Genome-wide DNA methylation profiles and ribosomal DNA copy number at birth

Ribosomal DNA copy number (rDNAcn) and DNA methylation are important modulators of the human genome, both studied in relation to overall cellular function, biological ageing, and disease development. Despite the overlapping roles, their relationship remains poorly understood, especially in the early stages of life, characterized by rapid growth and high cellular demands. Even though previous studies have associated rDNA methylation with cancer and ageing, no study to date has examined the interplay between rDNAcn and whole-genome DNA methylation. In an epigenome-wide association study of 45S rDNAcn variation in 194 newborns, we show strong positive associations between rDNAcn and single DNA methylated CpGs, measured with the Illumina EPIC array. Out of the 122 Bonferroni-significant CpGs, 63.5% were also Bonferroni-significant in a replication cohort of 167 newborns, in which a second EWAS was conducted using DNA methylation data from the Illumina 450K array. The identified CpGs were dispersed over the autosomes and were not functionally related to the rDNA-forming nucleolar-associated domains. The top CpGs were annotated to genes (<i>GFI1, USP46</i>, <i>ABHD14B</i>, <i>CHL1</i>, <i>CGREF1</i>) that are functionally linked to cancer and cellular proliferation. In downstream analyses, the 122 rDNAcn-related CpGs revealed 31 differentially methylated regions and 253 nominally significant correlations with cord blood gene transcripts in an eQTM analysis. Pathway enrichment analyses showed an overrepresentation of the following pathways: 'RNA Polymerase III transcription' (R-HSA-76071, R-HSA-76046, R-HSA-74158, R-HSA-749476, R-HSA-73780, R-HSA-73980, R-HSA-76066, R-HSA-76061, hsa03020), 'cytosolic sensors of pathogen-associated DNA' (R-HSA-1834949), 'RNA polymerase II transcribes snRNA genes' (R-HSA-6807505), and 'translation initiation' (R-HSA-72613, R-HSA-72737). Our findings reveal a close link between rDNAcn variation and DNA methylation in early life. Disruptions in this interplay may influence cellular functions critical for early development, potentially shaping health and disease trajectories later in life.