytes, and microglia. Tooley KB, Chucair-Elliott AJ, Ocañas SR, Machalinski AH, Pham KD, Hoolehan W, Kulpa AM, Stanford DR, Freeman WM. Epigenetics Chromatin. 2023; 16(1):45.
Epigenetics
Our scientific premise is that changes in genomic patterns of DNA modifications – methylation (mC) and hydroxymethylation hmC – are central regulators of altered genome function and gene expression with aging. Our studies are determining changes in mC and hmC patterns with aging in specific hippocampal cell populations (neurons, microglia, and astrocytes) across the lifespan in both female and male mice, testing whether a ‘youthful’ modification pattern can be retained through caloric restriction, and identifying how altered DNA modification patterns regulate gene expression. The long-term goal of this research is to prevent or reverse age-related changes in DNA modification patterns to maintain healthy brain function and prevent neurodegenerative diseases such as Alzheimer’s.
Age-related differentially methylated CGs (aDMCGs) in male and female hippocampus. aDMCGs are presented by chromosomal location in males (top) and females (bottom) and the difference in mean methylation (Old-Young) on the inner axis. Each point represents one aDMCG meeting false discovery rate (FDR) cut off of q < 0.05. Sites ≥ 5 % absolute change in methylation with age (hypermethylated) are in green, while in red are sites ≤ -5% change with age (hypomethylated). These sites were used in a principle component analysis of the samples. Samples clustered by group and separated by age in the 1st component and by sex in the 2nd component.
From: Sexually divergent DNA methylation patterns with hippocampal aging. Masser DR, Hadad N, Porter HL, Mangold CA, Unnikrishnan A, Ford MM, Giles CB, Georgescu C, Dozmorov MG, Wren JD, Richardson A, Stanford DR, Freeman WM. Aging Cell. 2017 Dec;16(6):1342-1352.