InteractiveFly: GeneBrief

Bub3 interacting GLEBS and Zinc finger domain protein: Biological Overview | References

Exploring the role of phase separation in intracellular compartment formation is an active area of research. However, the associations of phase separation with intestinal stem cell (ISC)-dependent regeneration and aging remain unclear. This study demonstrates that BuGZ, a coacervating mitotic effector, shows age- and injury-associated condensation in Drosophila ISC nuclei during interphase. BuGZ condensation promotes ISC proliferation, affecting Drosophila gut repair and longevity. Moreover, m(6)A reader YT521-B acts as the transcriptional and functional downstream of BuGZ. The binding of YT521-B promotor or m(6)A writer Ime4/ Mettl14 to BuGZ controls its coacervation, indicating that the promotor may accelerate the phase transition of its binding transcription factor. Hence, it is proposed that phase separation and m(6)A regulators may be critical for ameliorating ISC-dependent gut regeneration and aging and requires further study (Zhang, 2023).

Biological liquid-liquid phase separation (LLPS) triggers the formation of condensed liquid-like droplets, resembling intracellular compartmentations that spatiotemporally facilitate or attenuate biochemical reactions. The biophysical properties of these macromolecular condensates are precisely controlled by their concentrations, structural features including intrinsically disordered regions (IDRs) and post-translational modifications (PTMs), contiguous environment and interacting chaperones containing DNA, RNA, and proteins DNA-protein complexes such as centrosomes, mitotic spindle, actin polymerization complex, telomere, chromatin, heterochromatin, and nucleolus form droplet-like condensates. Despite super-enhancer, enhancer, exogenous pathogen, and telomere DNA have been shown to induce phase separation of their corresponding associated proteins, whether the promotor influences the phase transition of its targeted transcription factor (TF) during the transcription process remains ambiguous. Biological functions and impacts of phase separation have been ill-defined and fueled by the ease of visualizing the droplet-shaped coacervations and disrupting the reversible condensate assembly in vivo and in vitro, which have advanced various biological fields. Most studies on phase separation referred to stem cells focus on embryonic stem cells (ESCs) during organ development instead of adult stem cells (ASCs, also known as resident stem cells) during tissue homeostasis and aging. A recent finding revealing that phase separation regulates the asymmetric localization of Numb and PAR complexes and contributes to neuroblast division in neural stem cells (NSC), suggests a possible regulatory role of LLPS on some ASCs. However, no protein has been shown to undergo functionally relevant phase transition during ASCs-mediated tissue regeneration or aging, typically regulated by genetic mutations, epigenetic changes, and extrinsic factors, including glucose, lipid, Ca+ metabolism, and niche environment. To evaluate this phenomenon, this study utilized intestinal stem cells (ISCs) in the midgut of Drosophila as the ASCs model to study tissue regeneration and aging, which divide asymmetrically and differentiate into enteroblasts (EBs) or enteroendocrine mother cells (EMCs) to maintain intestinal homeostasis upon subjected to external injuries or the aging process (Zhang, 2023).

N6-methyladenine mRNA (m6A) modification is the most prevalent posttranscriptional RNA modification of the common RRACH (R=G or A, H=A, C or U) sequences. The m6A modification modulated by writers (RNA methyltransferases), readers (m6A recognizers), and erasers (RNA demethylases), is required for distinct cellular processes under different cellular contexts20. Although previous studies have shown that m6A writer Mettl14 and reader YTHDF1 separately facilitate ISC maintenance and gut homeostasis, no m6A regulator has been shown to function in ISC aging. Beyond its mitotic functions in microtubule (MT)-kinetochore interaction, spindle assembly checkpoint (SAC), and spindle assembly, BuGZ is also required for proper mRNA splicing, synaptic vesicles (SV) cycling, and Human embryonic stem cell (hESC) reprogramming during interphase. BuGZ shows the condensation behavior involved in the MT polymerization during mitosis. Nonetheless, whether BuGZ undergoes phase separation during interphase, and whether BuGZ and its condensation behavior contribute to ISC proliferation and aging are still unknown. This study uncovers the role of phase separation on ASC-dependent regeneration and aging, the mechanism for controlling the corresponding TF coacervation [a liquid-liquid phase separation process where a colloidal solution splits into a dense, polymer-rich phase (the coacervate) and a dilute phase] via its downstream promotor DNA, and the effect of m6A regulators on modulating the phase transition of the key driver in ASCs (Zhang, 2023).

Due to the impediments of observing the protein condensation processes in vivo and difficulties in verifying its physiological functions, the functions of protein phase separation in vivo, especially in tissue homeostasis, organ regeneration, and individual aging, are largely unknown. Based on the current investigation, the functions of BuGZ coacervation in regulating ISC activation and proliferation during aging and gut repair, imply that protein phase separation participates in tissue homeostasis, regeneration, and aging processes mediated by ASCs. Accordingly, this study found that inhibiting BuGZ phase transition in ISCs of aged Drosophila improves intestinal functions and prolongs the lifespan of Drosophila, revealing that manipulating protein phase transition may delay or antagonize ASC-associated aging. Moreover, these findings reveal the physiological significance of the BuGZ condensate formation in DNA binding and transcriptional regulation in aging and gut repair and provide insights into the regulatory mechanisms underlying the BuGZ-mediated modulation of gene expression. Additionally, this study reports a mechanism that another cis-regulatory element (CRE) of genes the promotor facilitates the compartmentalization of its binding transcriptional condensate beyond enhancer and super-enhancer, other CREs including silencer and insulator may also possess feedback on regulating the coacervation behavior of their associated transcription factors (Zhang, 2023).

Since RNA m6A modification behaves as the most common nucleotide modification of the eukaryotic mRNAs, previous studies have demonstrated that mRNA m6A modification widely affects various aspects of metabolism, homeostasis, ontogenesis, and pathema. One previous study indicates that m6A reader YTHDF1 maintains stem cell traits during regeneration and tumorigenesis in intestines by mediating Wnt activation. In addition, since YTHDF1 upregulation is observed in both human and mouse intestinal cancer, YTHDF1 may behave as a biomarker for CRC21. Recently, another study uncovered that the m6A writer Mettl14, is required for modulating the homeostatic self-renewal in colonic stem cells. Mettl14 depletion in mouse colon results in colonic stem cell apoptosis, mucosal barrier dysfunction and severe colitis by modulating the NF-kappaB pathway. In addition, previous reports indicated Mettl14-mediated GsdmC N6-adenomethylation is essential for the survival of Lgr5+ intestinal stem cells and the maintenance of normal colonic epithelial regeneration. However, whether and how m6A regulators function in ISC-mediated gut regeneration upon injury and organ aging, remains largely unexplored. In the current study, by systematically investigating the function of YT521-B in ISCs during aging in Drosophila,m6A reader YT521-B was found to play an indispensable role in the ISC proliferation, gut homeostasis and lifespan. Under the transcriptional regulation of BuGZ, YT521-B functions as the upstream regulator of the EGFR/RAS/MAPK signaling pathway to mediate ISC proliferation. Moreover, BuGZ coacervation negatively regulates the mRNA transcription of YT521-B, whose promotor reversely enhances the phase separation of BuGZ. Meanwhile, this study validated that m6A writers, Ime4 and Mettl14, inhibit the proliferation of ISC by suppressing the phase separation of BuGZ to regulate the transcription of YT521-B. Thus, these data reveals the relationship between m6A regulators and phase separation in controlling ISC proliferation during gut regeneration and aging. Meanwhile, a mechanism is reported that controls the candidate TF coacervation by the downstream promotor and a way of probing the roles of m6A regulators in phase transition and regulation of ISCs. Additionally, this study not only deciphers a new regulatory mechanism of m6A reader YT521-B transcription but also a new function of m6A writer (Ime4, Mettl14) or reader (YT521-B) in ASCs homeostasis and aging (Zhang, 2023).

Despite previous studies showing that m6A writer Mettl14 and reader YTHDF1 separately facilitate stem cell maintenance and regeneration in the m6A-dependent manner, some other studies propose that m6A regulators exert some physiological functions independent of their m6A modification activities. The m6A writer METTL3 plays a catalytic-independent role in recruiting eIF3 to the translation initiation complex to regulate the translation of oncogenic transcripts including EGFR and TAZ. This activity also enhances the PES1 mRNA translation in the cytoplasm of chronic myeloid leukemia cells and amplifies p53 signaling in response to cellular stress Another m6A writer, METTL16, also interacts with eIF3 to enhance mRNA translation during tumorigenesis in an m6A-independent manner. Thus, future studies should evaluate whether the roles of m6A regulators (Ime4, Mettl14, and YT521-B) on ISC proliferation via the EGFR signaling pathways are mediated by the m6A modification. Future studies should also determine whether BuGZ and its phase separation act as the upstream regulator of m6A modification to affect m6A levels during gut regeneration and aging. Recently, the core m6A reader, YTHDF1/3, has been reported to promote stress granule formation through its phase transition. Phase separation of m6A reader YTHDF1 promotes mRNA degradation by interacting with AGO259, while the m6A reader YTHDF2 undergoes m6A-mediated phase separation in mESCs60. Additionally, the phase separation of the m6A writer, METTL3, regulates the dynamic assembly of the mRNA m6A methyltransferase complex. The predicted IDR domains within the m6A writers Ime4 (Drosophila ortholog of METTL3) and Mettl14 (data not shown) suggest that Ime4 and Mettl14 may adjust the ISC proliferation via their property of phase separation, and their interferences in BuGZ coacervation also may rely on the interactions with BuGZ through their phase transition (Zhang, 2023).

Splicing function of mitotic regulators links R-loop-mediated DNA damage to tumor cell killing

Although studies suggest that perturbing mitotic progression leads to DNA damage and p53 activation, which in turn lead to either cell apoptosis or senescence, it remains unclear how mitotic defects trigger p53 activation. This study shows that BuGZ and Bub3, which are two mitotic regulators localized in the interphase nucleus, interact with the splicing machinery and are required for pre-mRNA splicing. Similar to inhibition of RNA splicing by pladienolide B, depletion of either BuGZ or Bub3 led to increased formation of RNA-DNA hybrids (R-loops), which led to DNA damage and p53 activation in both human tumor cells and primary cells. Thus, R-loop-mediated DNA damage and p53 activation offer a mechanistic explanation for apoptosis of cancer cells and senescence of primary cells upon disruption of the dual-function mitotic regulators. This demonstrates the importance of understanding the full range of functions of mitotic regulators to develop antitumor drugs (Wan 2015).

A tubulin-binding protein that preferentially binds to GDP-tubulin and promotes GTP exchange

alpha- and beta-tubulin form GTPase heterodimers and assemble into microtubules. Like other GTPases, the tubulin heterodimer's nucleotide-bound state regulates its activity. In the dimer, alpha-tubulin is constitutively bound to GTP, while beta-tubulin can bind to either GDP (GDP-tubulin) or GTP (GTP-tubulin). Following assembly into microtubules, GTP-tubulin hydrolyzes GTP to GDP, triggering microtubule disassembly. This generates free GDP-tubulin, which must exchange GDP for GTP to undergo assembly again. Tubulin dimers undergo rapid nucleotide exchange in vitro, leading to a commonly accepted belief that a tubulin guanine nucleotide exchange factor (GEF) may be unnecessary for microtubule assembly in cells. This study used quantitative binding assays to show that BuGZ, a spindle assembly factor, binds tightly to GDP-tubulin, less tightly to GTP-tubulin, and weakly to microtubules. BuGZ promotes the incorporation of GTP into tubulin using a nucleotide exchange assay. The discovery of a tubulin GEF suggests a mechanism that may aid rapid microtubule assembly dynamics in cells (Yon, 2025).

Parthenolide disrupts mitosis by inhibiting ZNF207/BUGZ-promoted kinetochore-microtubule attachment

Parthenolide is a natural compound that has shown highly promising anticancer activity. Even though its mode of action has been studied for decades, its antimitotic activity has been largely overlooked, limiting the understanding of its full anticancer potential. This study combined click-chemistry with quantitative mass spectrometry and cell biology to elucidate the mechanism of action of parthenolide in mitosis. Parthenolide does not act as a microtubule-targeting agent in cells. Instead, it binds to the kinetochore protein ZNF207/BUGZ, preventing the establishment of proper kinetochore-microtubule attachment. These results show that parthenolide covalently binds to Cys54 of BUGZ via Michael addition to its alpha-methylene-gamma-lactone moiety. Since Cys54 is located within the second zinc-finger domain of the BUGZ microtubule-targeting region, it is proposed that parthenolide interferes with the microtubule-binding ability of BUGZ, consequently preventing kinetochore-microtubule attachments required for accurate chromosome congression to the spindle equator (Eibes, 2025).

Aurora-A condensation mediated by BuGZ aids its mitotic centrosome functions

Centrosomes composed of centrioles and the pericentriolar material (PCM), serve as the platform for microtubule polymerization during mitosis. Despite some centriole and PCM proteins have been reported to utilize liquid-liquid phase separation (LLPS) to perform their mitotic functions, whether and how centrosomal kinases exert the coacervation in mitosis is still unknown. This study reveals that Aurora-A, one key centrosomal kinase in regulating centrosome formation and functions, undergoes phase separation in vitro or in centrosomes from prophase, mediated by the conserved positive-charged residues inside its intrinsic disordered region (IDR) and the intramolecular interaction between its N- and C-terminus. Aurora-A condensation affects centrosome maturation, separation, initial spindle formation from the spindle pole and its kinase activity. Moreover, BuGZ interacts with Aurora-A to enhance its LLPS and centrosome functions. Thus, it is proposed that Aurora-A collaborates with BuGZ to exhibit the property of LLPS in centrosomes to control its centrosome-dependent functions from prophase (Zheng, 2024).

SETD1A function in leukemia is mediated through interaction with mitotic regulators BuGZ/BUB3

The H3K4 methyltransferase SETD1A plays a crucial role in leukemia cell survival through its noncatalytic FLOS domain-mediated recruitment of cyclin K and regulation of DNA damage response genes. This study identified a functional nuclear localization signal in and interaction partners of the FLOS domain. The screen for FLOS domain-binding partners reveals that the SETD1A FLOS domain binds mitosis-associated proteins BuGZ/BUB3. Inhibition of both cyclin K and BuGZ/BUB3-binding motifs in SETD1A shows synergistic antileukemic effects. BuGZ/BUB3 localize to SETD1A-bound promoter-TSS regions and SETD1A-negative H3K4me1-positive enhancer regions adjacent to SETD1A target genes. The GLEBS motif and intrinsically disordered region of BuGZ are required for both SETD1A-binding and leukemia cell proliferation. Cell-cycle-specific SETD1A restoration assays indicate that SETD1A expression at the G1/S phase of the cell cycle promotes both the expression of DNA damage response genes and cell cycle progression in leukemia cells (Perlee, 2023).

A role for the mitotic proteins Bub3 and BuGZ in transcriptional regulation of catalase-3 expression

The spindle assembly checkpoint factors Bub3 and BuGZ play critical roles in mitotic process, but little is known about their roles in other cellular processes in eukaryotes. In aerobic organisms, transcriptional regulation of catalase genes in response to developmental or environmental stimuli is necessary for redox homeostasis. This study demonstrates that Bub3 and BuGZ negatively regulate cat-3 transcription in the model filamentous fungus Neurospora crassa. The absence of Bub3 caused a significant decrease in BuGZ protein levels. These data indicate that BuGZ and Bub3 interact directly via the GLEBS domain of BuGZ. Despite loss of the interaction, the amount of BuGZ mutant protein negatively correlated with the cat-3 expression level, indicating that BuGZ amount rather than Bub3-BuGZ interaction determines cat-3 transcription level. Further experiments demonstrated that BuGZ binds directly to the cat-3 gene and responses to cat-3 overexpression induced by oxidative stresses. However, the zinc finger domains of BuGZ have no effects on DNA binding, although mutations of these highly conserved domains lead to loss of cat-3 repression. The deposition of BuGZ along cat-3 chromatin hindered the recruitment of transcription activators GCN4/CPC1 and NC2 complex, thereby preventing the assembly of the transcriptional machinery. Taken together, these results establish a mechanism for how mitotic proteins Bub3 and BuGZ functions in transcriptional regulation in a eukaryotic organism (Zhou, 2022).

Phylogenetic convergence of phase separation and mitotic function in the disordered protein BuGZ

Intrinsically disordered proteins (IDPs) effect biological function despite their sequence-encoded lack of preference for stable three-dimensional structure. Among their many functions, IDPs form membraneless cellular compartments through liquid-liquid phase separation (LLPS), also termed biomolecular condensation. The extent to which LLPS has been evolutionarily selected remains largely unknown, as the complexities of IDP evolution hamper progress. Unlike structured proteins, rapid sequence divergence typical of IDPs confounds inference of their biophysical or biological functions from comparative sequence analyses. This study leveraged mitosis as a universal eukaryotic feature to interrogate condensate evolutionary history. It was observed that evolution has conserved the ability for six homologs of the mitotic IDP BuGZ to undergo LLPS and to serve the same mitotic function, despite low sequence conservation. It was also observed that cellular context may tune LLPS. The phylogenetic correlation of LLPS and mitotic function in one protein raises the possibility of an ancient evolutionary interplay between LLPS and biological function, dating back at least 1.6 billion years to the last common ancestor of plants and animals (Chin, 2022).

BuGZ facilitates loading of spindle assembly checkpoint proteins to kinetochores in early mitosis

BuGZ is a kinetochore component that binds to and stabilizes Bub3, a key player in mitotic spindle assembly checkpoint signaling. Bub3 is required for kinetochore recruitment of Bub1 and BubR1, two proteins that have essential and distinct roles in the checkpoint. Both Bub1 and BubR1 localize to kinetochores through interactions with Bub3, which are mediated through conserved GLEBS domains in both Bub1 and BubR1. BuGZ also has a GLEBS domain, which is required for its kinetochore localization as well, presumably mediated through Bub3 binding. Although much is understood about the requirements for Bub1 and BubR1 interaction with Bub3 and kinetochores, much less is known regarding BuGZ's requirements. This study used a series of mutants to demonstrate that BuGZ kinetochore localization requires only its core GLEBS domain, which is distinct from the requirements for both Bub1 and BubR1. Furthermore, the kinetics of Bub1, BubR1, and BuGZ loading to kinetochores differ, with BuGZ localizing prior to BubR1 and Bub1. To better understand how complexes containing Bub3 and its binding partners are loaded to kinetochores, size-exclusion chromatography was carried out and Bub3-containing complexes from cells under different spindle assembly checkpoint signaling conditions were examined. Prior to kinetochore formation, Bub3 is complexed with BuGZ but not Bub1 or BubR1. These results point to a model in which BuGZ stabilizes Bub3 and promotes Bub3 loading onto kinetochores in early mitosis, which, in turn, facilitates Bub1 and BubR1 kinetochore recruitment and spindle assembly checkpoint signaling (Shirnekhi, 2020).

Search PubMed for articles about Drosophila BugZ

Chin, A. F., Zheng, Y., Hilser, V. J. (2022). Phylogenetic convergence of phase separation and mitotic function in the disordered protein BuGZ. Protein Sci, 31(4):822-834 PubMed ID: 34984754

Eibes, S., Lakshmi, R. B., Rajendraprasad, G., Weinert, B. T., Kamounah, F. S., Gamon, L. F., Rodriguez-Calado, S., Meldal, M., Davies, M. J., Pittelkow, M., Choudhary, C., Barisic, M. (2025). Parthenolide disrupts mitosis by inhibiting ZNF207/BUGZ-promoted kinetochore-microtubule attachment. EMBO J, 44(13):3764-3793 PubMed ID: 40425854

Perlee, S., Kikuchi, S., Nakadai, T., Masuda, T., Ohtsuki, S., Matsumoto, M., Rahmutulla, B., Fukuyo, M., Cifani, P., Kentsis, A., Roeder, R. G., Kaneda, A., Hoshii, T. (2023). SETD1A function in leukemia is mediated through interaction with mitotic regulators BuGZ/BUB3. EMBO Rep, 24(10):e57108 PubMed ID: 37535603

Shirnekhi, H. K., Herman, J. A., Paddison, P. J., DeLuca, J. G. (2020). BuGZ facilitates loading of spindle assembly checkpoint proteins to kinetochores in early mitosis. J Biol Chem, 295(43):14666-14677 PubMed ID: 32820050

Wan, Y., Zheng, X., Chen, H., Guo, Y., Jiang, H., He, X., Zhu, X., Zheng, Y. (2015). Splicing function of mitotic regulators links R-loop-mediated DNA damage to tumor cell killing. J Cell Biol, 209(2):235-246 PubMed ID: 25918225

Yon, W. J., Ha, T., Zheng, Y., Pedersen, R. T. A. (2025). A tubulin-binding protein that preferentially binds to GDP-tubulin and promotes GTP exchange. J Biol Chem, 301(8):110401 PubMed ID: 40543590

Zhang, Q., Deng, K., Liu, M., Yang, S., Xu, W., Feng, T., Jie, M., Liu, Z., Sheng, X., Chen, H., Jiang, H. (2023). Phase separation of BuGZ regulates gut regeneration and aging through interaction with m(6)A regulators. Nat Commun, 14(1):6700 PubMed ID: 37872148

Zheng, H., Zhang, Q., Liu, X., Shi, F., Yang, F., Xiang, S., Jiang, H. (2024). Aurora-A condensation mediated by BuGZ aids its mitotic centrosome functions. iScience, 27(5):109785 PubMed ID: 38746663

Zhou, Y., Shen, S., Du, C., Wang, Y., Liu, Y., He, Q. (2022). A role for the mitotic proteins Bub3 and BuGZ in transcriptional regulation of catalase-3 expression. PLoS Genet, 18(6):e1010254 PubMed ID: 35666721

date revised: March 3, 2026

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