Ribosomal DNA and the rDNA-binding protein Indra mediate non-random sister chromatid segregation in Drosophila male germline stem cells
Preprint posted on 16 December 2018 https://www.biorxiv.org/content/early/2018/12/16/498352?%3Fcollection=
Sisters are not always identical: Chromosomal immortality in germline stem cells rests on rDNA loci and their binding partner Indra
Selected by Maiko Kitaoka
Categories: cell biology, genetics
Background
In a canonical cell cycle, replicated sister chromatids are assumed to be identical and segregated randomly into daughter cells. However, sisters can be epigenetically distinct and can undergo non-random segregation. This has thought to contribute to asymmetric cell divisions, particularly in stem cells where one daughter maintains a stem cell state and the other can differentiate.
Here, Watase and Yamashita use the Drosophila male germline stem cells to investigate the non-random sister segregation of X and Y chromosomes. The group has previously shown that autosomes segregate randomly, while X and Y chromosomes do not, but how and why this phenomenon occurs is still unknown.
Key findings
The authors first identified ribosomal DNA (rDNA) to be a key locus on the X and Y chromosomes. D. melanogaster autosomes do not contain any rDNA loci and also segregate randomly, suggesting that this feature on the sex chromosomes is an important aspect. Deletion strains with no rDNA loci on the X or Y chromosomes showed random sister chromatid segregation, demonstrating the requirement of rDNA loci for non-random segregation.
Since rDNA loci are very large and contain several individual and distinct elements, the authors compared D. melanogaster to D. simulans, whose Y chromosome still segregates non-randomly despite lacking several rDNA elements. Interestingly, both species had intergenic spacer sequence (IGS) repeats, so they attempted to identify any potential IGS-binding proteins by mass spectrometry. This allowed them to uncover 18 proteins enriched for IGS sequence binding, including an uncharacterized zinc finger protein. They named the gene indra after a Hindu god who lost immortality. Consistent with its proposed role with rDNA, Indra localizes to the nucleolus and to the rDNA loci on metaphase chromosomes.
In characterizing indra’s function, Watase and Yamashita discovered that RNAi knockdown of indra led to random sister chromatid segregation. A stronger loss of indra in the germline revealed severe fertility defects due to the rapid loss of germ cells (and loss of cellular immortality). Using DNA FISH, they found that X and Y chromosomes frequently have inter-homolog exchange at the rDNA loci when indra is depleted, indicating indra’s role in preventing recombination between the sex chromosomes. This could be accomplished by preventing DNA double stranded breaks at rDNA loci or by encouraging sister chromatid DNA repair, rather than homolog repair.
Interestingly, when indra is depleted from male germline, progeny exhibited a bobbed phenotype, where the stripes on the adult fly’s back are not continuous and straight across, which has been shown to be a hallmark of rDNA insufficiency. This further implicates the role of rDNA, so how does Indra help to maintain rDNA copy number? Unequal sister chromatid exchange is a proposed mechanism for rDNA copy number expansion, where one chromatid gains copy number at the expense of the other. The authors propose that non-random sister chromatid segregation may reflect non-random segregation of higher vs. lower rDNA copy number after unequal sister chromatid exchange. While they could not measure the absolute copy number at rDNA loci, the authors observed multiple sister chromatid exchanges at rDNA loci in indra-depleted GSCs, potentially equalizing the rDNA copy number between sister chromatids. This indicates that indra limits the number of sister chromatid exchanges, pointing to a mechanism where indra acts to ensure that unequal exchange is productive and allows for unequal expansion of rDNA in the stem cell.
To conclude, Watase and Yamashita have discovered a mechanistic explanation to non-random sister chromatid segregation in asymmetric cell division, proving not only the importance of rDNA loci and the newly-named IGS binding protein Indra but also shedding light on the unique identities of sister chromatids. This exciting work opens many more questions for future investigation. Most immediately, it would be interesting to see whether an Indra binding site/IGS repeats are sufficient to induce non-random segregation on the D. melanogaster autosomes. Although, given that the rDNA locus is already very large, artificially adding it to autosomes may prove to be a technical challenge. Second, and more physiologically, what advantage do GSCs maintain by inheriting the sister chromatid with an expanded rDNA copy number? Perhaps the ability of GSCs to undergo repeated rounds of asymmetric cell division may contribute to this, since they would add rDNA copies gradually over time instead of all at once. Finally, the mass spectrometry pull-down experiment identified several other proteins as well that bound to IGS repeats. While some candidates are probably essential for cell survival, it will be interesting to investigate these further and see how they also interact with the rDNA loci and/or Indra.
Questions for the authors
The D. simulans Y chromosome has the Indra binding site and IGS repeats, but no other rDNA loci elements, so do they still show rDNA expansion? If not, how might the sister chromatids segregate non-randomly without the unequal rDNA expansion?
Can you speculate on Indra’s mechanism of action after binding to IGS repeats? How does the binding prevent inter-homolog exchange and/or multiple sister chromatid exchanges?
In somatic stem cells, do the sex chromosomes segregate non-randomly and the autosomes randomly as well?
Posted on: 20 December 2018
doi: https://doi.org/10.1242/prelights.6514
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