Zoller JA et al. (2023), DNA methylation clocks for clawed frogs reveal ...

XB-ART-59782

Geroscience 2023 Feb 04;461:945-960. doi: 10.1007/s11357-023-00840-3.

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DNA methylation clocks for clawed frogs reveal evolutionary conservation of epigenetic aging.

Zoller JA , Parasyraki E , Lu AT , Haghani A , Niehrs C , Horvath S .

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To address how conserved DNA methylation-based epigenetic aging is in diverse branches of the tree of life, we generated DNA methylation data from African clawed frogs (Xenopus laevis) and Western clawed frogs (Xenopus tropicalis) and built multiple epigenetic clocks. Dual species clocks were developed that apply to both humans and frogs (human-clawed frog clocks), supporting that epigenetic aging processes are evolutionary conserved outside mammals. Highly conserved positively age-related CpGs are located in neural-developmental genes such as uncx, tfap2d as well as nr4a2 implicated in age-associated disease. We conclude that signatures of epigenetic aging are evolutionary conserved between frogs and mammals and that the associated genes relate to neural processes, altogether opening opportunities to employ Xenopus as a model organism to study aging.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: atf5.2 clock cnot1 dcx dlx6 dnmt1 dnmt3a eed ehbp1 fbxl13 foxl1 gclm gria3 hibadh hoxa10 ikzf1 irx2 kmt2c mef2c metap1d miga1 mon2 nr4a2 olig3 pdlim5 pprc1 prox1 shox2 siae six2 skida1 sox4 tet2 tet3 tfap2d tshz3 uncx znf503

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	Fig. 1. Unsupervised hierarchical clustering of Xenopus tissues. The clustering height (y-axis) can be interpreted as distance based on pairwise correlation coefficients. Color bands underneath indicate clustering branch (corresponding to a height cut-off of 0.29 on the y-axis), frog species (blue, tropicalis; turquoise, laevis), tissue type (see the labels), age (red corresponds to old age), and sex (pink, female; blue, male; grey, unknown). Sex was unknown for samples where the animals were too young to determine their sex.
	Fig. 2. Cross-validation study of epigenetic clocks for Xenopus. Leave-one-sample-out estimate of DNA methylation age (y-axis, in units of years) versus chronological age (units of years) for A all Xenopus tissues (both species), B all tissues from young Xenopus (age < 2 years), D all tissues from X. laevis, E all tissues from X. tropicalis. C Leave-one-sample-out estimate of DNA methylation relative age (y-axis, values range from 0 to 1) versus relative age for all Xenopus tissues (both species). Relative age was determined by dividing chronological age (measured in years) by the maximum lifespan (also expressed in years). All clocks are pan tissue clocks, i.e., apply to all considered tissues Each panel reports the sample size (in parenthesis), correlation coefficient, median absolute error (MAE).
	Fig. 3. Cross-validation study of epigenetic clocks for Xenopus and humans. Ten-fold cross validation analysis of the human-clawed frog clocks for A, B chronological age and C, D relative age, respectively. A, C Human samples are colored in black and Xenopus samples are colored by species and tissue type, and analogous in B, D but restricted to Xenopus samples (colored by Xenopus tissue type). Relative age was determined by dividing chronological age (measured in years) by the maximum lifespan (also expressed in years). The relative age clock allows for alignment and biologically meaningful comparison between species with different lifespan (clawed frogs and humans). Each panel reports the sample size (in parenthesis), correlation coefficient, median absolute error (MAE)
	Fig. 4. EWAS of age in Xenopus. The top panels display Manhattan plots for EWAS of age A Stouffer’s method meta-analysis that combines B X. laevis, and C X. tropicalis across all tissue types. The red dash lines indicate suggestive levels of significance at P < 1.0E-05. CpGs are colored in red or blue for positive or negative age correlations, respectively. The y-axis displays -log base 10 transformed P-value and the x-axis displays chromosome number based on the X. tropicalis genome (v9.1.102). Chromosome KV denotes the alias names for the CpGs with unspecified chromosomes. D–F Display the scatter plots between frog EWAS of age and Eutherian EWAS of age, based on Z statistics. Each of the 4239 points in the scatter plot correspond to a CpG that is present on our mammalian array and maps to the X. tropicalis genome. The title presents the Pearson correlation and its P-value between the two EWAS Z statistics. The CpG cg17865363 exhibiting highly significant P-value in frog EWAS is annotated by its nearby gene sox4 and marked in purple. Labels are provided for the top 10 hypermethylated/hypomethylated CpGs according to the product of Z scores in x- and y-axis.
	Fig. 5. Genomic region-based GREAT functional enrichment analysis. GREAT functional enrichment analysis was based on the top 500 CpGs that increased or decreased with age from EWAS in (1) meta-analysis, (2) X. laevis, and (3) X. tropicalis, respectively (Suppl. Table 4). The background was based on the genomic regions of the 4239 mammalian CpGs and the assembly in hg19. The y-axis lists the name of a functional gene set/biological pathway, sorted by ontology and the most significant hypergeometric P-value within each ontology. The bar plots in the first column report the total number of genes at each studied gene set adjusted based on our background. The left and right panels of the x-axis list the enrichment results based on the top 500 CpGs with positive and negative age correlation. We list unadjusted hypergeometric P-value (number of overlap genes) at each cell, provided P < 0.1. The heatmap color codes -log10 (P-value). Abbreviations: BENPORATH_EED_TARGETS denotes “EED targets: genes identified by ChIP on chip as targets of the Polycomb protein EED (GeneID = 8726) in human embryonic stem cells.
	Fig. 6. Chromatin state analysis of age-related CpGs. The heatmap color-codes the hypergeometric overlap analysis between age-related CpGs (columns) and two groupings of CpGs (a) binding by Polycomb repressive complex 1 and 2 (PRC1, PRC2) defined based on ChipSeq datasets in ENCODE [64] and (b) universal chromatin states analysis [61], see the first two rows. The background is based on the 4239 mammalian CpGs that can map to the X. tropicalis genome (v9.1.102). The first column shows a bar plot that reports the proportion of CpGs bound by PRC2 that ranges from zero (RPC1) to one (PRC2). For each row (chromatin state or PRC annotation), the table reports odds ratios (OR) from hypergeometric test results for the top 500 CpGs that increased/decreased with age from meta-EWAS, X. laevis EWAS and X. tropicalis EWAS, respectively. The heatmap color gradient is based on -log10 (unadjusted hypergeometric P-value) multiplied by the sign of OR greater than one. Red colors denote OR greater than one in contrast with blue colors for OR less than one. Legend lists states based on their group category and PRC group. The y-axis lists chromatin states and PRC2 target sites. The left/right panel lists the results based on the top 500 CpGs with positive/negative age correlation. We display 16 universal chromatin states that show significant enrichment/depletion at P < 0.001 in any of the EWAS

References [+] :

Ammous, Epigenome-wide association study identifies DNA methylation sites associated with target organ damage in older African Americans. 2021, Pubmed