DNA methylation-based age prediction and sex-specific epigenetic aging in a lizard with female-biased longevity

Sex differences in lifespan are widespread across animal taxa, but their causes remain unresolved. Alterations to the epigenome are hypothesized to contribute to vertebrate aging, and DNA methylation-based aging clocks allow for quantitative estimation of biological aging trajectories. Here, we investigate the influence of age, sex, and their interaction on genome-wide DNA methylation patterns in the brown anole (Anolis sagrei), a lizard with pronounced female-biased survival and longevity. We develop a series of age predictor models and find that contrary to our predictions, rates of epigenetic aging were not slower in female lizards. However, methylation states at loci acquiring age-associated changes appear to be more “youthful” in young females, suggesting that female DNA methylomes are preemptively fortified in early life in opposition to the direction of age-related drift. Collectively, our findings provide new insights into epigenetic aging in reptiles and suggest that early-life epigenetic profiles are more informative than rates of change over time for predicting sex biases in longevity.

Methods
Organism: Anolis sagrei

Tissue: Red blood cells

Genotype: WT

Sampling protocol: Animals were immediately euthanized by decapitation. Blood was collected from the trunk and stored on ice prior to separation of plasma and blood cells by centrifugation. All tissue samples and blood components were snap-frozen in liquid nitrogen.

Husbandry protocol: Animals used in this study were captive-bred descendants of stock originally collected from native A. sagrei populations near Georgetown, Great Exuma, in the Commonwealth of the Bahamas (23º29’N, 75º45’W) and imported under permits from the Bahamas Environment, Science, and Technology Commission, the Bahamas Ministry of Agriculture, and the United States Fish and Wildlife Service. Animals were housed at the University of Virginia and all procedures were approved under UVA ACUC protocol 3896. The exact hatch date of each captive-bred animal was recorded such that age was known with certainty.

Extracted molecule: genomic DNA

Extraction protocol: DNA was isolated from the blood cell pellet (majority nucleated red blood cells) using Qiagen’s PureGene CELL extraction kit (method described in Lindsey and Schwartz 2022, "DNA Isolation from Reptile Blood using Gentra Puregene (Qiagen) DNA Isolation Kit" published on protocols.io).

Sequencing protocol: Libraries were prepared using the NEBNext Enzymatic Methyl-seq kit from New England BioLabs with 200 ng genomic DNA as input. Prior to enzymatic conversion, libraries were sheared to a target fragment size of 300 bp using a Covaris sonication instrument. After assessing library qualities using Bioanalyzer and Qubit instruments, 40 enzymatically converted libraries were sequenced on two lanes of an Illumina Novaseq 6000 instrument at the University of Florida’s Interdisciplinary Center for Biotechnology and Research to yield an average of ~120 million paired-end, 150bp reads per sample.

Bioinformatic protocol: Reads were trimmed and quality-checked with Trim_Galore!, a wrapper script for CutAdapt and FastQC using a PHRED score cutoff of 20 in addition to a 4 bp hard clip on both read ends in order to reduce methylation bias. Alignment to the A. sagrei reference genome, AnoSag2.1 (Refseq: GCF_025583915.1), was conducted using Bismark with default parameters, and duplicate reads resulting from PCR bias were removed using the deduplicate_bismark tool. Alignment files were sorted and converted to .sam files using SAMtools. Three samples were excluded from downstream analysis due to low alignment rates (<5%), leaving a total of 37 samples. SAM files were imported into R and converted to a methylation matrix and filtered using the R package methylKit.