RNA-mediated epigenetic regulation of DNA copy number

Abstract

Increasing evidence suggests that parentally supplied RNA plays crucial roles during eukaryotic development. This epigenetic contribution may regulate gene expression from the earliest stages. Although present in a variety of eukaryotes, maternally inherited characters are especially prominent in ciliated protozoa, in which parental noncoding RNA molecules instruct whole-genome reorganization. This includes removal of nearly all noncoding DNA and sorting the remaining fragments, producing extremely gene-rich somatic genomes. Chromosome fragmentation and extensive replication produce variable DNA copy numbers in the somatic genome. Understanding the forces that drive and regulate copy number change is fundamental. We show that RNA molecules present in parental cells during sexual reproduction can regulate chromosome copy number in the developing nucleus of the ciliate Oxytricha. Experimentally induced changes in RNA abundance can both increase and decrease the levels of corresponding DNA molecules in progeny, demonstrating epigenetic inheritance of chromosome copy number. These results suggest that maternal RNA, in addition to controlling gene expression or DNA processing, can also program DNA amplification levels.

Fig. 1.

Microinjection of RNA increases chromosome copy number in sexual progeny. Southern analysis of whole-cell DNA isolated from pooled progeny of 40 cells (20 conjugating pairs) in each experiment injected (inj.) with TEBPβ sense (s) or antisense (as) RNA or both strands. Hybridization to a TEBPβ probe (lanes 1–5) shows increased levels of the WT TEBPβ chromosome in progeny of injected cells (lanes 3–5). Gray bars indicate relative amount of TEBPβ probe signal. Lanes 6 to 10 and 11 to 15 are hybridization to a TBE1 transposase and ActinI probe, respectively, as DNA loading controls, in addition to ethidium bromide staining (lanes 16–20) on a 0.9% agarose gel. ActinI hybridization and ethidium bromide staining indicate that the effect of TEBPβ RNA microinjection is sequence-specific, as it does not increase the levels of ActinI chromosomes or of macronuclear DNA in general.

Fig. 2.

Microinjection of RNA or DNA increases chromosome copy number in sexual progeny. Southern analysis of total DNA from progeny of cells injected with sense RNA copies of the complete ActinI nanochromosome. Lanes 1 to 9 and 28 to 36 contain DNA from the progeny of individually injected mating pairs hybridized to an ActinI probe. F₁ cells (lanes 3–7) show increased levels of the ActinI nanochromosome. F₂ cells (lanes 8 and 9) that were derived from the progeny of individual mating pairs isolated from F₁ cells also show higher amounts of the ActinI chromosome, although slightly less than their parents (lanes 3 and 4). Lanes 30 to 33 contain DNA from four different clonal cultures derived from noninjected parents. They do not exhibit differences in ActinI chromosome copy number. Lanes 34 to 36 contain DNA from the progenies of cells injected with the full-length synthetic DNA chromosome. Two show increased levels of ActinI chromosomes. To compare relative signal intensities on both blots, samples 1 and 4 (Left) were duplicated in lanes 28 and 29. Gray bars indicate relative levels of ActinI probe signal. TBE1 transposase probe provided a loading control (lanes 10–18 and 37–45) in addition to ethidium bromide staining (lanes 19–27 and 46–54). The ethidium staining also confirms that ActinI injection did not influence the overall levels of macronuclear DNA, helping to exclude non-sequence-specific effects.

Fig. 3.

RNAi during conjugation reduces chromosome copy number in progeny. Southern analysis of whole-cell DNA from progeny of cells treated with RNAi against pol-α (lanes 1–4) and TEBPα (lanes 5–8). RNAi during conjugation reduces the levels of corresponding chromosomes in sexual progeny (lanes 2 and 5). Gray bars indicate relative levels of pol-α and TEBPα probe signals. TBE1 transposase probe signal (lanes 9–12), as well as ethidium bromide staining of whole-cell DNA (lanes 13–16), provided a loading control.

Fig. 4.

Model for RNA-guided epigenetic inheritance of chromosome copy number in Oxytricha. During sexual conjugation, all DNA in the maternal somatic nucleus produces transcripts in approximately similar abundance to the chromosome copy number. After transport to the developing nucleus, the maternal RNAs guide DNA rearrangements, yielding a quantity of mature chromosomes proportional to the level of available templates, or alternatively, the template RNAs may protect correctly processed DNA molecules from degradation. Numbered DNA and RNA segments represent regions that are retained during genome rearrangement, whereas yellow boxes are deleted. Black boxes represent telomeres.