CENP-C functions in centromere assembly, the maintenance of CENP-A asymmetry and epigenetic age in Drosophila germline stem cells

Abstract

Germline stem cells divide asymmetrically to produce one new daughter stem cell and one daughter cell that will subsequently undergo meiosis and differentiate to generate the mature gamete. The silent sister hypothesis proposes that in asymmetric divisions, the selective inheritance of sister chromatids carrying specific epigenetic marks between stem and daughter cells impacts cell fate. To facilitate this selective inheritance, the hypothesis specifically proposes that the centromeric region of each sister chromatid is distinct. In Drosophila germ line stem cells (GSCs), it has recently been shown that the centromeric histone CENP-A (called CID in flies)-the epigenetic determinant of centromere identity-is asymmetrically distributed between sister chromatids. In these cells, CID deposition occurs in G2 phase such that sister chromatids destined to end up in the stem cell harbour more CENP-A, assemble more kinetochore proteins and capture more spindle microtubules. These results suggest a potential mechanism of 'mitotic drive' that might bias chromosome segregation. Here we report that the inner kinetochore protein CENP-C, is required for the assembly of CID in G2 phase in GSCs. Moreover, CENP-C is required to maintain a normal asymmetric distribution of CID between stem and daughter cells. In addition, we find that CID is lost from centromeres in aged GSCs and that a reduction in CENP-C accelerates this loss. Finally, we show that CENP-C depletion in GSCs disrupts the balance of stem and daughter cells in the ovary, shifting GSCs toward a self-renewal tendency. Ultimately, we provide evidence that centromere assembly and maintenance via CENP-C is required to sustain asymmetric divisions in female Drosophila GSCs.

Fig 1. CENP-C is assembled between S-phase and G₂/prophase in female GSCs.

(A) Schematic of the Drosophila ovary (created by B. L. Carty), composed of 16 ovarioles (one ovariole is highlighted in grey) organised into developing egg chambers. The GSC niche is located in the anterior-most chamber of the ovariole, the germarium (boxed). (B) Schematic of the GSC niche and 2- and 4-cell cysts in the germarium. G₂/prophase GSCs can be identified with a round spectrosome attached to the cap cells. CB = cystoblast, CC = cystocyte. (C-G’) Immunofluorescent image of a wild type GSC (circled) in S-phase with a bridged spectrosome (white arrow) (G) and in G₂/prophase with a round spectrosome (white arrow) (G’) stained with DAPI (cyan), EdU (blue), spectrosome (1B1, red) and CENP-C (yellow). The circled GSC is a projection of z-stacks that displays the spectrosome morphology (round or bridged) and all centromere foci of that specific cell. (H) Quantitation of total CENP-C fluorescent intensity (integrated density) in GSCs at S-phase and G₂/prophase. ***p<0.001. Scale bar = 5 μm. Error bars = Standard Error of the Mean (SEM).

Fig 2. CENP-C is required for CID assembly in GSCs.

(A-E’) nanos-GAL4, (F-J’) CENP-C RNAi, (L-P’) HA-CENP-C and (R-V’) HA-CENP-C; CENP-C RNAi (rescue) stained with DAPI (cyan), EdU (blue), 1B1 (red) and CID (yellow). S-phase GSCs (A-E, F-J, L-P, R-V) are positive for EdU, contain a bridge spectrosome and clustered centromeres. G₂/prophase GSCs (A’-E’, F’-J’, L’-P’, R’-V’) are EdU negative, contain a round spectrosome and dispersed centromeres. The circled GSC is a projection of z-stacks that displays the spectrosome morphology (round or bridged) and all centromere foci of that specific cell. * denotes cap cells. Scale bar = 5 μm. Quantitation of total CID fluorescent intensity (integrated density) in GSCs at S-phase and G₂/prophase in nanos-GAL4 and (K) CENP-C RNAi, (Q) HA-CENP-C and (W) HA-CENP-C; CENP-C RNAi. ***p<0.001, **p<0.01, ns = non-significant. Error bars = SEM.

Fig 3. CENP-C is asymmetrically distributed in mitosis and its depletion enhances the asymmetric CID distribution between GSCs and CBs at S-phase.

Control (nanos-GAL4) GSC at anaphase (A-D) and telophase (A’-D’) of mitosis stained for H3S10P (red), CENP-C (yellow), and DAPI (cyan). Values indicate fold differences in CENP-C intensities between GSC and CB centromeres. (EI-V) nanos-GAL4, (FI-V) CENP-C RNAi, (GI-V) HA-CENP-C and (HI-V) HA-CENP-C; CENP-C RNAi (rescue) stained with DAPI (cyan), EdU (blue), 1B1 (red) and CID (yellow). S-phase GSCs and CBs are positive for EdU, contain a bridge spectrosome and clustered centromeres. Dashed white line outlines GSC/CB pairs. Images are projections of z-stacks that display the bridged spectrosome morphology and all centromere foci of each GSC/CB pair. * denotes cap cells. Scale bar = 5 μm. Quantitation of the ratio of total CID fluorescent intensity (integrated density) between GSC/CB S-phase pairs in nanos-GAL4 and (I) CENP-C RNAi, (J) HA-CENP-C and (K) HA-CENP-C; CENP-C RNAi (rescue). Each point represents the ratio of total CID between GSC versus its corresponding CB. ns = non-significant. *p<0.05. Error bars = SEM.

Fig 4. CENP-C depletion disrupts GSC proliferation and maintenance over time.

(A-D) Characterisation of the phenotypes arising in CENP-C depleted germaria. (A, A’) Normal germarium are healthy with the expected lineage of germ cysts and spectrosome/fusome development. (B, B’) Germline tumours are characterised by an increased number of germ cells (GSCs, CBs, cysts) in the germaria, often displaced from their normal position with abnormal spectrosome/fusome morphology. (C, C’) The differentiation defect is characterised by a pool of GSCs/CBs in the apical end of the germaria, separated from later stage developing cysts. (D, D’) GSC loss is characterised by the absence of GSCs (and often CBs and early germ cysts) at the apical end of the germarium. * denotes cap cells. Scale bar = 20 μm. Continued egg chamber development in CENP-C RNAi ovarioles displaying normal germaria (E), germline tumours (F), differentiation defects (G) or GSC loss (H). Scale bar = 20 μm. (I) Quantitation of the frequency of the above phenotypes observed in germaria at 5-days (5d) and 10-days (10d) post-eclosion, and in the HA-CENP-C; CENP-C RNAi rescue at 5-days (5d) post-eclosion. Charts each represent 3 biological replicates (50 germaria analysed per replicate).

Fig 5. CID and CENP-C level is reduced in aged GSCs and CENP-C depletion accelerates CID loss.

(A-C) Wild type germaria (5-, 10- and 20-day old) stained with DAPI (cyan), 1B1 (red) and CID (yellow) or (E-G) CENP-C (yellow). GSCs are boxed and inset. * denotes cap cells. Scale bar = 10 μm. Quantitation of total CID (D) or CENP-C (H) integrated density in wild type GSCs at 5-, 10- and 20-days post eclosion. *p<0.05, ***p<0.001, ns = non-significant. Error bars = SEM. (I-L) Germaria of nanos-GAL4 (5d, 10d) and CENP-C RNAi (5d, 10d differentiation defect phenotype) stained with DAPI (cyan), 1B1 (red) and CID (yellow). GSCs are boxed and inset. * denotes cap cells. Scale bar = 10 μm. (M) Quantitation of total CID integrated density per GSC in nanos-GAL4 (5d, 10d), CENP-C RNAi (5d, 10d differentiation defect phenotype). *p<0.05, ****p<0.0001. Error bars = SEM.

Fig 6. CENP-C depletion shifts GSCs toward a self-renewal tendency.

(A-F) nanos-GAL4 (5d, 10d), CENP-C RNAi (5d, 10d), HA-CENP-C (5d) and HA-CENPC;CENPC RNAi rescue (5d) germaria and (G) nanos-GAL4; tubGAL80ts (15d), (H) CENP-C RNAi; nanos-GAL4; tubGAL80ts (15d) germaria stained with DAPI (cyan), SEX-LETHAL (SXL, red) and pMad (yellow). Scale bar = 10 μm. * denotes cap cells. White dashed circles highlight SXL or pMad positive cells. Images are projections of z-stacks that capture total pMad/SXL signal per germarium. (I) Quantitation of the number of pMad positive (yellow) and SXL positive (red) cells per germarium (n = 40–45). (J) Ratio of the number of SXL:pMad positive cells per germarium. **** p<0.0001, ** p<0.01. ns = non-significant. Error bars = SEM.