Chromatin Structure of Ribosomal RNA Genes in Dipterans and Its Relationship to the Location of Nucleolar Organizers

Abstract

Nucleoli, nuclear organelles in which ribosomal RNA is synthesized and processed, emerge from nucleolar organizers (NORs) located in distinct chromosomal regions. In polytene nuclei of dipterans, nucleoli of some species can be observed under light microscopy exhibiting distinctive morphology: Drosophila and chironomid species display well-formed nucleoli in contrast to the fragmented and dispersed nucleoli seen in sciarid flies. The available data show no apparent relationship between nucleolar morphology and location of NORs in Diptera. The regulation of rRNA transcription involves controlling both the transcription rate per gene as well as the proportion of rRNA genes adopting a proper chromatin structure for transcription, since active and inactive rRNA gene copies coexist in NORs. Transcription units organized in nucleosomes and those lacking canonical nucleosomes can be analyzed by the method termed psoralen gel retarding assay (PGRA), allowing inferences on the ratio of active to inactive rRNA gene copies. In this work, possible connections between chromosomal location of NORs and proportion of active rRNA genes were studied in Drosophila melanogaster, and in chironomid and sciarid species. The data suggested a link between location of NORs and proportion of active rRNA genes since the copy number showing nucleosomal organization predominates when NORs are located in the pericentric heterochromatin. The results presented in this work are in agreement with previous data on the chromatin structure of rRNA genes from distantly related eukaryotes, as assessed by the PGRA.

Chromosomal organization of the ribosomal RNA genes in the genus Chironomus (Diptera, Chironomidae)

“…The share of transcriptionally active copies in the population of ribosomal genes is tissue-specific, amounting to 80% in the fat body cells, to 50% in the salivary glands, and only 20% in the Malpighian tube cells (Sanz et al 2007). An analogous ratio is observed in the Chironomus tentans salivary gland cells, where 40% of the ribosomal genes are in a transcriptionally active state (Madalena et al 2012). Since silver staining predominantly detects active NORs, the variation in NOR number observed in chironomids using this technique may be actually determined by the variation in the transcriptional activity of their ribosomal genes.…”

Dramatic nucleolar dispersion in the salivary gland of Schwenkfeldina sp. (Diptera: Sciaridae)

“…Heterochromatin may also appear in the form of chromosome breakpoints as a result of local DNA under-replication. As seen in R. americana , T. pubescens as well as in many organisms, heterochromatin is in addition characterised by the enrichment with methylation of histone H3 in lysine 9 (H3K9Me1-3) 26 , 27 . In Schwenkfeldina sp., rather than lack of banding pattern in condensed pericentric heterochromatin, its occurrence is immediately suggested by polytene chromosome breakpoints localised in several interstitial regions.…”

“…Also, the polytene chromosome structure of R. americana is well defined with particular regard to the pericentric heterochromatin. These regions are distinguished by a significant enrichment with H3K9Me1-3 visualised as strong signals from the antibody binding ( 26 , 27 ; Fig. S12 ).…”

Beyond DNA puffs: What can we learn from studying sciarids?

“…Not all rRNA encoding genes in the NOR are transcribed at the same time, or in the same ratio, due either to differences in transcriptional rates per gene or to epigenetic silencing of rDNA units. PGRA (psoralen gel retarding assay) was employed to estimate the ratio of both active and inactive rRNA gene copies in five genera from three dipteran families and revealed a relationship between chromosomal location of NORs and the proportion of active rRNA genes (Madalena et al ., ). In Chironomidae, about 40% of the rDNA copies, which reside in euchromatic regions, are structured as transcriptionally active chromatin as revealed by PGRA.…”

“…In Chironomidae, about 40% of the rDNA copies, which reside in euchromatic regions, are structured as transcriptionally active chromatin as revealed by PGRA. In contrast, in the salivary gland polytene chromosomes of both D. melanogaster and the three sciarid species that were investigated, in which the rDNA repeats are located in heterochromatic regions, the copy number of transcribing rDNA estimated through PGRA is lower than 10% (Madalena et al ., ). Further studies in sciarids could contribute to the understanding of how transcriptionally active rRNA genes are selected and how they escape inactivation in heterochromatic domains.…”