Centromeres are epigenetically defined by CENP-A–containing chromatin and are essential for cell division. Previous studies suggest asymmetric inheritance of centromeric proteins upon stem cell division; however, the mechanism and implications of selective chromosome segregation remain unexplored. We show that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres after replication and before segregation. Specifically, CENP-A deposition is promoted by CYCLIN A, while excessive CENP-A deposition is prevented by CYCLIN B, through the HASPIN kinase. Furthermore, chromosomes inherited by GSCs incorporate more CENP-A, making stronger kinetochores that capture more spindle microtubules and bias segregation. Importantly, symmetric incorporation of CENP-A on sister chromatids via HASPIN knockdown or overexpression of CENP-A, either alone or together with its assembly factor CAL1, drives stem cell self-renewal. Finally, continued CENP-A assembly in differentiated cells is nonessential for egg development. Our work shows that centromere assembly epigenetically drives GSC maintenance and occurs before oocyte meiosis.

Tandemly-repeated DNAs, or satellites, are enriched in heterochromatic regions of eukaryotic genomes and contribute to nuclear structure and function. Some satellites are transcribed, but we lack direct evidence that specific satellite RNAs are required for normal organismal functions. Here, we show satellite RNAs derived from AAGAG tandem repeats are transcribed in many cells throughout Drosophila melanogaster development, enriched in neurons and testes, often localized within heterochromatic regions, and important for viability. Strikingly, we find AAGAG transcripts are necessary for male fertility, and that AAGAG RNA depletion results in defective histone-protamine exchange, sperm maturation and chromatin organization. Since these events happen late in spermatogenesis when the transcripts are not detected, we speculate that AAGAG RNA in primary spermatocytes ‘primes’ post-meiosis steps for sperm maturation. In addition to demonstrating essential functions for AAGAG RNAs, comparisons between closely related Drosophila species suggest that satellites and their transcription evolve quickly to generate new functions.

Author for correspondence elaine.dunleavy@nuigalway.ie; phone number: +353 (0)91 494046 SUMMARY Centromeres, chromosomal loci essential for genome integrity, are epigenetically defined by CENP-Acontaining chromatin. Recent studies suggest that parental CENP-A is asymmetrically distributed upon stem cell asymmetric division. However, a direct link between centromeres and stem cell identity has not been demonstrated. We show that Drosophila female germline stem cells (GSCs) and neuroblasts assemble centromeres between G2-phase and prophase, requiring CYCLIN A. Intriguingly, chromosomes that will be inherited by GSCs incorporate more CENP-A and capture more spindle fibers at pro-metaphase.Furthermore, over-expression of CAL1 (Drosophila CENP-A assembly factor) causes GSC-like tumours, while over-expression of both CENP-A and CAL1 promotes stem cell self-renewal. Finally, once centromeres have been assembled in GSCs, continued CENP-A assembly is not required in differentiating cells outside of the niche and CAL1 becomes dispensable. According to our results CENP-A regulates stem cell identity/maintenance. Moreover, crucial centromere assembly occurs in the niche prior to oocyte meiosis.

The centromere is the constricted chromosomal region required for the correct separation of the genetic material at cell division. The kinetochore protein complex assembles at the centromere and captures microtubules emanating from the centrosome to orchestrate chromosome segregation in mitosis and meiosis. Asymmetric cell division (ACD) is a special type of mitosis that generates two daughter cells with different fates. Epigenetic mechanisms operating at the centromere have been proposed to contribute to ACD. Recent studies have shown that an asymmetric distribution of CENP-A—the centromere-specific histone H3 variant—between sister chromatids can bias chromosome segregation in ACD. In stem cells, this leads to non-random sister chromatid segregation, which can affect cell fate. These findings support the ‘silent sister' hypothesis, according to which the mechanisms of ACD are epigenetically regulated through centromeres. Here, we review the recent data implicating centromeres in ACDs and cell fate in Drosophila melanogaster female and male germline stem cells.

Stem cells can undergo asymmetric cell division (ACD) giving rise to one new stem cell and one differentiating daughter cell. In Drosophila germline stem cells (GSCs), the centromeric histone CENP-A - CID in flies - is asymmetrically distributed between sister chromatids such that chromosomes that end up in the GSC harbour more CID at centromeres. A model of ‘mitotic drive’ has been proposed in GSCs such that stronger and earlier centromere and kinetochore interactions with microtubules bias sister chromatid segregation. Here we show that in Drosophila males, centromere proteins CID, CAL1 and CENP-C are asymmetrically distributed in newly divided GSCs and daughter cells in S-phase. We find that CID overexpression, overexpression of CID together with CAL1 or CENP-C depletion disrupts CID asymmetry, with an increased pool of GSCs relative to daughter cells detectable in the niche. This result suggests a shift toward GSC self-renewal rather than differentiation, important for maintaining tissue homeostasis. Overexpression of CAL1 does not disrupt asymmetry, but instead drives germ cell proliferation in the niche. Our results in male GSCs are comparable to female GSCs, indicating that despite differences in signaling, organisation and niche composition, effects of centromere proteins on GSC maintenance are conserved between the sexes.

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