InteractiveFly: GeneBrief

wuho: Biological Overview | References

WD40 proteins control many cellular processes via protein interactions. Drosophila Wuho controls fertility, although the involved mechanisms are unclear. This study shows that Wh promotion of Mei-p26 (a human TRIM32 ortholog) function maintains ovarian germ cell homeostasis. Wh and Mei-p26 are epistatically linked, with wh and mei-p26 mutants showing nearly identical phenotypes, including germline stem cell (GSC) loss, stem-cyst formation due to incomplete cytokinesis between GSCs and daughter cells, and overproliferation of GSC progeny. Mechanistically, Wh interacts with Mei-p26 in different cellular contexts to induce cell type-specific effects. In GSCs, Wh and Mei-p26 promote BMP stemness signaling for proper GSC division and maintenance. In GSC progeny, Wh and Mei-p26 silence nanos translation, downregulate a subset of microRNAs involved in germ cell differentiation and suppress ribosomal biogenesis via dMyc to limit germ cell mitosis. This study also found that the human ortholog of Wh (WDR4) interacts with TRIM32 in human cells. These results show that Wh is a regulator of Mei-p26 in Drosophila germ cells and suggest that the WD40-TRIM interaction may also control tissue homeostasis in other stem cell systems (Rastegari, 2020).

Stem cell self-renewal and differentiation must be balanced for proper tissue homeostasis. This balance is known to be coordinated by transcriptional and post-transcriptional mechanisms, which have not been fully described at a molecular level. An excellent model for studying genetic regulation of the cell fate transition from stem cell to differentiated progeny is the Drosophila ovary, as its germline stem cells (GSCs) and differentiated progeny are well characterized in cell biology. Although the physiology of Drosophila egg production is well described at a cellular level, the molecular regulatory mechanisms are still an area of active investigation (Rastegari, 2020).

Wuho (Wh; meaning 'no progeny' in Chinese) is an evolutionarily conserved protein comprising five WD40 domains (Cheng, 2016; Wu, 2006), which mediate protein-protein interactions. Homologs of Wh have been shown to exert a wide variety of functions via interactions with m7G46 tRNA methyltransferase, Flap endonuclease 1 (FEN1) and Culin-Ring ubiquitin ligase 4. Interestingly, wh mutant males are sterile because their spermatids are not properly elongated to make functional spermatozoa, and female flies are semi-sterile for unknown reasons (Wu, 2006), suggesting that Wh may potentially play an important role in the molecular regulatory circuitry of the GSC lineage (Rastegari, 2020).

Interestingly, the ovarian phenotypes reported in Wh mutants are strikingly similar to those in Mei-p26 mutants. Mei-p26 is a member of the tripartite motif and Ncl-1, HT2A and Lin-41 domain (TRIM- NHL) family of proteins, which is highly conserved among metazoans. TRIM-NHL proteins are known to control developmental transitions through mechanisms such as the promotion of stem cell differentiation by suppressing proliferation. The molecular action of TRIM-NHL proteins is typically ubiquitination and translation silencing via E3 ligase RING domains; meanwhile, the NHL domains mediate protein-protein interactions. In the Drosophila germline, Mei-p26 controls GSC maintenance and differentiation depending on its expression level. However, the regulators of Mei-p26 in the GSC lineage are not known. This study shows that Wh is a key regulator of Mei-p26 and that these proteins function together in multiple contexts to control GSC maintenance and differentiation for germline homeostasis. These results document a potentially generalizable role for WD40 proteins as a bridge between TRIM-NHL proteins and other cellular components, a function that is necessary to balance self-renewal and differentiation in the GSC lineage (Rastegari, 2020).

Homeostatic regulation of stem cells, the very foundation of tissue homeostasis, remains poorly understood at a molecular level. Using the Drosophila GSC lineage as an in vivo model to study stem cell biology, this study found that Wh, a WD40 protein, controls GSC self- renewal and differentiation via Mei-p26, a TRIM-NHL protein. An interaction between the proteins was identified in the ovary, and striking similarities between wh⁷ and mei-p26 mutants were observed at both phenotypic and molecular levels. Based on these findings and published results regarding Mei-p26 function, it is proposed that, in wild-type GSCs, Wh, Mei-p26 and Nos form a complex to inhibit brat translation, allowing Mad to be stabilized and phosphorylated by BMP signaling. pMad then translocates to the nucleus, where it suppresses transcription of bam, a master regulator of differentiation. On the other hand, Wh also promotes differentiation of GSC progeny by multiple mechanisms. First, Wh, Mei-p26, Bgcn, Sxl and Bam form a complex that binds to the 3' UTR of nos to silence its translation, possibly helping to turn off BMP signaling in the differentiating GSC progeny. Second, Wh interacts with Mei-p26, an E3 ubquitin ligase, to control dMyc protein levels and allow proper ribosomal biogenesis. Third, the interaction between Wh and Mei-p26 also limits expression of a subset of microRNAs, which may contribute to differentiation. These functions of Wh appear to be especially important for the last step of cytokinesis (abscission), which is completed between the GSC and its daughter cell after early G2 phase, as revealed by closure of the ring canal (Rastegari, 2020).

In wh mutant GSCs, brat translation is not suppressed, decreasing the level of pMad and increasing the expression of the differentiation factor Bam. This sequence of events causes premature differentiation of GSCs and leads to GSC loss, consistent with the known roles of Brat and Bam in germ cell differentiation. Mutation of brat or bam increases GSC number, whereas overexpression of brat or bam in germ cells causes germ cell depletion by forcing GSC differentiation. Mutation of wh in germ cells also results in incomplete abscission between GSCs and daughter cells, leaving open ring canals that create stem-cysts. Although this study did not determine the molecular mechanism by which Wh controls GSC abscission, removing a copy of bam significantly reduced stem-cyst number in wh mutant germaria, which suggests a role for Bam in Wh control of GSC abscission. In wh mutant germaria, stem-cysts simultaneously express Nos (a GSC maintenance factor) and Bam; thus, the growths display characteristics of both GSCs and daughter cells. In addition, ribosomal biogenesis is promoted via upregulated dMyc and drives germ cell overproliferation. Lastly, some differentiation-associated microRNAs are increased in the mutant ovaries, although their functions are not yet clear. It is not known whether a defect in meiosis is a consequence of overproliferative wh mutant germ cells, or whether Wh has a separate role in meiosis (Rastegari, 2020).

The results show that Wh is required for Mei-p26 function in germ cell homeostasis. However, it is unclear whether Wh directly interacts with Mei-p26 and which domains in the two proteins mediate the interaction. It is possible that Wh serves as a bridge for Mei-p26 to interact with its known partners. This study observed an interaction between human orthologs of Wh (WDR4) and Mei-p26 (TRIM32), suggesting that the interaction is evolutionarily conserved. Thus, the interaction between WD40 and TRIM-NHL proteins may be crucial for stem cell regulation in other organisms (Rastegari, 2020).

The last step in cell division, cytokinesis, is completed by abscission, which physically separates the two daughter cells. Cytokinesis starts by ingression of the cleavage furrow, constricting the plasma membrane onto the spindle midzone to form an electron- dense structure, the midbody, which comprises a thin membrane channel bridging two nascent daughter cells. The stem-cyst forms owing to a failure of GSCs to separate from daughter cells. Two possible mechanisms may produce such an abscission failure. First, a stem-cell-specific defect may prevent GSC-CB abscission. Second, GSCs may exhibit characteristics of differentiating cells that cause them to adopt incomplete cell cytokinesis programs. In addition to controlling chromosome orientation and segmentation, Aurora B is known to intrinsically regulate the timing of cell abscission, including in Drosophila female GSCs. During abscission, Aurora B in GSCs is targeted to the midbody and triggers membrane abscission via Endosomal sorting complex required for transport III (ESCRT-III) machinery. Aurora B negatively controls ESCRT-III, i.e. when Aurora B is active, ESCRT-III activity is low and abscission is delayed, and vice versa. It has also been shown that ribosomal biogenesis coordinates with ESCRT-III in GSCs to promote GSC abscission. Increasing Aurora B activity or disrupting ESCRT-III generates stem-cysts with germ cells that undergo synchronous division, yielding 32 germ cells in most egg chambers. However, wh mutant germ cells within stem-cysts divide asynchronously with elevated ribosomal biogenesis, and wh mutant egg chambers carry various numbers of germ cells. In addition, in wh mutant stem-cysts, decreased pMad expression, upregulated Bam expression and branched fusomes are all hallmarks of differentiating cysts. These results suggest that wh mutant stem-cysts may adopt the abscission program of differentiating cysts. Interestingly, removing a copy of bam from wh mutants or from shrub (a subunit of ESCRT-III) mutants partially rescues stem-cysts, suggesting that GSC abscission is coupled with cell fate. Further studies will be required to understand the molecular targets of Wh that control GSC abscission, and how GSCs and differentiating cysts acquire different abscission programs. Nevertheless, this study has shown that Wh participates with Mei-p26 to regulate fate determination in stem cells and daughter cells. This novel interaction may be conserved in other species and introduces the idea that WD40 proteins may participate with TRIM-NHL proteins in cell fate decision (Rastegari, 2020).

Wuho is a new member in maintaining genome stability through its interaction with Flap endonuclease 1

Replication forks are vulnerable to wayward nuclease activities. This paper reports the discovery of a new member in guarding genome stability at replication forks. Previously a Drosophila mutation was isolated, wuho (wh, no progeny), characterized by a severe fertility defect and affecting expression of a protein (WH) in a family of conserved proteins with multiple WD40 repeats. Knockdown of WH by siRNA in Drosophila, mouse, and human cultured cells results in DNA damage with strand breaks and apoptosis through ATM/Chk2/p53 signaling pathway. Mice with mWh knockout are early embryonic lethal and display DNA damage. The flap endonuclease 1 (FEN1) was identified as one of the interacting proteins. Fluorescence microscopy showed the localization of WH at the site of nascent DNA synthesis along with other replication proteins, including FEN1 and PCNA. WH is able to modulate FEN1's endonucleolytic activities depending on the substrate DNA structure. The stimulatory or inhibitory effects of WH on FEN1's flap versus gap endonuclease activities are consistent with the proposed WH's functions in protecting the integrity of replication fork. These results suggest that wh is a new member of the guardians of genome stability because it regulates FEN1's potential DNA cleavage threat near the site of replication (Cheng, 2016).

A new Drosophila gene wh (wuho) with WD40 repeats is essential for spermatogenesis and has maximal expression in hub cells

Through mutagenesis by P-element transposition, this study identified a series of mutants with deletions in topoisomerase 3beta gene (top3beta) and an adjacent, previously uncharacterized gene CG15897, named wuho (wh) in this study. Whereas top3beta truncation does not affect viability or fertility, wh null mutants display male sterile and female semi-sterile phenotypes. Furthermore, wh mutants can be fully rescued by wh transgenes, but not by top3beta transgenes, suggesting that the fertility phenotypes are caused by wh deletion. The alignment of WH protein sequence with other eukaryotic putative homologues shows they are evolutionarily conserved proteins with 5 WD40 repeats in the middle portion of the protein, and a bipartite nuclear localization signal at the carboxyl terminus. Yeast homologue with 5 WD40 repeats, Trm82, is the non-catalytic subunit of a tRNA methylase. Immunostaining shows that WH has the highest expression in hub cells, a niche for germline stem cells of testis. However, WH is not required for the maintenance of hub cells or the germline stem cells. In wh mutant males, spermatogenesis is arrested at the elongating stage of the developing spermatids, resulting in an absence of mature sperms in the seminal vesicles. The decreased fertility in wh mutant females is mostly due to defects in oogenesis. There are abnormal egg chambers present in the mutant females, in which the cystocytes fail to arrest their cell division at the fourth mitotic cycle, resulting in more than 16 cells in a single egg chamber. Additionally, these abnormal cystocytes do not undergo multiple rounds of endoreplication as the nurse cells do in a normal egg chamber. Therefore, the cytological analyses demonstrate that wh has a critical function in cellular differentiation for germline cells during gametogenesi (Wu, 2006).

Search PubMed for articles about Drosophila Wuho

Cheng, I. C., Chen, B. C., Shuai, H. H., Chien, F. C., Chen, P. and Hsieh, T. S. (2016). Wuho is a new member in maintaining genome stability through its interaction with Flap endonuclease 1. PLoS Biol 14(1): e1002349. PubMed ID: 26751069

Lee, C. C. and Hsieh, T. S. (2018). Wuho/WDR4 deficiency inhibits cell proliferation and induces apoptosis via DNA damage in mouse embryonic fibroblasts. Cell Signal 47: 16-26. PubMed ID: 29574139

Rastegari, E., Kajal, K., Tan, B. S., Huang, F., Chen, R. H., Hsieh, T. S. and Hsu, H. J. (2020). WD40 protein Wuho controls germline homeostasis via TRIM-NHL tumor suppressor Mei-p26 in Drosophila. Development 147(2). PubMed ID: 31941704

Wu, J., Hou, J. H. and Hsieh, T. S. (2006). A new Drosophila gene wh (wuho) with WD40 repeats is essential for spermatogenesis and has maximal expression in hub cells. Dev Biol 296(1): 219-230. PubMed ID: 16762337

date revised: 12 January 2022

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