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Parafibromin governs cell polarity and centrosome assembly in Drosophila neural stem cells [1]

['Qiannan Deng', 'Neuroscience', 'Behavioral Disorders Programme', 'Duke-Nus Medical School', 'Cheng Wang', 'Chwee Tat Koe', 'Jan Peter Heinen', 'Institute For Research In Biomedicine', 'The Barcelona Institute Of Science', 'Technology']

Date: 2022-10 

Neural stem cells (NSCs) divide asymmetrically to balance their self-renewal and differentiation, an imbalance in which can lead to NSC overgrowth and tumor formation. The functions of Parafibromin, a conserved tumor suppressor, in the nervous system are not established. Here, we demonstrate that Drosophila Parafibromin/Hyrax (Hyx) inhibits ectopic NSC formation by governing cell polarity. Hyx is essential for the asymmetric distribution and/or maintenance of polarity proteins. hyx depletion results in the symmetric division of NSCs, leading to the formation of supernumerary NSCs in the larval brain. Importantly, we show that human Parafibromin rescues the ectopic NSC phenotype in Drosophila hyx mutant brains. We have also discovered that Hyx is required for the proper formation of interphase microtubule-organizing center and mitotic spindles in NSCs. Moreover, Hyx is required for the proper localization of 2 key centrosomal proteins, Polo and AurA, and the microtubule-binding proteins Msps and D-TACC in dividing NSCs. Furthermore, Hyx directly regulates the polo and aurA expression in vitro. Finally, overexpression of polo and aurA could significantly suppress ectopic NSC formation and NSC polarity defects caused by hyx depletion. Our data support a model in which Hyx promotes the expression of polo and aurA in NSCs and, in turn, regulates cell polarity and centrosome/microtubule assembly. This new paradigm may be relevant to future studies on Parafibromin/HRPT2-associated cancers.

Human Parafibromin/Cell division cycle 73 (Cdc73)/hyperparathyroidism type 2 (HRPT2) is a tumor suppressor that is linked to several cancers, including parathyroid carcinomas and hyperparathyroidism–jaw tumor syndrome, head and neck squamous cell carcinomas, as well as breast, gastric, colorectal, and lung cancers [ 45 – 48 ]. Somatic mutations in parafibromin have been found in 67% to 100% of sporadic parathyroid carcinomas [ 45 ]. Parafibromin is part of a conserved polymerase-associated factor complex that primarily regulates transcriptional events and histone modification [ 49 , 50 ]. Hyrax (Hyx), Drosophila Parafibromin, is essential for embryonic and wing development and is known to positively regulate Wnt/Wingless signaling pathway in wing imaginal discs by directly interacting with β-catenin/Armadillo [ 51 ]. Human Parafibromin, but not yeast Cdc73, rescues defects in wing development and the embryonic lethality caused by hyx loss-of-function alleles [ 51 ], suggesting that Parafibromin functions during development are conserved across metazoans. Interestingly, Parafibromin is expressed in both mouse and human brains, including the cortex, basal ganglia, cerebellum, and the brainstem [ 52 ], suggesting that Parafibromin may play a role in central nervous system (CNS) functions. However, the specific functions of Parafibromin in the nervous system are not established. Here, we investigate the role of Parafibromin/Hyx in the asymmetric division of NSCs during Drosophila larval brain development.

The dysregulation of a few cell cycle regulators, such as Aurora-A kinase (AurA), Polo kinase (Polo), and Serine/Threonine protein phosphatase 2A (PP2A) results in disruption to NSC asymmetry and microtubule functions, leading to NSC overgrowth and brain tumor formation [ 27 , 29 , 35 , 37 – 43 ]. Moreover, ADP ribosylation factor like-2 (Arl2), a major regulator of microtubule growth, localizes Mini spindles (Msps)/XMAP215/ch-TOG and Transforming acidic coiled-coil containing (D-TACC) to the centrosomes to regulate microtubule growth and the polarization of NSCs [ 44 ].

The asymmetric division of stem cells is a fundamental strategy for balancing self-renewal and differentiation in diverse organisms including humans. The Drosophila neural stem cells (NSCs), also known as neuroblasts, have emerged as an excellent model for the study of stem cell self-renewal and tumorigenesis [ 1 – 5 ]. During asymmetric division, each NSC generates a self-renewing NSC and a neural progenitor that can produce neurons and glial cells [ 2 ]. Cell polarity is established by the apically localized Par complex, including atypical PKC (aPKC), Bazooka (Baz, the Drosophila homologue of Par3), and Par6 [ 6 – 8 ], as well as the Rho GTPase Cdc42 [ 9 ]. This protein complex displaces the cell fate determinants Prospero (Pros), Numb and their adaptor proteins Miranda (Mira) and Partner of Numb (Pon) to the basal cortex [ 10 – 14 ]. Another protein complex, including Partner of inscuteable (Pins), heterotrimeric G protein subunit Gαi, and their regulators, which is linked to the Par proteins by Inscuteable (Insc), is recruited to the apical cortex during mitosis [ 15 – 21 ]. Upon division, apical proteins segregate exclusively into the larger NSC daughter cell to sustain self-renewal, and basal proteins segregate into the smaller progenitor daughter cell to promote neuronal differentiation [ 1 , 22 ]. Such asymmetric protein segregation is facilitated by the orientation of the mitotic spindle along the apicobasal axis [ 2 , 23 – 29 ]. A failure in asymmetric divisions during development may result in cell fate transformation, leading to the formation of ectopic NSCs or the development of brain tumors [ 24 , 26 , 27 , 30 – 36 ].

Results

The disruption of NSC polarity and centrosome assembly is a direct consequence of hyx depletion, but not aging To rule out the possibility that the disruption of NSC polarity and centrosome assembly was due to consequence of aging in late larval stages, we examined NSC polarity proteins and centrosomal proteins at 24 h ALH, a time point when NSCs exit quiescence and reenter the cell cycle [77]. At 24 h ALH, Hyx was dramatically lost in NSCs upon knocking down hyx by RNAi under the control of insc-Gal4 driver in hyxHT622/+ background, suggesting an efficient knockdown of Hyx (S9A Fig). In hyx RNAi hyxHT622/+ at 24 h ALH, aPKC was delocalized in 96.8% of NSCs and Mira in 91.9% of NSCs, compared with a control that both aPKC and Mira formed proper crescent in all metaphase NSCs (S9B Fig). Likewise, centrosome protein γ-tub was severely reduced at the centrosomes in 82.4% of interphase NSCs and 85.0% of metaphase NSCs from hyx RNAi hyxHT622/+ (S9C–S9E Fig). Similarly, Polo was largely delocalized from the centrosomes in NSCs from hyx RNAi hyxHT622/+, which led to a significant decrease of Polo protein levels at the centrosomes in both interphase and metaphase NSCs (S9F–S9H Fig). In addition, in late larval stages, hyx RNAi hyxHT622/+ showed a stronger NSC overproliferation phenotype than that observed in hyx knockdown alone (Fig 1B and 1C); 84.3% of type I lineages and 93.3% of type II lineages with multiple NSCs were observed in hyx RNAi hyxHT622/+ compared with the control with a single NSC per lineage (S10A Fig). Moreover, Hyx protein was diminished in 89.1% of hyx RNAi hyxHT622/+ NSCs, while it was strongly detected in the control (S10B Fig). Consistent with these observations, strong reduction of γ-tub and Polo protein levels at the centrosomes was observed in both interphase and metaphase NSCs from hyx RNAi hyxHT622/+ (S10C–S10H Fig). Taken together, the disruption of NSC polarity and centrosome assembly is a direct consequence of hyx loss of function instead of aging.

Hyx is required for centrosome assembly in S2 cells in vitro To investigate whether Hyx plays a role in centrosome assembly in nonneuronal cells, we knocked down hyx in S2 cells by dsRNA treatment. We found that a centriolar protein, Ana2, remained localized at the centrosomes in metaphase cells (S8G Fig). This suggests that Hyx is not essential for the localization of centriolar proteins in both S2 cells and NSCs. Next, we examined the localization of other centrosomal proteins in S2 cells. Remarkably, D-TACC intensity was significantly decreased at the centrosomes, upon hyx knockdown, in metaphase S2 cells (Fig 6A and 6B). Consistent with these observations, the intensity of α-tub was also decreased by 0.65-fold on mitotic spindles (Fig 6D and 6E). These in vitro data support our observations in the larval brain and indicate that Hyx regulates microtubule growth and the localization of centrosomal proteins. Polo is undetectable in interphase S2 cells, unlike its robust localization in NSCs during the interphase. Consistent with our in vivo observations, we found that the overall intensity of Polo was significantly reduced to 0.67-fold in the dividing metaphase cells upon hyx knockdown (Fig 6A and 6C). Also, γ-tub intensity at the centrosomes marked by Ana2 was similar to that observed in the control (S8G and S8H Fig). The different observations in S2 cells and larval brains are likely due to incomplete depletion of hyx in S2 cells and/or different underlying mechanisms in vitro. PPT PowerPoint slide

PNG larger image

TIFF original image Download: Fig 6. Hyx is required for the recruitment of centrosome-related proteins and directly regulates their expression in vitro. (A) Metaphase cells from ds-egfp-treated S2 cells and ds-hyx-treated S2 cells were labeled for DTACC, Polo, Asl, and DNA. (B) Quantification graph of the fold change of DTACC intensity (with SD) in S2 cells from A. ds-egfp, 1-fold, n = 66; ds-hyx, 0.75 ± 0.05-fold, n = 69. (C) Quantification graph of the fold change of overall Polo intensity (with SD) in S2 cells from A. ds-egfp, 1-fold, n = 77; ds-hyx, 0.67 ± 0.08-fold, n = 81. (D) Metaphase S2 cells treated with ds-egfp and ds-hyx were labeled for α-tub, Asl, and DNA. (E) Quantification graph of the fold change of α-tub intensity in S2 cells from D. ds-egfp, 1-fold, n = 83; ds-hyx, 0.65 ± 0.10-fold, n = 88. (F) Spinning disc super-resolution imaging of Cnn, γ-tub, and Asl in metaphase S2 cells treated with ds-egfp (Cnn, n = 64; γ-tub, n = 55) and ds-hyx (Cnn, n = 58; γ-tub, n = 58). (G) Quantification graph of the percentage (with SD) of metaphase S2 cells forming “doughnut-like” shape of Cnn and γ-tub in F. ds-egfp: Pattern of Cnn, Normal, 94.9% ± 5.60%; abnormal, 5.1 ± 5.60%. ds-hyx: Pattern of Cnn, Normal, 48.7 ± 21.86%; abnormal, 51.3 ± 21.86%. ds-egfp: Pattern of γ-tub, Normal, 92.8 ± 8.57%; abnormal, 7.2 ± 8.57%. ds-hyx: Pattern of γ-tub, Normal, 46.6 ± 22.41%; abnormal, 53.4 ± 22.41%. (H) Location of primer pairs used for ChIP-qPCR on the promoter of genes in I. The schematic represents the polo gene, with the arrow indicating the upstream fragment distance from the TSS and the center nucleotide position of the primer pair is given and AAA showing the approximate location of the cleavage and polyadenylation site. (I) Quantification graph of ChIP-qPCR for detecting occupancy by Hyx on various genes in S2 cells, with an intergenic region at 5 kb downstream of the numb gene as a negative control and orb2 as a positive control. After normalizing against “Pre-serum,” fold enrichment from “Pre-serum” was taken as 1-fold for all primer sets. Fold enrichment (with SD) in “Hyx”: negative control, 1.37 ± 0.18-fold; positive control orb2, 2.94 ± 1.24-fold; polo (−587), 2.95 ± 1.47-fold; polo (−224), 2.63 ± 1.68-fold; aurA, 2.85 ± 1.77-fold; numb, 1.64 ± 0.31-fold. Minimum of 3 biological replicates were performed. (J) Luciferase assay in S2 cells shows an increase of the pGL3-luciferase reporter coupled with polo promoter (poloPro, 643 bp of sequences upstream of TSS) by endogenous hyx expression. Vector (pAFW)+pGL3-basic: 1.0 ± 0.28; Vector (pAFW)+ poloPro: 122 ± 6.05. The relative luciferase activity was normalized to Renilla luciferase activity. (K) Luciferase assay in S2 cells shows down-regulation of the pGL3-luciferase reporter coupled with poloPro ds-hyx treatment. The relative luciferase activity was normalized to Renilla luciferase activity. ds-egfp: 1-fold; ds-hyx: 0.5 ± 0.13-fold. (L) Luciferase assay in S2 cells shows no consistent alterations on the pGL3-luciferase reporter coupled with actin5c promoter between ds-egfp and ds-hyx treatment. The relative luciferase activity was normalized to Renilla luciferase activity. ds-egfp: 1-fold; ds-hyx: 0.2 ± 0.51-fold. (M) Quantification graph of RT-qPCR analysis in 48 h ALH brains from control (UAS-β-gal RNAi; UAS-β-gal RNAi) and hyx RNAi with UAS-Dicer2 driven by actin5C-Gal4. Minimum 3 repeats were conducted. After normalization against control (with SD): control, 1-fold; hyx, 0.23 ± 0.05-fold; polo, 0.33 ± 0.08-fold; aurA, 0.24 ± 0.07-fold; γ-tub, 0.59 ± 0.20-fold; cnn, 0.68 ± 0.17-fold; msps, 0.86 ± 0.15-fold; tacc, 0.45 ± 0.15-fold. (N) 48 h ALH larval brains from control (UAS-β-Gal RNAi; UAS-β-Gal RNAi) and hyx knockdown with UAS-Dicer2 (hyx RNAi; UAS-Dicer2 RNAi) under the control of actin5C-Gal4 were labelled with Hyx, Dpn, and Phalloidin. (O) Quantification graph for (N) showing the fluorescence intensity of Hyx throughout the whole brain and in the NSCs, respectively. Overall Hyx intensity in control: 41.5 ± 13.25, n = 34 ROI (region of interest excluded neuropil region) from 10 BL; hyx RNAi: 19.7 ± 8.99, n = 36 ROI from 8 BL. Hyx intensity in NSCs in control: 107.1 ± 29.18, n = 96 NSCs; hyx RNAi: 20.4 ± 12.92, n = 99 NSCs. (P) Western blotting analysis of larval brain protein extracts of control (UAS-β-Gal RNAi; UAS-β-Gal RNAi) and hyx knockdown with UAS-Dicer2 (hyx RNAi; UAS-Dicer2 RNAi) driven by actin5C-Gal4 at 48 h ALH. Blots were probed with anti-Hyx antibody and anti-GAPDH antibody. (Q) Fold change of Hyx protein after normalization against GAPDH in brain extracts from control (UAS-β-gal RNAi; UAS-β-gal RNAi) and hyx RNAi with UAS-Dicer2 driven by actin5C-Gal4 at 48 h ALH. Control, 1-fold; hyx RNAi, 0.34 ± 0.07-fold. (R) Western blotting analysis of 48 h ALH larval brain extracts of control (UAS-β-Gal RNAi; UAS-β-Gal RNAi) and hyx knockdown with UAS-Dicer2 (hyx RNAi; UAS-Dicer2 RNAi) under the control of actin5C-Gal4. Blots were probed with anti-Polo antibody, anti-AurA antibody, and anti-GAPDH antibody. (S) Fold change of Polo and AurA protein intensity after normalization against GAPDH in brain extracts from control (UAS-β-gal RNAi; UAS-β-gal RNAi) and hyx RNAi with UAS-Dicer2 driven by actin5C-Gal4 at 48 h ALH. Polo: control, 1-fold; hyx RNAi, 0.25 ± 0.01-fold. AurA: control, 1-fold; hyx RNAi, 0.43 ± 0.00-fold. Two individual repeats were conducted (P and R). Error bars indicate standard deviation in J-S. Arrows indicate the centrosomes in A, D and NSCs in N. Statistical significances were determined by unpaired two-tailed Student t test in B, C, E, K-M, O, Q, and S. ****p < 0.0001 for B, J, M, O, and S; ***p = 0.0004 for tacc in M;**p = 0.0017 for C, **p = 0.0063 for γ-tub in M, and **p = 0.0084 for cnn in M; *p = 0.0391 for E; D, F; **p = 0.0065 for E; **p = 0.0020 for K; ns = 0.6287 for L; ns = 0.1224 for msps in M; **p = 0.0036 for Q; ***p = 0.0002 for S. Scale bars: 5 μm. The underlying data for this figure can be found in the S1 Data. ALH, after larval hatching; Asl, Asterless; AurA, Aurora-A; ChIP-qPCR, chromatin immunoprecipitation coupled with quantitative PCR; Cnn, centrosomin; Hyx, Hyrax; msps, mini spindles; NSC, neural stem cell; RNAi, RNA interference; ROI, region of interest; RT-qPCR, reverse transcription quantitative real-time PCR; TSS, transcription start site; α-tub, α-tubulin; γ-tub, γ-tubulin. https://doi.org/10.1371/journal.pbio.3001834.g006 To further probe how Hyx regulates centrosome assembly, we examined the ultrastructure of Cnn and γ-tub using super-resolution imaging. Cnn and γ-tub formed “doughnut-like” rings surrounding the centriolar protein Asl, at the centrosomes, in 94.9% and 92.9% of control metaphase cells, respectively (Fig 6F and 6G). Remarkably, Cnn and γ-tub failed to form the ring patterns or formed a ring with reduced inner size at the centrosomes in 51.3% and 53.4% of hyx knockdown mitotic cells, respectively (Fig 6F and 6G). These observations suggest that Hyx is required for the proper recruitment of Cnn and γ-tub at the centrosomes in S2 cells.

Hyx directly regulates the expression of polo and aurA in vitro Next, we investigated whether Hyx directly regulates the expression of polo and aurA, the 2 key centrosomal proteins. We performed chromatin immunoprecipitation (ChIP) coupled with quantitative PCR (ChIP-qPCR) in S2 cells. After normalizing against “Pre-serum” (1-fold), only a 1.37-fold increase was seen for the negative control. In contrast, 2.94-fold enrichment was observed for orb2 promoter, a positive control. Moreover, we found Hyx binds to the promoter region of polo (new Fig 6I; 2.63-fold and 2.95-fold using 2 pairs of primers). Hyx also binds to the promoter region of aurA (Fig 6I; 2.85-fold), but not numb (Fig 6I; 1.64-fold). Therefore, Hyx binds to the promoter region of both polo and aurA. We performed the luciferase assay to verify the direct binding of Hyx to the polo promoter. The endogenous Hyx in S2 cells was able to induce the luciferase reporter activity under the control of polo-promoter (poloPro) normalized against Renilla luciferase activity, but not with the vector control (Fig 6J). We attempted to overexpress Hyx in S2 cells to test if it further enhances the luciferase activity under the control of the polo promoter. However, overexpression of Venus-tagged full-length hyx (hyx-FL) resulted in severe cell death (54.3%) detected by active Caspase-3 (S10I and S10J Fig; 11.2% cell death in the control), which precluded us from testing the effect of Hyx overexpression on the transcription of polo in the luciferase assay. Next, we sought to knock down hyx with dsRNA treatment in S2 cells and analyze the relative luciferase activity under the control of the polo promoter. The relative luciferase activity from the ds-hyx treatment group was significantly reduced to 0.5-fold compared with 1-fold from the control group (ds-egfp) (Fig 6K) The relative luciferase activity driven by actin5c promoter induced by ds-hyx treatment and ds-egfp groups was indistinguishable (Fig 6L; 1.0-fold versus 1.2-fold). We conclude that hyx can directly bind to the polo promoter region and promotes its transcription.

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[1] Url: https://journals.plos.org/plosbiology/article?id=10.1371/journal.pbio.3001834

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