InteractiveFly: GeneBrief

Bonus: Biological Overview | References

The canonical function of the Hippo signaling pathway is the regulation of organ growth. How this pathway controls cell-fate determination is less well understood. This study identified a function of the Hippo pathway in cell-fate decisions in the developing Drosophila eye, exerted through the interaction of Yorkie (Yki) with the transcriptional regulator Bonus (Bon), an ortholog of mammalian transcriptional intermediary factor 1/tripartite motif (TIF1/TRIM) family proteins. Instead of controlling tissue growth, Yki and Bon promote epidermal and antennal fates at the expense of the eye fate. Proteomic, transcriptomic, and genetic analyses reveal that Yki and Bon control these cell-fate decisions by recruiting transcriptional and post-transcriptional co-regulators and by repressing Notch target genes and activating epidermal differentiation genes. This work expands the range of functions and regulatory mechanisms under Hippo pathway control (Zhao, 2023).

Tissue growth and cell-fate determination are critical developmental processes controlled by multiple signaling pathways, including the evolutionarily conserved Hippo pathway, whose dysregulation leads to developmental abnormalities and diseases. The core Hippo (Hpo, MST1/2 in mammals)/Warts (Wts, LATS1/2 in mammals) kinase cascade inhibits the activity of the transcriptional coactivator Yorkie (Yki, YAP/TAZ in mammals) by phosphorylation and cytoplasmic retention, whereas unphosphorylated nuclear Yki associates with DNA-binding proteins such as Scalloped (Sd, TEAD1-4 in mammals) to activate gene expression (Zhao, 2023).

The canonical transcriptional targets of the Yki-Sd complex in Drosophila include Cyclin E (CycE), Death-associated inhibitor of apoptosis 1 (Diap1), bantam microRNA (mir-ban), and expanded (ex), which promote proliferation, inhibit apoptosis, and enable negative feedback regulation. Although increasing evidence supports the essential role of the Hippo pathway in cell-fate determination, the cellular mechanisms remain poorly understood (Zhao, 2023).

The Drosophila eye is an excellent model to study gene regulatory networks controlling cell-fate determination. ost of the Drosophila adult head structures develop from the larval eye-antennal disc, with the compound eye and ocelli originating from the eye disc compartment, the antenna and maxillary palp from the antennal compartment, and the head epidermis from the tissues surrounding the two compartments (Zhao, 2023).

Segregation of the mutually antagonistic eye, antennal, and head epidermal fates, which begins at the second instar larval stage (L2), is regulated by several signaling pathways, including Notch, EGFR, Wingless, and Hedgehog, and retinal determination genes such as eyeless (ey) and homothorax (hth). Alteration of these regulatory inputs can cause a switch from one fate to another, leading to partial or, in some cases, complete homeotic transformations of the affected structures (Zhao, 2023).

Key patterning events in the eye are linked to a wave of differentiation called the morphogenetic furrow (MF) that starts in the early third instar larval stage (L3) and proceeds from the posterior to the anterior of the eye disc, resulting in the differentiation of an array of optical units called ommatidia, each consisting of photoreceptor cells, cone cells, primary pigment cells, interommatidial bristles, and secondary and tertiary pigment cells (also called interommatidial cells) (Zhao, 2023).

Previous studies of the Hippo pathway in Drosophila eye differentiation focused on MF progression, terminal differentiation of photoreceptor cells, and formation of peripodial epithelium. Mutant analyses of the Hippo pathway components ex, Merlin (Mer), and mob as tumor suppressor (mats) have suggested an earlier and broader impact of the Hippo pathway in eye specification (Zhao, 2023).

However, the involvement of the Hippo pathway in controlling major cell-fate decisions among the eye, antenna, and head epidermis remains elusive, and the underlying transcriptional mechanisms are unknown. It was reasoned that the Hippo pathway may function in controlling the eye-antenna-epidermis fate determination through yet unknown interactors that regulate the transcriptional output of the Yki-Sd complex. To identify such interactors, proteomic analyses was performed and a Yki-interacting protein, Bonus (Bon) was identified. Bon is the only Drosophila ortholog of mammalian TIF1 family proteins TIF1α (TRIM24), TIF1β (TRIM28/KAP1), TIF1γ (TRIM33), and TIF1δ (TRIM66) TIF1/Bon proteins are chromatin-associated factors that activate or repress transcription by binding to co-regulators and controlling chromatin state. TIF1 proteins play various roles during vertebrate development and are implicated in cancer. Drosophila Bon is essential for nervous system development, embryo patterning, metamorphosis, and cell survival (Zhao, 2023).

Evidence is presented that Bon and the Hippo pathway co-regulate major cell-fate decisions during the development of the Drosophila eye. Yki and Bon bind via WW domain-PPxY motif interactions and cooperate to produce epidermal cells in the eye at the expense of ommatidial cells, while the loss of bon induces ectopic eye markers, suggesting that the Hippo pathway and Bon control the choice between the eye and epidermal fates. The Hippo pathway and Bon also regulate the eye-antennal specification, with Yki and Bon inhibiting the eye fate and promoting the antennal fate. Through the analysis of Bon and Yki protein interactors, multiple transcriptional and post-transcriptional regulators have been identified that are necessary for their control of cell-fate decisions. Transcriptomic and genetic analyses have revealed that Bon and Yki exert their functions by jointly activating epidermal differentiation genes and, unexpectedly, repressing Notch target genes. Overall, this study has identified a function of the Hippo pathway in the eye/antenna/epidermis cell-fate decisions during Drosophila eye development. This function requires the interaction of Yki with Bon, their recruitment of co-regulators, and the joint transcriptional control of a non-canonical set of target genes (Zhao, 2023).

Instead of mediating the previously described independent functions of Yki (growth control) and Bon (PNS differentiation), the Yki-Bon module regulates proper segregation of the eye, epidermal, and antennal fates in the developing eye. This function involves promotion of the epidermal and antennal fates, and suppression of the eye fate, via transcriptional regulation of a distinct set of target genes. This study thus provides a molecular mechanism for the biological function of the Hippo pathway and Bon in cell-fate determination during eye development (Zhao, 2023).

The results suggest that the Hippo pathway and Bon regulate the developmental cell-fate decisions in the eye at two levels. First, the Yki-Bon complex promotes antennal and epidermal fates and suppresses the eye fate during early eye field specification, before the L3 larval stage. This is supported by the phenotypes observed under various genetic manipulations of Bon, Wts, Yki, and Sd during the L1 and L2 larval stages, including the reciprocal transformations of eye and antenna, epidermal outgrowths in the eye, and ectopic eye fate. The Yki-Bon module is thus an essential component of the extensive gene regulatory network that controls these cell-fate decisions in early eye development (Zhao, 2023).

Previous studies showed that ex, Mer, and mats mutants exhibit eye-to-epidermal transformation and an occasional eye-to-antenna transformation (in an ex mutant combination), suggesting that the upstream Hippo pathway may also regulate the Yki-Bon module in fate determination at this stage. Second, after the segregation of the eye/antenna/epidermis fields and the start of MF in L3, the Yki-Bon complex promotes the epidermal cell fate while suppressing ommatidia, whereas Wts counteracts this activity. This is evidenced by the formation of epidermal trichomes in the retina and the suppression of ommatidial cell types, especially cone cells, upon knockdown of wts or overexpression of Bon or Yki with the late eye driver GMR-GAL4. Furthermore, RNA-seq data also revealed cell-fate regulation at the molecular level: the Yki-Bon complex activates epidermal differentiation genes (sha, f, nyo, and neo) and represses Notch targets (E(spl)-C, ct, and TfAP-2) that are required for eye fate establishment and are expressed in ommatidial cells (e.g., ct in cone cells and E(spl)mdelta-HLH in primary pigment cells). Although activation of the Yki-Bon complex at this stage did not exhibit an eye-to-antenna transformation phenotype, certain antennal genes (Gr64s and Or47b) were upregulated, suggesting transformation at the level of gene expression (Zhao, 2023).

This work shows that these fates are not completely defined during the early stages, as the retina could still transform into epidermal tissue and express epidermal and even antennal genes when the Hippo pathway and Bon were modulated after MF formation. Notably, conditional knockout of eya after the MF results in suppression of ommatidia and formation of trichomes in the eye. This suggests that the retinal determination genes are also involved in eye-epidermal fate decisions during later stages of eye development and that trichome induction may be a general biological outcome of interference with the eye vs. epidermis specification after the start of MF. Thus, it is concluded that the eye is still developmentally plastic at late stages, with a latent epidermal fate that is normally inhibited. Interestingly, this fate is revealed in the insect order Strepsiptera, whose compound eyes are composed of optical units that are separated by epidermal tissue bearing trichome-like extensions (Zhao, 2023).

Given Bon's role in promoting the epidermal fate in the eye, it was asked whether Bon is involved in epidermal differentiation in other tissues. Knockdown of bon by RNAi in the wing with the C5-GAL4 driver did not affect the number of trichomes, but trichome morphology was abnormal, with bon-RNAi wing cells growing thinner trichomes. bon^21B mutant sensory bristles on the notum showed a similar thinning effect, although the Sb clonal marker precluded genotyping surrounding epidermal cells. These results suggest that Bon may contribute to epidermal differentiation in other contexts in addition to its role in the eye (Zhao, 2023).

This study has identified an unexpected layer of control over eye specification exerted by Yki and Bon at the level of Notch target genes. The Hippo pathway has been reported to control cell-fate determination in other biological contexts through regulation of the Notch receptor or ligands. Although several Notch targets were identified that are repressed by Bon and Yki, Notch and its ligands, Serrate and Delta, were not jointly regulated or found in high-confidence Yki or Bon protein interactomes. Therefore, it is proposed that during cell-fate determination in the eye, Bon and Yki repress Notch targets (such as E(spl)-C genes) independently from upstream Notch signaling. It is noted that not all E(spl)-C genes are under Bon and Yki control, implying context-dependent regulation and functional divergence of E(spl)-C genes, as suggested by previous studies (Zhao, 2023).

Both Hippo and Notch contribute to cell proliferation and growth of the eye. The data suggest that Bon is not required for the growth-controlling function of the Hippo pathway. Instead, the Bon-Yki complex directs the acquisition of appropriate cell fates in the eye through the regulation of Notch targets. It is speculated that Bon may function as a switch that redirects some of Hippo pathway activities from growth regulation to cell-fate determination. So far, Drosophila Yki has only been implicated in transcriptional activation. However, studies in mammalian systems have shown that the YAP/TAZ-TEAD complex can also function as a transcriptional repressor on non-canonical target genes (Zhao, 2023).

The repression of Notch targets reported in this study suggests that Drosophila Yki can also function in transcriptional repression, likely via the recruitment of corepressors mediated by Bon. HDAC1 and its associated corepressor complexes repress gene transcription, including Notch targets (Zhao, 2023).

This study identified HDAC1 and its corepressor, CoRest, in the Bon interactome, raising the possibility that Bon and Yki repress Notch target genes in part via recruiting this repressor complex. The involvement of epigenetic regulators is further exemplified by Su(var)2-10, which has a role in chromatin SUMOylation and piRNA target silencing. Interestingly, Su(var)2-10 can suppress eye fate and even induce antennal fate in a sensitized background. Due to the strong genetic interaction between Su(var)2-10 and the Bon-Yki complex, and the identification of the Drosophila SUMO (smt3) in the Bon interactome, chromatin SUMOylation may be involved in gene repression by Bon and Yki. Future studies of chromatin status and epigenetic marks may reveal the mechanistic details of gene repression by Bon and Yki. The Hippo pathway and TIF1 family proteins are conserved and broadly expressed in higher eukaryotes, raising the possibility that they may also function together in other species and developmental processes, such as retinogenesis and hematopoiesis. Thus, the biological functions controlled by the Hippo pathway and Bon, and the underlying molecular mechanisms reported in this study here, may be evolutionarily conserved (Zhao, 2023).

For this study, RNA-seq was performed using pupal eyes when trichomes initiate; however, the pupal eye patterning defects were detectable before trichome initiation. Thus, there might be additional differentially expressed genes at earlier stages of cell-fate determination that were have missed. Cells mutant for a null allele of bon tend to be eliminated, potentially masking additional cell differentiation defects in bon mutant clones. Although this study has largely focused on eye development, it is possible that Bon and Yki interaction has additional functions in other tissues. Further studies are needed to analyze the precise composition of multiprotein complexes involving Yki and Bon, as well as their effects on the target genes that were identified in this study (Zhao, 2023).

SUMOylation of Bonus, the Drosophila homolog of Transcription Intermediary Factor 1, safeguards germline identity by recruiting repressive chromatin complexes to silence tissue-specific genes

The conserved family of Transcription Intermediary Factors (TIF1) proteins consists of key transcriptional regulators that control transcription of target genes by modulating chromatin state. Unlike mammals that have four TIF1 members, Drosophila only encodes one member of the family, Bonus. Bonus has been implicated in embryonic development and organogenesis and shown to regulate several signaling pathways, however, its targets and mechanism of action remained poorly understood. This study found that knockdown of Bonus in early oogenesis results in severe defects in ovarian development and in ectopic expression of genes that are normally repressed in the germline, demonstrating its essential function in the ovary. Recruitment of Bonus to chromatin leads to silencing associated with accumulation of the repressive H3K9me3 mark. Bonus associates with the histone methyltransferase SetDB1 and the chromatin remodeler NuRD and depletion of either component releases Bonus-induced repression. This study further established that Bonus is SUMOylated at a single site at its N-terminus that is conserved among insects and this modification is indispensable for Bonus's repressive activity. SUMOylation influences Bonus's subnuclear localization, its association with chromatin and interaction with SetDB1. Finally, Bonus SUMOylation was shown to be mediated by the SUMO E3-ligase Su(var)2-10, revealing that although SUMOylation of TIF1 proteins is conserved between insects and mammals, both the mechanism and specific site of modification is different in the two taxa. Together, this work identified Bonus as a regulator of tissue-specific gene expression and revealed the importance of SUMOylation as a regulator of complex formation in the context of transcriptional repression (Godneeva, 2023).

Impact of Germline Depletion of Bonus on Chromatin State in Drosophila Ovaries

Gene expression is controlled via complex regulatory mechanisms involving transcription factors, chromatin modifications, and chromatin regulatory factors. Histone modifications, such as H3K27me3, H3K9ac, and H3K27ac, play an important role in controlling chromatin accessibility and transcriptional output. In vertebrates, the Transcriptional Intermediary Factor 1 (TIF1) family of proteins play essential roles in transcription, cell differentiation, DNA repair, and mitosis. This study focused on Bonus, the sole member of the TIF1 family in Drosophila, to investigate its role in organizing epigenetic modifications. The findings demonstrated that depleting Bonus in ovaries leads to a mild reduction in the H3K27me3 level over transposon regions and alters the distribution of active H3K9ac marks on specific protein-coding genes. Additionally, through mass spectrometry analysis, novel interacting partners of Bonus in ovaries were identified, such as PolQ, providing a comprehensive understanding of the associated molecular pathways. Furthermore, this research revealed Bonus's interactions with the Polycomb Repressive Complex 2 and its co-purification with select histone acetyltransferases, shedding light on the underlying mechanisms behind these changes in chromatin modifications (Godneeva, 2023).

TIF1 family members play an important role in remodeling chromatin and the modulation of underlying transcriptional mechanisms. Bon is the only member of the TIF1 subfamily of TRIM/RBCC proteins in Drosophila, setting it apart from its mammalian counterparts, where four distinct members exhibit diverse functions and mechanisms of action. A genome-wide analysis was performed of several activating and silencing histone modifications in germline Bon-depleted ovaries. Bon's depletion did not significantly alter the genome-wide abundance of H3K27me3, although subtle reductions in H3K27me3 were observed in certain transposon-associated regions. Previous research indicated that Bon is not involved in transposon repression. This can be explained that despite the mild reduction in H3K27me3, transposon expression remained largely unchanged due to the persistent presence of the repressive H3K9me3 mark. While it has been well-established that H3K9me3 primarily marks transposable elements and satellite sequences, several studies have revealed the coexistence both the H3K9me3 and H3K27me3 marks on the same subset of genes, and in some the H3K27me3 mark is primarily responsible for the repression of transposons. In mammals, TIF1β/KAP-1 is known to repress endogenous retroviruses in an H3K9me3-dependent way. Although relatively few studies have showed the connection between the TIF1 family proteins and the H3K27me3 mark, one study did reveal that KAP-1 depletion resulted in a decreased level of H3K27me3 on the HIV-1 promoter. Therefore, it is proposed that in Drosophila, Bon participates in the repression of transposons through its association with the H3K27me3 mark, indicating a new role within the TIF1 subfamily (Godneeva, 2023).

Additionally, the association between Bon and the PRC2 complex, revealed through co-immunoprecipitation assays, raises intriguing questions regarding the functional consequences of this interaction. Bon has not previously been described in connection with Polycomb complexes, apart from a potential interaction between Bon and unknown members of a Polycomb group that was mentioned in the discussion of an early work. Interestingly, in mammals, KAP-1 was shown to interact with EZH2/E(z) in a PRC2-independent manner to coordinately regulate genes with roles in breast stem cell maintenance. In another study, KAP-1 associates with the PRC1 complex to repress differentiation-inducible genes in embryonic stem cells. Therefore, Bon may be involved in facilitating the recruitment of the PRC2 complex to specific genomic loci or can also associate with the PRC1 complex to repress the subsets of several genes (Godneeva, 2023).

The members of the TIF1 family are involved in both transcriptional activation and repression. Analysis of the active marks H3K9ac and H3K27ac indicates that Bon is directly regulating some of its targets by facilitating their H3K9 acetylation. However, the levels of these activation marks remained unchanged following Bon depletion on most of its targets, suggesting the involvement of other activation marks. Indeed, the Gcn5 histone acetyltransferase responsible for the deposition of the H3K9ac mark was not identified in mass-spec analysis for Bon interactors. The presence of HATs MOF, nej, and Taf1 in the Bon-interacting complex raises the possibility that Bon's modulation of gene expression may be accomplished through diverse acetylation marks. Additionally, indirect regulation through other transcriptional regulators remains a plausible mechanism. Notably, due to their C-terminal PHD/bromodomain, TIF1 proteins can function as 'readers' of histone modifications. Thus, mammalian TIF1γ/TRIM33 specifically recognizes an H3 tail that is acetylated at lysines K18 and K23, and TIF1α/TRIM24 was shown to recognize acetylated H3K23. Since the current results could not pinpoint a specific acetylation mark involved in Bon-dependent regulation, future studies will be necessary to precisely identify the acetylation mark associated with this process (Godneeva, 2023).

The search for interacting partners of Bon revealed an interesting interaction with PolQ, a specialized DNA polymerase (Pol θ) participating in DNA repair, which suggests Bon's potential involvement in end-joining repair of DSBs. In mammals, TIF1 members are known to play a major role in DSB repair mechanisms. DSBs are serious threats to cell survival and genome stability; therefore, in the future, it will be interesting to determine whether Bon is required for the repair activities or whether it can enhance the efficiency and accuracy of this repair process, ensuring the preservation of genomic integrity (Godneeva, 2023).

In summary, this study expands understanding of Bon's roles in the epigenetic landscape and offers a valuable resource of Bon's interactors in Drosophila ovaries. Although this study has demonstrated Bon's influence on the distribution of activating marks (H3K9ac and H3K27ac) and a repressive mark (H3K27me3), it is essential to acknowledge the complexity of chromatin regulation. Therefore, it will be necessary to study other histone modifications and effector molecules to better understand the mechanisms underlying Bon's impact on gene expression that controls the function of chromatin (Godneeva, 2023).

In conclusion, this study of the specific role of Bon has provided significant insights into its diverse contributions to the epigenetic landscape. Depletion of Bon affects the distribution of histone marks H3K27me3, H3K9ac, and H3K27ac, revealing its complex involvement in the regulation of chromatin states. The interaction with the PRC2 complex suggests potential involvement in Polycomb-mediated gene regulation, raising questions about the functional consequences of this cooperation. This study reveals previously unknown interactions between Bon and chromatin-modifying complexes, including histone acetyltransferases. Furthermore, the dual role of Bon in transcriptional activation and repression suggests the existence of a complex regulatory network. While this study provides valuable insights into Bon's interactions with key chromatin-modifying complexes and its role in DNA repair, the complex nature of chromatin regulation requires further exploration of additional histone modifications and effector molecules. Future studies, with a focus on identifying specific acetylation marks associated with Bon-dependent regulation and elucidating Bon's role in DNA repair mechanisms, will enhance understanding of Bon's influence on gene expression and genome stability (Godneeva, 2023).

New gene evolution in the bonus-TIF1-gamma/TRIM33 family impacted the architecture of the vertebrate dorsal-ventral patterning network

Uncovering how a new gene acquires its function and understanding how the function of a new gene influences existing genetic networks are important topics in evolutionary biology. This study demonstrates nonconservation for the embryonic functions of Drosophila Bonus and its newest vertebrate relative TIF1-gamma/TRIM33. Previous work showed that TIF1-gamma/TRIM33 functions as an ubiquitin ligase for the Smad4 signal transducer and antagonizes the Bone Morphogenetic Protein (BMP) signaling network underlying vertebrate dorsal-ventral axis formation. This study shows that Bonus functions as an agonist of the Decapentaplegic (Dpp) signaling network underlying dorsal-ventral axis formation in flies. The absence of conservation for the roles of Bonus and TIF1-gamma/TRIM33 reveals a shift in the dorsal-ventral patterning networks of flies and mice, systems that were previously considered wholly conserved. The shift occurred when the new gene TIF1-gamma/TRIM33 replaced the function of the ubiquitin ligase Nedd4L in the lineage leading to vertebrates. Evidence of this replacement is the demonstration that Nedd4 performs the function of TIF1-gamma/TRIM33 in flies during dorsal-ventral axis formation. The replacement allowed vertebrate Nedd4L to acquire novel functions as a ubiquitin ligase of vertebrate-specific Smad proteins. Overall these data reveal that the architecture of the Dpp/BMP dorsal-ventral patterning network continued to evolve in the vertebrate lineage, after separation from flies, via the incorporation of new genes (Wisotzkey, 2014).

Fruitless recruits two antagonistic chromatin factors to establish single-neuron sexual dimorphism

The Drosophila fruitless (fru) gene encodes a set of putative transcription factors that promote male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. However, the mechanism whereby fru establishes the sexual fate of neurons remains enigmatic. This study shows that Fru forms a complex with the transcriptional cofactor Bonus (Bon), which, in turn, recruits either of two chromatin regulators, Histone deacetylase 1 (HDAC1), which masculinizes individual sexually dimorphic neurons, or Heterochromatin protein 1a (HP1a), which demasculinizes them. Manipulations of HDAC1 or HP1a expression change the proportion of male-typical neurons and female-typical neurons rather than producing neurons with intersexual characteristics, indicating that on a single neuron level, this sexual switch operates in an all-or-none manner (Ito, 2012).

Sex-switching of the Drosophila brain by two antagonistic chromatin factors

In Drosophila melanogaster, the fruitless (fru) gene encoding BTB-Zn-finger transcription factors organizes male sexual behavior by controlling the development of sexually dimorphic neuronal circuitry. However, the molecular mechanism by which fru controls the sexual fate of neurons has been unknown. Z recent study represents a first step toward clarification of this mechanism. It was shown that: (1) Fru forms a complex with the transcriptional cofactor Bonus (Bon), which recruits either of two chromatin regulators, Histone deacetylase 1 (HDAC1) or Heterochromatin protein 1a (HP1a), to Fru-target sites; (2) the Fru-Bon complex has a masculinizing effect on single sexually-dimorphic neurons when it recruits HDAC1, whereas it has a demasculinizing effect when it recruits HP1a; (3) HDAC1 or HP1a thus recruited to Fru-target sites determines the sexual fate of single neurons in an all-or-none manner, as manipulations of HDAC1 or HP1a expression levels affect the proportion of male-typical neurons and female-typical neurons without producing neurons of intersexual characteristics. It is hypothesized that chromatin landscape changes induced by ecdysone surges direct the HDAC1- or HP1a-containing Fru complex to distinct targets, thereby allowing them to switch the neuronal sexual fate in the brain (Ito, 2013).

Trim24 targets endogenous p53 for degradation

Numerous studies focus on the tumor suppressor p53 as a protector of genomic stability, mediator of cell cycle arrest and apoptosis, and target of mutation in 50% of all human cancers. The vast majority of information on p53, its protein-interaction partners and regulation, comes from studies of tumor-derived, cultured cells where p53 and its regulatory controls may be mutated or dysfunctional. To address regulation of endogenous p53 in normal cells, a mouse and stem cell model was created by knock-in (KI) of a tandem-affinity-purification (TAP) epitope at the endogenous Trp-53 locus. Mass spectrometry of TAP-purified p53-complexes from embryonic stem cells revealed Tripartite-motif protein 24 (Trim24), a previously unknown partner of p53. Mutation of TRIM24 homolog, bonus, in Drosophila led to apoptosis, which could be rescued by p53-depletion. These in vivo analyses establish TRIM24/bonus as a pathway that negatively regulates p53 in Drosophila. The Trim24-p53 link is evolutionarily conserved, as TRIM24 depletion in human breast cancer cells caused p53-dependent, spontaneous apoptosis. Trim24 was found to ubiquitylate and negatively regulates p53 levels, suggesting Trim24 as a therapeutic target to restore tumor suppression by p53 (Allton, 2009).

Bonus, a Drosophila TIF1 homolog, is a chromatin-associated protein that acts as a modifier of position-effect variegation

Bonus, a Drosophila TIF1 homolog, is a nuclear receptor cofactor required for viability, molting, and numerous morphological events. This study established a role for Bonus in the modulation of chromatin structure. Weak loss-of-function alleles of bonus have a more deleterious effect on males than on females. This male-enhanced lethality is not due to a defect in dosage compensation or somatic sex differentiation, but to the presence of the Y chromosome. Additionally, it was shown that bonus acts as both an enhancer and a suppressor of position-effect variegation. By immunostaining, it was demonstrated that Bonus is associated with both interphase and prophase chromosomes and through chromatin immunoprecipitation it was shown that two of these sites correspond to the histone gene cluster and the Stellate locus (Beckstead, 2005).

Oncogenic activation of c-Myb correlates with a loss of negative regulation by TIF1beta and Ski

The c-myb proto-oncogene product (c-Myb) regulates proliferation of hematopoietic cells by inducing the transcription of a group of target genes. Removal or mutations of the negative regulatory domain (NRD) in the C-terminal half of c-Myb leads to increased transactivating capacity and oncogenic activation. This study reports that TIF1beta directly binds to the NRD and negatively regulates the c-Myb-dependent trans-activation. In addition, three corepressors (Ski, N-CoR, and mSin3A) bind to the DNA-binding domain of c-Myb together with TIF1beta and recruit the histone deacetylase complex to c-Myb. Furthermore, the Drosophila TIF1beta homolog, Bonus, negatively regulates Drosophila Myb activity. The Ski corepressor competes with the coactivator CBP for binding to c-Myb, indicating that the selection of coactivators and corepressors is a key event for c-Myb-dependent transcription. Mutations or deletion of the NRD of c-Myb and the mutations found in the DNA-binding domain of v-Myb decrease the interaction with these corepressors and weaken the corepressor-induced negative regulation of Myb activity. These observations have conceptual implications for understanding how the nuclear oncogene is activated (Nomura, 2004).

Bonus, a Drosophila homolog of TIF1 proteins, interacts with nuclear receptors and can inhibit betaFTZ-F1-dependent transcription

The Drosophila bonus (bon) gene encodes a homolog of the vertebrate TIF1 transcriptional cofactors. bon is required for male viability, molting, and numerous events in metamorphosis including leg elongation, bristle development, and pigmentation. Most of these processes are associated with genes that have been implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development. Bon is associated with sites on the polytene chromosomes and can interact with numerous Drosophila nuclear receptor proteins. Bon binds via an LxxLL motif to the activator function AF-2 domain present in the ligand binding domain of betaFTZ-F1 and behaves as a transcriptional inhibitor in vivo (Beckstead, 2001).

bon was isolated in a screen for mutations affecting embryonic peripheral nervous system (PNS) development. Three independent P-element alleles, bonS024108 (bon²⁴¹), bonS024912 (bon²⁴⁹), and bonS048706 (bon⁴⁸⁷), which mapped to 92E8-14, fail to complement each other. An additional bon allele, bon^21B, was generated by imprecise excision of bon²⁴¹. This allele fails to complement all bon alleles and a deficiency, Df(3R)H^B79, which removes chromosomal region 92E (Beckstead, 2001).

To establish the strength of each allele, complementation tests were performed and the lethal phase associated with each allelic combination was defined. Df(3R)H^B79/bon^21B animals exhibit the earliest stage of lethality, while homozygous bon²⁴¹/bon²⁴¹ animals display the least severe phenotype, with 34% of the expected animals surviving to pharate adults. In all genetic combinations, some first instar larvae survive up to a week and fail to molt into second instar larvae. No male third instar larvae, pharate adults or adults survived in any bon genetic background, indicating that loss of bon has a more deleterious effect on males than females. Based on the complementation data, the bon alleles were ordered as follows: Df(3R)H^B79 > bon^21B > bon⁴⁸⁷ > bon²⁴¹ = bon²⁴⁹ (Beckstead, 2001).

To pinpoint the phenotypes associated with bon mutations, morphological defects associated with different allelic combinations were analyzed. Df(3R)H^B79/bon^21B mutant embryos fully develop. However, many are unable to hatch from their egg case, and both Df(3R)H^B79/bon^21B embryos and first instar larvae exhibit disrupted fluid-filled trachea. The embryonic/first instar lethality probably corresponds to the zygotic null or a severe loss of function phenotype as there is very little maternal protein remaining in mature Df(3R)H^B79/bon^21B embryos (Beckstead, 2001).

Less severe loss of Bon function results in pupal defects. The majority of bon⁴⁸⁷/bon^21B mutant pupae display an almost complete lack of pigmentation. They initiate but fail to complete development of legs, wings, head, eyes, and cuticle. Salivary glands, which normally undergo apoptosis at 12 hr postpupariation, are present in 4 days post-pupariation bon⁴⁸⁷/bon^21B pupae and are similar in size to third instar larval glands. In bon²⁴¹/bon⁴⁸⁷ animals, defects in cuticle and bristle development are observed. The abdominal cuticle of these flies appears immature and the tergite and sternal bristles are severely reduced or absent. Bristles of the anterior wing margin of bon²⁴¹/bon⁴⁸⁷ pharate adults are almost entirely lacking. Finally, there is a dramatic reduction in pigmentation in the mutant wing cuticle. This data indicates that bon is required for numerous developmental processes, including control of larval molting, cuticle deposition and pigmentation, bristle development, and elimination of salivary glands by cell death (Beckstead, 2001).

To determine the effect of complete loss of bon on the development of adult tissues, the FLP/FRT system was used to create mutant clones in the developing eye imaginal disc with the eyeless enhancer driving FLP. Flies heterozygous for FRT bon^21B and a cell lethal gene marked with w⁺ (FRT w⁺ cl3R) expressing FLP in the eye disc generate clones of bon^21B/bon^21B. Loss of bon in the eye causes a loss of all mutant photoreceptors. A small patch of red photoreceptors remains because of a limited number of bon^21B/cl3R cells. This indicates that bon is required for cell viability or proliferation of photoreceptors. In addition, much of the head cuticle is missing, indicating that most or all cells of the eye disc that produce cuticle are also lacking. In addition, no adult mutant bon clones were observed in FLP/FRT experiments using heat shock-FLP; FRT82 bon^21B animals, even though numerous wild-type twin spots were observed. Hence, early and complete loss of bon is lethal to cells or disrupts proliferation (Beckstead, 2001).

To clone bon, genomic DNA flanking bon²⁴¹ was isolated and used to identify cDNAs. The bon cDNA (AF210315) permitted isolation of genomic phages and determination of the structure of the locus. Flanking sequences from bon²⁴¹, bon²⁴⁹, and bon⁴⁸⁷ were used to map the P-elements. Sequencing of bon^21B revealed a deletion of most of exon 1 and the 5' end of intron 1 (Beckstead, 2001).

Database searches have revealed that bon encodes the only Drosophila homolog of mammalian TIF1s. Bon exhibits 29% identity with mouse TIF1alpha and mouse TIF1beta, and 26% identity with human TIF1gamma. The overall identity between Bon and TIF1s is similar to the identity observed between the TIF1 members. A higher degree of identity is seen in the N- and C-terminal regions spanning the conserved domains. At the N terminus, a C3HC4 zinc-finger motif or RING finger is followed by two cysteine-rich zinc binding regions (B-boxes) and a coiled coil domain forming a tripartite motif designated RBCC. At the C terminus, a bromodomain is preceded by a C4HC3 zinc-finger motif or PHD finger (Beckstead, 2001 and references therein).

Northern analysis demonstrates that bon produces one predominant 6 kb transcript and two 4 kb transcripts, which each encode a protein of ~140 kDa. The two 4 kb transcripts are only present in 0-3 hr embryos and adult females. It is therefore possible that the 4kb mRNAs are maternal components. bon is expressed throughout embryogenesis and in first instars. Its levels increase in 9-12 hr embryos and are low during the second instar stage. bon is upregulated in late third instar larvae. The upregulation of bon during midembryogenesis and prior to pupariation correlates well with known high titer pulses of ecdysone (Beckstead, 2001).

Immunohistochemical staining of numerous tissues show that Bon is a nuclear protein expressed in most and possibly all cells during embryogenesis, in fat body, imaginal discs, salivary glands, brain, gut, Malpighian tubules, and trachea. Bon is a chromatin-associated protein that localizes to ~10%-15% of the polytene chromosome bands. This pattern is highly reproducible (Beckstead, 2001).

To determine whether the defects seen in bon mutants are due to disruptions in the ecdysone-regulated pathway, the expression of several ecdysone-regulated genes were examined in y w and bon²⁴¹/bon²⁴¹ larvae, prepupae, and pupae. In bon²⁴¹/bon²⁴¹ animals, levels of betaFTZ-F1, EcR-A, EcR-B, E74A, E74B, and BR-C are reduced. It appears that each gene is upregulated in response to the ecdysone pulse, but is unable to maintain expression in the bon mutants. However, DHR3 transcripts are prematurely expressed and the overall level of expression is elevated in bon²⁴¹/bon²⁴¹ animals when compared to y w control animals. In addition, the EcR-A transcript levels appear slightly reduced in bon²⁴¹/bon²⁴¹ animals, while the EcR-B transcript levels are severely reduced when compared to controls. Similar observations were made for all of the above genes in bon^21B/bon⁴⁸⁷ animals, except that DHR3 transcript levels are also reduced. Based on these effects on gene expression, defects in larval molting and metamorphosis, and the temporal expression pattern of Bon, it is proposed that Bon plays an important role in the regulation of genes in the ecdysone response pathway (Beckstead, 2001).

To better characterize the function of Bon, interacting proteins were sought. A Drosophila embryonic cDNA library was screened using Bon as bait. Isolated cDNAs were classified as positive when retested in another version of the two-hybrid system using the DNA binding domain of the estrogen receptor fused to Bon (DBD-Bon) and an ERE-URA3 reporter gene. One positive clone encoded the 488 C-terminal residues of betaFTZ-F1 (amino acids 315-802). Coexpression of DBD-Bon with AAD-betaFTZ-F1(315-802) transactivates the URA3 reporter. Hence, Bon is able to interact with betaFTZ-F1(315-802) in yeast cells (Beckstead, 2001).

To test whether Bon interacts with betaFTZ-F1 as well as other Drosophila nuclear receptors in vitro, binding assays were performed using purified recombinant proteins. Glutathione-S transferase (GST)-fused betaFTZ-F1, alphaFTZ-F1 (amino acids 154-1029), Seven-up (SVP), DHR3, USP, and EcR were immobilized on glutathione-Sepharose and incubated with purified N-terminally His-tagged Bon (His-Bon). His-Bon binds to GST-betaFTZ-F1, GST-alphaFTZ-F1, GST-DHR3, GST-SVP, GST-USP, and GST-EcR, but not to GST alone. Thus, Bon can bind directly to many members of the nuclear receptor family in vitro (Beckstead, 2001).

To define the domain(s) of betaFTZ-F1 responsible for Bon interaction, a deletion analysis of betaFTZ-F1 was performed using the yeast two-hybrid system. Various segments of betaFTZ-F1 were fused to the VP16 AAD and assayed for DBD-Bon interaction. No increase in reporter activity was observed with the N-terminal A/B region or with a fusion protein containing residues 270-631, which include the DNA binding domain and the hinge region of the receptor. In contrast, a 7-fold activation was detected in the presence of AAD-betaFTZ-F1(555-802), indicating that the E region encompassing the putative ligand binding domain (LBD) is sufficient for interaction with Bon (Beckstead, 2001).

Sequence analysis of the E region of betaFTZ-F1 has revealed a conserved transcriptional activation domain 2 core motif (AF-2 AD core) between residues 791 and 797. To investigate its activity, an expression vector encoding the E region of betaFTZ-F1 fused to the yeast GAL4 DNA binding domain was cotransfected into Drosophila Schneider (S2) cells together with a GAL4 reporter plasmid. An increase in reporter gene activity was observed, whereas no transactivation was detected with a GAL4-betaFTZ-F1 construct lacking the AF-2 AD core. Deletion of the AF-2 AD core also abolished Bon interaction with the betaFTZ-F1 E region in yeast. Thus, the LBD of betaFTZ-F1 contains an AF-2 activation domain, whose integrity is required for Bon interaction (Beckstead, 2001).

To determine which domain of Bon interacts with betaFTZ-F1, a series of DBD-Bon deletion constructs were generated and assayed for interaction with the E region of betaFTZ-F1. No significant increase in reporter activity was observed when fusion proteins of the RBCC motif (1-450) and the PHD/bromodomain (891-1133) of Bon were coexpressed with AAD-betaFTZ-F1 E region. In contrast, a 15-fold enhancement was observed in the presence of DBD-Bon (527-700) domain. Analysis of this region has revealed a predicted alpha-helical segment extending from residues 561 to 570. This domain contains an LxxLL consensus sequence, originally identified in the nuclear receptor-interacting domain of TIF1alpha and subsequently found in many other AF-2 mediators. In the presence of AAD-betaFTZ-F1 (E) wild-type, but not AAD-betaFTZ-F1(E)DeltaAF-2 AD-core, residues 561 to 570 of Bon fused to the ERalpha DBD activate the reporter gene ~8-fold above the level of unfused AAD. Thus, Bon contains an LxxLL motif that is sufficient to interact with the LBD of betaFTZ-F1 in an AF-2-integrity-dependent manner. To investigate whether Bon actually binds betaFTZ-F1 through this LxxLL motif, mutations in Bon were generated that eliminate the conserved leucine residues at positions 566 and 567. The replacement of these leucines by alanine residues abolishes the interaction with the LBD of betaFTZ-F1 in yeast. Hence, Bon interacts with the AF-2 of betaFTZ-F1 through an LxxLL motif (Beckstead, 2001).

betaFTZ-F1 plays an important role in the stage-specific response to the prepupal ecdysone pulse by positively regulating the expression of E74A, E75B, BR-C, EDG84A, and E93, and negatively regulating its own expression. Mutant betaFTZ-F1 animals display variable defects in early pupal events such as adult head eversion, leg elongation, and salivary gland cell death. Similar phenotypes are observed in bon mutant pupae (Beckstead, 2001).

The phenotypes associated with betaFTZ-F1^ex17/Df(3L)Cat^Dh104 mutants have been categorized into three lethal pupal classes: 38% die as pharate adults with short malformed legs; 45% undergo head eversion, but arrest early in pupal development; and 17% fail to undergo head eversion, but continue developing into cryptocephalic pharate adults. All betaFTZ-F1 mutants have deformed legs (Beckstead, 2001).

Because Bon is able to interact with betaFTZ-F1 in vitro, attempts were made to establish whether Bon interacts with betaFTZ-F1 in vivo. Flies were generated with either bon²⁴¹ or bon⁴⁸⁷ in the Df(3L)Cat^Dh104/betaFTZ-F1^ex17 or betaFTZ-F1^ex17/betaFTZ-F1^ex17 mutant backgrounds and assessed for their effect on betaFTZ-F1 phenotypes. Loss of one copy of bon is able to suppress the phenotypes associated with loss of betaFTZ-F1. In the Df(3L)Cat^Dh104/betaFTZ-F1^ex17 background, partial loss of Bon rescues the majority of mutant animals to pharate adult stages: 87% for bon⁴⁸⁷ and 70% for bon²⁴¹. In the betaFTZ-F1^ex17 homozygotes, partial loss of Bon dramatically increases the number of adult escapers: 72% for bon⁴⁷⁸ and 60% for bon²⁴¹, compared to 31% in a wild-type background. In addition, one mutant copy of bon also strongly suppresses the leg phenotypes associated with loss of betaFTZ-F1. In summary, these data indicate that partial loss of Bon suppresses the phenotypes associated with a partial loss of betaFTZ-F1 (Beckstead, 2001).

betaFTZ-F1^ex17 has been shown to be a hypomorphic allele that is the result of a deletion of a positive regulatory element. Northern analysis has demonstrated that betaFTZ-F1^ex17/betaFTZ-F1^ex17 animals exhibit low levels of betaFTZ-F1 transcripts. It was therefore hypothesized that bon suppression of the betaFTZ-F1^ex17 phenotypes may be the result of betaFTZ-F1 upregulation. To test this hypothesis, Northern analysis was performed on betaFTZ-F1^ex17/+, betaFTZ-F1^ex17/betaFTZ-F1^ex17, and betaFTZ-F1^ex17 bon⁴⁸⁷/betaFTZ-F1¹⁷ staged prepupae and the levels of betaFTZ-F1 expression was estimated. One mutant copy of bon⁴⁸⁷ results in a 1.8- and a 1.9-fold upregulation of betaFTZ-F1 in betaFTZ-F1^ex17 mutants. These results suggest that suppression by bon is at least partially due to the upregulation of betaFTZ-F1 and that Bon seems to play a direct role in repressing betaFTZ-F1 expression. These data appear in contrast to, but are not inconsistent with the general loss of Bon function that affects the transcription of most nuclear receptors negatively. Because betaFTZ-F1 is a downstream effector in the ecdysone pathway, the specificity of the interaction between betaFTZ-F1 and Bon is probably masked in a severe loss of function bon animal (Beckstead, 2001).

To determine whether Bon is able to repress transcription, the coding sequence of Bon was fused to the yeast GAL4 DNA binding domain. The resulting fusion protein was tested for its ability to repress transcription activated by ER(C)-VP16, a chimeric activator containing the DBD of ERalpha fused to VP16. GAL4-Bon and ER(C)-VP16 were transiently transfected into S2 cells with a reporter containing a GAL4 binding site (17M) and an estrogen response element (ERE) in front of a thymidine kinase (tk) promoter-CAT fusion (17M-ERE-tk-CAT). GAL4-Bon efficiently represses transcription in a dose-dependent manner. In contrast, coexpression of Bon without the GAL4 DNA binding domain causes a reproducible increase in CAT activity, indicating that repression by Bon is entirely dependent on DNA binding (Beckstead, 2001).

To map the domain of Bon responsible for transcriptional repression, a set of N- and C-terminally truncated derivatives were assayed for their ability to repress VP16-activated transcription in S2 cells. In the absence of the RBCC motif, the GAL4-Bon fusion protein, GAL4-Bon [471-1133]) fails to repress transcription, indicating that the N-terminal region of Bon is required for repression. However, this region is not sufficient for full repression. Consistent with this, a C-terminal truncation, GAL4-Bon (1-890), is a less potent repressor, indicating that the C-terminal residues of the protein including the PHD finger and the bromodomain also contribute to the repression potential of Bon. However, this domain on its own exhibits little repression. A 3- to 4-fold increase in CAT activity is observed with the central region between the coiled-coil and the PHD finger, suggesting that Bon may also contain a 'masked' activation domain. Note, however, that no significant activation was observed with GAL4-Bon (471-890) tested in the absence of ER(C)-VP16. Taken together, these results indicate that most of the repression activity of Bon resides within the N-terminal RBCC domain (Beckstead, 2001).

To investigate functional consequences of the Bon-betaFTZ-F1 interaction, the transcriptional activity of betaFTZ-F1 AF-2 was assayed alone or in combination with overexpressed Bon in transiently transfected cells. Bon and the GAL4-betaFTZ-F1(E) derivative were cotransfected into S2 cells together with the GAL4-responsive reporter, 17M-ERE-tk-CAT. GAL4-betaFTZ-F1(E) exerts a trans-stimulation activity that is repressed by the addition of Bon. Taken together, these results provide support for the hypothesis that Bon plays a role in downregulating betaFTZ-F1-dependent transcription (Beckstead, 2001).

Bon and TIF1s contain an N-terminal RBCC (RING finger/B boxes/coiled coil) motif. In the absence of the RBCC motif, the GAL4-Bon protein, unlike the full-length protein, fails to repress transcription. The TIF1beta RBCC domain has been shown to be necessary for the oligomerization of TIF1beta and KRAB binding. Because Bon is able to homodimerize, this domain may be involved in formation of protein complexes (Beckstead, 2001).

The PHD finger and bromodomain are characteristic features of nuclear proteins known to be associated with chromatin and/or to function at the chromatin level. For instance, the chromosomal proteins Trithorax and Polycomb-like contain multiple PHD fingers, while the histone acetyltransferases CBP and GCN5 as well as the chromatin-remodeling factor SWI2/SNF2 are also bromodomain containing proteins. Bromodomains have been shown to bind to acetyl-lysine and specifically interact with the amino-terminal tails of histones H3 and H4, suggesting a chromatin-targeting function for this highly evolutionarily conserved domain. Because Bon is localized to hundreds of chromatin bands on Drosophila polytene chromosomes, it is probably involved in chromatin-mediated regulation of transcription of numerous genes (Beckstead, 2001).

Bon can repress both basal and activated transcription when recruited to the promoter region of a target gene, similar to TIF1alpha, -beta, and -gamma. For TIF1alpha and TIF1beta, a link between silencing and histone modification has been established, and TIF1beta is part of a large multiprotein complex that possesses histone deacetylase activity. Moreover, TIF1beta was also reported to colocalize and interact directly with members of the heterochromatin protein 1 (HP1) family. Similar to TIF1beta, TIF1alpha can bind the HP1 proteins in vitro. However, TIF1alpha-mediated repression in transfected cells does not require the integrity of the HP1 interaction domain, nor is there any significant subnuclear colocalization of HP1alpha and TIF1alpha. No interactions were observed between Bon and HP1 in a yeast two-hybrid assay, nor was any evidence found for genetic interactions. However, in a yeast two-hybrid screen, Bon interacted with members of the Polycomb group, suggesting that Bon may also be part of heterochromatin-like complexes and/or may require some of the members of the Polycomb group genes to repress transcription. This would imply that Bon has a dual role, similar to some members of the Polycomb group family: transcriptional repression and heterochromatin formation. Both of these roles may be required in transcriptional repression (Beckstead, 2001).

Upon ecdysone binding, the EcR/USP complex upregulates the expression of a group of transcription factors, many of which are nuclear receptors. During this ecdysone regulatory cascade, both induction and repression of transcription are required to regulate the timing and the response to the ecdysone signal. Bon is able to interact with many members of the nuclear receptor family, suggesting it may have a role in multiple steps during metamorphosis and affect expression of many ecdysone regulated genes. For example, DHR3, a key component of the ecdysone response, is required for patterning and integrity of the adult cuticle, and DHR3 mutant clones exhibit a loss of pigmentation, cuticle defects, and missing bristles, similar to a partial loss of Bon. In addition, mutations in betaFTZ-F1, E74B, and BR-C exhibit malformed legs, which are a result of failure in the ecdysone response pathway. Again, very similar defects are observed in bon mutants. Salivary glands in betaFTZ-F1, BR-C, and bon mutant pupae also fail to undergo apoptosis. The ability of bon mutations to cause phenotypes that resemble defects associated with mutations with multiple members of the pathway suggests that Bon is interacting with several members of the pathway at several stages, in agreement with the biochemical observations (Beckstead, 2001).

The interaction of Bon with nuclear receptors is similar to TIF1alpha but unlike TIF1beta and TIF1gamma. This interaction requires the integrity of the nuclear receptor AF-2 activation domain and is mediated by the Bon/TIF1alpha LxxLL motif. These observations suggest that Drosophila nuclear receptors and Bon have co-evolved to maintain their interaction. It is therefore likely that the biological role of this interaction has been conserved in mammals (Beckstead, 2001).

The data provide genetic evidence for the biological relevance of the interaction between Bon and the nuclear receptor betaFTZ-F1. Reduction in the level of Bon, but not the complete loss of Bon, which affects the entire pathway, suppresses the phenotypes associated with a regulatory loss of function mutation of betaFTZ-F1. This suppression is likely to be the result of an increase in the transcription of betaFTZ-F1, suggesting that Bon plays a role in the repression of betaFTZ-F1. Because betaFTZ-F1 represses its own transcription, it is likely that a protein complex containing betaFTZ-F1 and Bon is required for this repression. Removal of a copy of Bon may therefore lead to an up-regulation of betaFTZ-F1 transcription. Although these data appear to contrast the loss of betaFTZ-F1 transcription in bon²⁴¹/bon²⁴¹ mutants, they are not inconsistent. In the bon²⁴¹/bon²⁴¹ mutant background, loss of two copies of bon severely affects the entire ecdysone pathway. This is clearly not the case when one copy of bon is mutated. Therefore, removal of one copy of bon in the betaFTZ-F1 mutant background allows for the detection of protein:protein interactions between betaFTZ-F1 and Bon. Thus the phenotypic suppression and the S2 cell transcription data are in agreement with Bon functioning as a negative regulator of betaFTZ-F1-dependent transcription. It is therefore tempting to speculate by analogy that TIF1alpha may also interact with and inhibit transactivation by nuclear receptors in mammals. A model is favored in which Bon (or TIF1alpha), once recruited to particular regions of chromatin containing acetylated histones via Bon's bromodomain, interacts via Bon's bromodomain LxxLL motif with the AF-2 domain of DNA-bound nuclear receptors. This complex then represses transcription from cognate target genes, possibly via an effect on chromatin structure (Beckstead, 2001).

Search PubMed for articles about Drosophila Bonus

Allton, K., Jain, A. K., Herz, H. M., Tsai, W. W., Jung, S. Y., Qin, J., Bergmann, A., Johnson, R. L., Barton, M. C. (2009). Trim24 targets endogenous p53 for degradation. Proc Natl Acad Sci U S A, 106(28):11612-11616 PubMed ID: 19556538

Beckstead, R., Ortiz, J. A., Sanchez, C., Prokopenko, S. N., Chambon, P., Losson, R., Bellen, H. J. (2001). Bonus, a Drosophila homolog of TIF1 proteins, interacts with nuclear receptors and can inhibit betaFTZ-F1-dependent transcription. Mol Cell, 7(4):753-765 PubMed ID: 11336699

Beckstead, R. B., Ner, S. S., Hales, K. G., Grigliatti, T. A., Baker, B. S., Bellen, H. J. (2005). Bonus, a Drosophila TIF1 homolog, is a chromatin-associated protein that acts as a modifier of position-effect variegation. Genetics, 169(2):783-794 PubMed ID: 15545640

Godneeva, B., Ninova, M., Fejes Toth, K., Aravin, A. A. (2023). SUMOylation of Bonus, the Drosophila homolog of Transcription Intermediary Factor 1, safeguards germline identity by recruiting repressive chromatin complexes to silence tissue-specific genes. bioRxiv, PubMed ID: 37645991

Godneeva, B., Fejes Toth, K., Quan, B., Chou, T. F., Aravin, A. A. (2023). Impact of Germline Depletion of Bonus on Chromatin State in Drosophila Ovaries. Cells, 12(22) PubMed ID: 37998364

Ito, H., Sato, K., Koganezawa, M., Ote, M., Matsumoto, K., Hama, C., Yamamoto, D. (2012). Fruitless recruits two antagonistic chromatin factors to establish single-neuron sexual dimorphism. Cell, 149(6):1327-1338 PubMed ID: 22682252

Ito, H., Sato, K., Yamamoto, D. (2013). Sex-switching of the Drosophila brain by two antagonistic chromatin factors. Fly (Austin), 7(2):87-91 PubMed ID: 23519136

Nomura, T., Tanikawa, J., Akimaru, H., Kanei-Ishii, C., Ichikawa-Iwata, E., Khan, M. M., Ito, H., Ishii, S. (2004). Oncogenic activation of c-Myb correlates with a loss of negative regulation by TIF1beta and Ski. J Biol Chem, 279(16):16715-16726 PubMed ID: 14761981

Wisotzkey, R. G., Quijano, J. C., Stinchfield, M. J., Newfeld, S. J. (2014). New gene evolution in the bonus-TIF1-gamma/TRIM33 family impacted the architecture of the vertebrate dorsal-ventral patterning network. Mol Biol Evol, 31(9):2309-2321 PubMed ID: 24881051

Zhao, H., Moberg, K. H. and Veraksa, A. (2023). Hippo pathway and Bonus control developmental cell fate decisions in the Drosophila eye. Dev Cell 58(5): 416-434. PubMed ID: 36868234

date revised: 3 May, 2024

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