Localization of rDNA-associated retrotransposons refines the heterochromatin map of the X chromosome in Drosophila melanogaster.

Abstract

Introduction: R1 and R2 retrotransposons specifically integrate into 28S rRNA genes, thereby disrupting many rDNA units within the nucleolar organizer region (NOR) of Drosophila. However, they also appear to play mutualistic roles, contributing to the maintenance of rDNA copy number and the regulation of nucleolar dominance. In addition to their presence in nucleolar rDNA, R1 elements are strongly enriched in the pericentromeric heterochromatin of the X chromosome, located distal to the NOR. This enrichment coincides with several enigmatic genetic phenomena-such as the ABO and cr phenotypes-whose molecular basis remains poorly understood. Notably, this region is one of the least characterized domains of the D. melanogaster genome, lying outside the reference assembly and unresolved in metaphase chromosome preparations.

Methods: We performed cytological mapping of R1 and R2 retrotransposons in D. melanogaster heterochromatin using polytene chromosomes from Rif11 mutant, which suppresses under-replication of all types of heterochromatic sequences. These were combined with classical eu-heterochromatic inversions of the X chromosome.

Results and conclusions: We identified distinct clusters of both R1 and R2 elements within the X chromosome heterochromatin outside the NOR. R1 elements are highly enriched in the region between the heterochromatic Stellate (hSte) gene cluster and the NOR. This zone exhibits a unique response to Su(var)3-9 mutations, characterized by pronounced decondensation and the formation of a pseudo-puff. Proximal to the R1-enriched domain and adjacent to hSte cluster, we observed a region enriched in R2 elements. The edges of the NOR also show R2 enrichment, likely corresponding to intra-nucleolar domains that accumulate transcriptionally inactive rDNA units. In contrast, nucleolar R1 elements-which also mark inactive rDNA units-are more evenly distributed across the entire NOR. Based on these findings, we propose a refined cytological map of X chromosome heterochromatin in D. melanogaster.