InteractiveFly: GeneBrief

multiprotein bridging factor 1: Biological Overview | References

Gene name - multiprotein bridging factor 1

Synonyms - MRE4

Cytological map position - 73A9-73A9

Function - transcription factor

Keywords - coactivator that translocates from the cytoplasm into the nucleus to induce stress-response genes - mRNA-binding protein - protects Enhancer of zeste mRNA from Pacman activity thereby ensuring Polycomb silencing - AP-1-dependent epithelial closure becomes sensitive to H2O2 in flies lacking MBF1 - co-activator for Tracheae Defective contributing to the formation of tracheal system and nervous system

Symbol - mbf1

FlyBase ID: FBgn0262732

Genetic map position - chr3L:16,574,436-16,576,408

Classification - Multiprotein bridging factor 1; Helix-turn-helix

Cellular location - cytoplasmic and nuclear

NCBI link: EntrezGene, Nucleotide, Protein
mbf1 orthologs: Biolitmine

Under stress conditions, the coactivator Multiprotein bridging factor 1 (Mbf1) translocates from the cytoplasm into the nucleus to induce stress-response genes. However, its role in the cytoplasm, where it is mainly located, has remained elusive. This study shows that Drosophila Mbf1 associates with E(z) mRNA and protects it from degradation by the exoribonuclease Pacman (Pcm), thereby ensuring Polycomb silencing. In genetic studies, loss of mbf1 function enhanced a Polycomb phenotype in Polycomb group mutants, and was accompanied by a significant reduction in E(z) mRNA expression. Furthermore, a pcm mutation suppressed the Polycomb phenotype and restored the expression level of E(z) mRNA, while pcm overexpression exhibited the Polycomb phenotype in the mbf1 mutant but not in the wild-type background. In vitro, Mbf1 protected E(z) RNA from Pcm activity. These results suggest that Mbf1 buffers fluctuations in Pcm activity to maintain an E(z) mRNA expression level sufficient for Polycomb silencing (Nishioka, 2018).

Polycomb silencing is essential for the developmental regulation of gene expression. The silencing needs to be robust to tightly repress the expression of developmental genes in undifferentiated cells, such as stem cells, but should also be flexible for rapid release upon differentiation. However, this paradoxical aspect of Polycomb silencing is not well understood (Nishioka, 2018).

Mbf1 was originally identified as an evolutionarily conserved coactivator that connects a transcriptional activator with the TATA element-binding protein (Li, 1994; Takemaru, 1997; Takemaru, 1998). Usually, Mbf1 is present in the cytoplasm; however, under stress conditions, Mbf1 translocates into the nucleus to induce stress-response genes. Previous studies have revealed roles for the coactivator in axon guidance, oxidative stress response, defense against microbial infection, and resistance to drugs such as tamoxifen. However, the cytoplasmic role of Mbf1 has remained elusive, except for mRNA or ribosomal binding (Nishioka, 2018).

Pacman (Pcm/Xrn1) is an evolutionarily conserved 5'-3' exoribonuclease that degrades decapped mRNA (Till, 1998; Jones, 2012). Genetic studies have demonstrated that Drosophila pcm is involved in epithelial closure, male fertility, apoptosis and growth control (Grima, 2008; Lim, 2009; Jones, 2012; Jones 2016; Waldron, 2015). Null mutants of pcm are lethal during early pupal stages, suggesting the enzyme plays an essential role in development (Waldron, 2015; Jones, 2016; Nishioka, 2018 and references therein).

Using a genetic approach in Drosophila, this study shows that cytoplasmic Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from degradation by Pcm. The results thus demonstrate an unexpected component of the regulatory mechanism underlying Polycomb silencing. This mechanism might also allow flexibility in Polycomb silencing, as Mbf1 protein expression declines upon differentiation (Nishioka, 2018).

To address the cytoplasmic role of Mbf1, novel genes were sought that interact with mbf1. Surprisingly, the mbf1 mutation enhanced a classical Polycomb phenotype of Psc and Pc mutants, namely the appearance of an ectopic sex comb tooth or teeth on the male mid-leg. Although mbf12/+ or mbf12/mbf12 flies never exhibited the Polycomb phenotype, penetrance of the phenotype in Psc1/+ increased significantly in Psc1/+; mbf12/+, and further increased in Psc1/+; mbf12/mbf12. The penetrance was restored to the Psc1/+ level by expressing wild-type Mbf1 protein from a transgene. Similar effects of the mbf12 allele were observed with the Pc6 mutation (Nishioka, 2018).

To gain insight into the mechanism underlying the genetic interaction between Psc and mbf1, the expression of the representative Polycomb group genes Pc, E(z) and pho was analyzed. Results of reverse transcription-quantitative PCR (RT-qPCR) analyses demonstrated a prominent reduction in the expression level of E(z) mRNA in Psc1/+; mbf12/+ larvae, whereas Pc and pho mRNA levels remained unchanged. Immunostaining of wing discs demonstrated that E(z) protein expression was severely compromised in Psc1/+; mbf12/+ compared with that in wild type, mbf12/+ or Psc1/+. By contrast, the expression of Pc and Pho proteins was not significantly affected. Western blot analyses confirmed the marked decrease in the E(z) protein level in both wing and leg discs from Psc1/+; mbf12/+. Consistently, Psc1/+; E(z)731/+ exhibited the extra sex comb phenotype, which was comparable to Psc1/+; mbf12/+ (Nishioka, 2018).

It is unlikely that Mbf1 affects E(z) transcription because no significant difference was detected in the E(z) mRNA level between wild-type and mbf12/mbf12 larvae. Consistently, it was not possible to detect any significant difference in the expression of E(z) in the wing disc upon knockdown or overexpression of Mbf1 using a posterior compartment-specific Gal4 driver. When cytoplasmic and nuclear RNA fractions from wing discs were analyzed by RT-qPCR, the nuclear E(z) mRNA level was similar between wild type and Psc1/+; mbf12/+. However, the cytoplasmic E(z) mRNA level in Psc1/+; mbf12/+ decreased to ~20% of the wild-type level. Collectively, these results suggest that mbf1 regulates the E(z) mRNA level post-transcriptionally in the cytoplasm (Nishioka, 2018).

Considering that Mbf1 binds to mRNA, it was hypothesized that cytoplasmic Mbf1 might bind to E(z) mRNA to protect it from degradation, and thereby regulates the E(z) mRNA level. Results of RNA-immunoprecipitation (RIP) experiments revealed a preferential binding of Mbf1 to E(z) mRNA. A ~10-fold enrichment of E(z) mRNA was found in the anti-Mbf1 antibody pull-down fraction from cytoplasmic extracts of embryos. The pull-down was clearly selective, as enrichment of abundant mRNAs, such as RpL32 and RpL30, was not observed. By contrast, E(z) mRNA was barely detectable in the anti-Mbf1 antibody pull-down fraction from embryonic extracts of the mbf1 mutant, used as a negative control. This is not due to absence of E(z) mRNA in the mbf1 mutant (Nishioka, 2018).

Following the observed preferential binding of Mbf1 to E(z) mRNA, this study focused on the Polycomb phenotype and reduced E(z) mRNA expression level, which were not caused by the mbf1 mutation alone. Enhancement of the Polycomb phenotype and the reduction of E(z) mRNA were only detected in the double mbf1 and Polycomb group gene mutant. To explain the synergistic effect of mbf1 and Polycomb group mutations, it was posited that a component of the mRNA degradation pathway was only activated in the Polycomb group mutant background. Therefore, attempts were made to identify the component of the pathway that was activated in the Psc or Pc mutants. Among the mRNAs tested, only pcm mRNA, which encodes the 5'-exoribonuclease, was upregulated in Psc1/+ and Pc6/+ larvae. Neither the decapping enzyme (Dcp2), components of the exosome [Dis3, Prp6 (CG6841) and Prp40 (CG3542)], nor components in the 3'-deadenylation-mediated pathway (twin and Nab2) appeared to be activated. Western blot analyses revealed a 2-fold increase in the Pcm protein level in wing discs from Psc1/+ or Pc6/+ larvae compared with that from wild type. These results led to an investigation of the effects of the pcm mutation on Polycomb silencing and E(z) mRNA expression (Nishioka, 2018).

Strikingly, the pcmΔ1 mutation resulted in significant suppression of the Polycomb phenotype in Psc1/+ and Psc1/+; mbf12/+. This suppression was rescued by expressing the wild-type Pcm protein from a transgene. Similar results were obtained using the Pc6 mutant. Consistent with this result, the pcmΔ1 mutation restored the E(z) mRNA levels in Psc1/+ and Psc1/+; mbf12/+ to near wild-type levels (Nishioka, 2018).

In addition to the extra sex comb phenotype, Psc1/+; mbf12/+ exhibited misexpression of Ubx in wing discs. The signals appeared as spots consisting of clusters of Ubx-positive cells. The pcmΔ1 mutation decreased the number of spots per wing disc. The misexpression occurred predominantly around the dorsoventral border in the posterior compartment. Consistently, adult wing defects were observed along the posterior wing margin, which was also suppressed by pcmΔ1 (Nishioka, 2018).

Importantly, the extra sex comb phenotype was detected under mild overexpression of pcm in mbf12/hs-pcm double heterozygotes at 25°C, even in the wild-type Polycomb group background. hs-pcm/+ exhibited an ~2.5-fold overexpression of Pcm at 25°C. Nevertheless, hs-pcm heterozygotes in the wild-type mbf1 background did not show any Polycomb phenotype. These results suggest that Mbf1 stabilizes Polycomb silencing against fluctuations in the Pcm protein level in vivo. Enhancement of the Polycomb phenotype was also observed in Psc1/+; hs-pcm/+ compared with that in Psc1/+ (Nishioka, 2018).

Biochemical analyses using purified recombinant Mbf1 and Pcm proteins revealed that Mbf1 protects E(z) RNA from degradation by Pcm. RNA protection assays were performed in which in vitro-synthesized E(z) RNA was treated with the RNA pyrophosphatase RppH to convert the 5'-triphosphoryl end into the 5'-monophosphoryl form, which is a Pcm substrate. The RNA was digested with Pcm in the presence or absence of Mbf1. Mbf1 inhibited the digestion of E(z) RNA. In the absence of RppH, RNA degradation was barely detectable, suggesting that the digestion was due to 5'-exoribonuclease activity. Gel filtration of a mixture of Pcm and Mbf1 resulted in the elution of each protein in a clearly separated peak. Furthermore, Mbf1 did not co-immunoprecipitate with Pcm and vice versa. These results suggest that Mbf1 does not inhibit Pcm activity through protein-protein interactions. Collectively, it is concluded that Mbf1 protects E(z) mRNA from degradation by Pcm both in vivo and in vitro (Nishioka, 2018).

It is proposed that cytoplasmic Mbf1 ensures Polycomb silencing by protecting E(z) mRNA from the activity of Pcm. In the mbf1 mutant, E(z) mRNA is free from Mbf1 protein, but pcm expression is downregulated by Polycomb group genes. In the Polycomb group mutant, Pcm expression is upregulated, but E(z) mRNA is partly protected by Mbf1. In the mbf1 Polycomb group double mutant, E(z) mRNA is free from Mbf1 protein and is subject to Pcm attack. Whereas Mbf1 is highly expressed in undifferentiated cells, such as those of embryos, larval testis, ovary, imaginal discs and neuroblasts, its expression is reduced in differentiated tissues, similar to the situation in the mbf1 mutant. This would facilitate the rapid release of developmental genes from Polycomb silencing upon differentiation. Interestingly, expression of mammalian Mbf1 [also termed endothelial differentiation-related factor 1 (Edf1)] and Ezh2 declines immediately after the onset of differentiation (Nishioka, 2018).

A recent study demonstrated that Pcm prevents apoptosis in imaginal discs and downregulates specific transcripts such as hid and reaper (Waldron, 2015). However, suppression of apoptosis did not rescue the lethality of a pcm null mutation at the early pupal stage. Therefore, there might be other targets of Pcm that are essential for early pupal development. The present study indicates that E(z) mRNA could be one such target (Nishioka, 2018).

The mRNA-binding activity of Mbf1 was selective, but might not be strictly specific to E(z) mRNA. Although Polycomb silencing is central to the developmental regulation of gene expression, there could be other mRNAs that bind to Mbf1 in a similar manner, thereby modulating another biological function. Therefore, RIP-seq analysis was conducted to identify Mbf1-bound mRNAs. To ensure robustness of the RIP-seq data, the results were compared independently with two publically available datasets and identified 804 commonly enriched mRNAs. Among these, the enrichment of four representative mRNAs (GstD5, Ide, Tep2 and Pebp1) was confirmed by RIP RT-qPCR analyses. Interestingly, the expression levels of these four mRNAs decreased in Psc1/+; mbf12/+ and increased in pcmΔ1/Y compared with those in wild type, suggesting that the model can be applied to a wider range of mRNAs than just E(z). However, dependency on the Mbf1/Pcm antagonism appears to differ among the mRNAs (Nishioka, 2018).

Gene ontology and pathway analyses of the 804 genes revealed some interesting properties of the Mbf1-associated mRNAs. The gene ontology terms 'glutathione metabolic process', 'oxidation-reduction process' and 'neurogenesis' which includes E(z), are consistent with the fact that previous studies found defects in oxidative stress defense and axon guidance in the mbf1 mutant (Liu, 2003; Jindra, 2004). Also of interest are the groups 'positive regulation of innate immune response' and 'defense response to Gram-negative bacterium', as Arabidopsis MBF1 is involved in host defense against microbial infection. Moreover, pathway analysis of the enriched genes implicated Mbf1 in 'drug metabolism', as previously suggested for tamoxifen resistance. This raises an intriguing possibility that Mbf1 contributes to various types of stress defense, metabolic processes and neurogenesis as both a nuclear coactivator and as a cytoplasmic mRNA-stabilizing protein. Although mbf1 null mutants are viable under laboratory conditions, evolutionary conservation of mbf1 suggests that it has essential role(s) under real-world stress conditions (Nishioka, 2018).

Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila

Basic leucine zipper proteins Jun and Fos form the dimeric transcription factor AP-1, essential for cell differentiation and immune and antioxidant defenses. AP-1 activity is controlled, in part, by the redox state of critical cysteine residues within the basic regions of Jun and Fos. Mutation of these cysteines contributes to oncogenic potential of Jun and Fos. How cells maintain the redox-dependent AP-1 activity at favorable levels is not known. This study shows that the conserved coactivator MBF1 is a positive modulator of AP-1. Via a direct interaction with the basic region of Drosophila Jun (D-Jun), MBF1 prevents an oxidative modification (S-cystenyl cystenylation) of the critical cysteine and stimulates AP-1 binding to DNA. Cytoplasmic MBF1 translocates to the nucleus together with a transfected D-Jun protein, suggesting that MBF1 protects nascent D-Jun also in Drosophila cells. mbf1-null mutants live shorter than mbf1+ controls in the presence of hydrogen peroxide (H2O2). An AP-1-dependent epithelial closure becomes sensitive to H2O2 in flies lacking MBF1. It is concluded that by preserving the redox-sensitive AP-1 activity, MBF1 provides an advantage during oxidative stress (Jindra, 2004).

Sensitivity of AP-1 to oxidation requires a mechanism to protect AP-1 activity. This study introduces MBF1 as a new player that allows cells to maintain adequate AP-1 activity under oxidative stress. Drosophila AP-1 components D-Jun and D-Fos undergo oxidative inactivation via the same cysteine residues as the human orthologs. MBF1 prevents this oxidation and preserves the DNA-binding activity. In mbf1 mutants, an AP-1-dependent developmental process becomes hypersensitive to oxidative stress, suggesting that MBF1 also protects D-Jun from an oxidative modification in vivo. The protection is unlikely to be complete because it relies on the binding of MBF1 to Jun. Thus, the AP-1 action may be in an equilibrium between acceleration by the MBF1 protection of Jun and brake by the oxidative inactivation of Jun (Jindra, 2004).

The mechanism by which MBF1 ensures the activity of AP-1 is different from that of the nuclear protein Ref-1, which reactivates oxidized AP-1 by reduction. MBF1 was a much stronger enhancer of AP-1 activity when coexpressed and copurified with D-Jun from E. coli than when it was added to the DNA-binding assay separately. Unlike Ref-1, MBF1 was unable to restore AP-1 activity once lost. Thus, rather than reactivating AP-1, MBF1 protects it from oxidation in a preventive manner. Protection from oxidation is however one of several stabilizing effects that MBF1 exerts on AP-1, because MBF1 can stimulate DNA binding even of mutant AP-1 proteins, possessing serine instead of the redox-sensitive cysteine residues (Jindra, 2004).

MBF1 enhanced the DNA-binding activity of AP-1 selectively through D-Jun. Since MBF1 bound D-Jun but not D-Fos in a direct interaction assay, it is proposed that the selectivity is based on an exclusive contact between MBF1 and D-Jun. This was unexpected as human MBF1 was shown to bind a GST-c-Fos fusion. On the other hand, D-Jun and c-Jun share more similarity than the Fos orthologs; in particular, the critical cysteine context KCR reads RCR in D-Fos (Jindra, 2004).

Although AP-1 regulation via the redox-sensitive cysteine residues was postulated more than a decade ago, the nature of the cysteine modification remained unknown. The prediction is that a regulatory oxidation may involve a reversible formation of sulfenic acid or a disulfide bond. To examine how the critical cysteine is modified, the molecular mass was determined of the bacterially expressed D-Jun used in DNA-binding assays. The E. coli system allowed expression of D-Jun in the absence of endogenous MBF1. Surprisingly, a previously undescribed modification was identified of the critical cysteine, S-cystenyl cystenylation. In a striking contrast, no such modification occurred in D-Jun coexpressed with MBF1 or in D-Jun lacking the critical cysteine residue. S-glutathiolation of the cysteine, a similar modification that was known to prevent binding of c-Jun to an AP-1 site, was not observed in D-Jun despite GSH:GSSG is an abundant redox system in E.coli. Whether S-cystenyl cystenylation is only a product of the prokaryotic expression system or whether it represents true physiological regulation of AP-1 activity remains to be tested. However, the aim of this study was to disclose the role for MBF1, and the ability of MBF1 to avert S-cystenyl cystenylation shows that this role is to protect D-Jun (Jindra, 2004).

While the data illuminate the role of MBF1 in the protection of the redox-sensitive cysteine in D-Jun, MBF1 also stimulated DNA binding of the serine mutant. Thus the effect of MBF1 on D-Jun is not limited to protecting the critical cysteine but includes a more general stabilizing effect on the basic region. This is consistent with the observation that yeast MBF1 enhanced DNA binding of GCN4, which harbors a serine in the position of the oxidation-sensitive cysteine. Analysis of yeast MBF1 and GCN4 indicates that this serine resides within the region contacted by MBF1. It is speculated that it is this evolutionarily ancient function of MBF1 to support the activity of bZIP proteins that permitted the acquisition of the redox regulation of AP-1 by oxidation of the critical cysteine; in the absence of MBF1, such mutation (serine to oxidation-sensitive cysteine) would be prone to the total destruction of the AP-1 activity even under mild oxidative conditions. Interestingly, the yeast counterpart of AP-1 (yAP-1) is also required for antioxidant defense and is accordingly regulated by the redox state, albeit at the level of nuclear export. The metazoan AP-1 may have introduced redox sensing at the DNA-binding step since it is directly involved in transcriptional regulation compared with the nuclear export (Jindra, 2004).

Despite the fact that evolutionary conservation of MBF1 suggests an essential role for the protein, null mutants lacking MBF1 proved to be viable in Drosophila and yeast under laboratory conditions. Strikingly, however, in both organisms, MBF1 is essential during stress situations encountered in the real world: Drosophila mbf1 mutants are sensitive to oxidative stress induced by H2O2, and yeast MBF1 mutants are unable to overcome nutritional stress due to their inability to maintain the activity of GCN4, a regulator of amino-acid synthesis. A comparative advantage provided by MBF1 under stress conditions is thus the likely cause of its evolutionary conservation. It is proposed that in both yeast and Drosophila, MBF1 achieves these functions via the same mechanism, through binding a bZIP transcription factor (Jindra, 2004).

The interaction between MBF1 and D-Jun, documented in this study, provides a molecular basis of the H2O2 sensitivity of mbf1 mutants. This is supported by the recently published evidence that JNK signaling is indeed required for oxidant resistance in Drosophila. A developmental defect that can occur in mbf1 mutants under oxidative stress is the failure to form a continuous cuticle at the dorsal midline. The cell shape changes of epithelia that occur at the dorsal closure during embryogenesis and adult morphogenesis are regulated by the JNK signaling pathway, culminating in the phosphorylation of D-Jun. Using a knockdown experiment, this study shows that also D-Jun is directly involved in the adult thorax closure. Because MBF1 exhibits a genetic interaction with AP-1 subunits under H2O2 challenge, it is likely that D-Jun requires its partner MBF1 to be protected from oxidation during its function in thorax closure. Necrotic wounds in mbf1 D-fos/mbf1 flies are a newly observed phenomenon, which may be connected with the exposure to H2O2 and may reflect a specific requirement for Fos in wound healing. Another phenotype that mbf1 mutants display is the reduced longevity when challenged with H2O2. Since AP-1 is known to trigger antioxidant defense, the idea is favored that H2O2 hypersensitivity of mbf1 mutants is also due to their failure to protect Drosophila AP-1 activity during oxidative condition. For either phenotype function, the possibility remains that MBF1 also supports functions of other transcription factors (Jindra, 2004).

MBF1 was first described as a coactivator that bridges bZIP transcription factors and the basal transcriptional machinery. Yeast MBF1 supports GCN4-dependent activation of the HIS3 gene and Drosophila MBF1 serves as a coactivator of a bZIP protein Tracheae defective/Apontic during morphogenesis of the tracheal and nervous systems (Liu, 2003). In either case, MBF1 facilitates the formation of a ternary complex consisting of the bZIP protein, MBF1 and the general transcription factor TBP. MBF1 has been recently shown to interact also with human AP-1 proteins and function as a novel transcriptional coactivator of c-Jun in a human cell line (Jindra, 2004).

Results presented in this study suggest a new function for coactivators. This study demonstrates that MBF1 can prevent an oxidative modification of D-Jun produced in bacteria, and that MBF1 activity becomes important under oxidative environmental conditions in vivo. Association of MBF1 with D-Jun in Drosophila cells and the D-Jun-dependent nuclear localization of MBF1 suggest that endogenous Jun, once synthesized, is quickly bound by MBF1. Thus it is possible that transcriptional coactivators may exert a stabilizing or protective effect on their partner transcription factors even before they engage in transcription, and that the formation of the ternary complex is a two-step phenomenon involving a preformed complex and TBP (Jindra, 2004).

Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems

During gene activation, the effect of binding of transcription factors to cis-acting DNA sequences is transmitted to RNA polymerase by means of co-activators. Although co-activators contribute to the efficiency of transcription, their developmental roles are poorly understood. Drosophila has been used to conduct molecular and genetic dissection of an evolutionarily conserved but unique co-activator, Multiprotein Bridging Factor 1 (MBF1), in a multicellular organism. Through immunoprecipitation, Drosophila Mbf1 was found to form a ternary complex including Mbf1, TATA-binding protein (TBP) and the bZIP protein Tracheae Defective (Tdf)/Apontic. A Drosophila mutant has been isolated. This mutant lacks the mbf1 gene; no stable association between TBP and TDF is detectable, and transcription of a TDF-dependent reporter gene is reduced by 80%. Although the null mutants of mbf1 are viable, tdf becomes haploinsufficient in mbf1-deficient background, causing severe lesions in tracheae and the central nervous system, similar to those resulting from a complete loss of tdf function. These data demonstrate a crucial role of MBF1 in the development of tracheae and central nervous system (Liu, 2003).

A cDNA encoding Drosophila Mbf1 was cloned from a larval CNS library. The predicted protein of 145 amino acids has 44%, 64% and 83% identity to MBF1 from yeast, human and silkmoth, respectively. MBF1 consists of two structural domains: a well-structured C-terminal half that binds the general transcription factor TBP; and a flexible N-terminal half that participates in binding to various activators. The region conserved in Drosophila Mbf1 includes both of these functional domains. Expression of Drosophila mbf1 cDNA partially rescued the yeast mbf1 mutant phenotype upon amino acid starvation, indicating that the ability to bind partner transcription factors is also conserved between yeast and Drosophila. In situ hybridization has revealed that a large amount of maternal mbf1 mRNA is deposited to the egg. Likewise, MBF1 protein is present in preblastoderm embryos and is later expressed in many tissues, including the CNS and the trachea. Widespread expression of MBF1 is also seen in post-embryonic stages, with particularly high levels in the larval salivary glands, gonads and adult gonads (Liu, 2003).

The relationship between Drosophila Mbf1 and Tdf is similar to that between yeast MBF1 and its partner transcription factor GCN4. Just as yeast MBF1 contacts GCN4 through its bZIP domain, Drosophila Mbf1 binds the bZIP domain of Tdf. Moreover, the lack of GCN4-dependent activation in yeast mbf1 mutant can be partially restored by expressing Drosophila Mbf1. The sequence and functional conservation between yeast and Drosophila Mbf1 indicates that the interaction with bZIP proteins is a conserved feature of the bridging factor MBF1 (Liu, 2003).

Genetic studies of mbf1 in yeast and Drosophila suggest that MBF1-associated transcription factors have two pathways for activation. In addition to the MBF1-mediated recruitment of TBP via its bZIP domain, GCN4 also recruits the SAGA complex with its N-terminal activation domain and effects transcription through chromatin modification. Likewise, Drosophila Tdf has a region similar to the glutamine-rich transactivation domain and may employ an activation pathway independent of recruiting TBP through Mbf1. Such a pathway may account for the residual expression of the TDS-lacZ reporter gene in the absence of Mbf1. Although Yeast MBF1 is essential for GCN4-dependent transcription of its target gene HIS3, low level of Tdf-dependent transcription of the TDS-lacZ gene can still occur in the absence of MBF1. This suggests that the relative importance of the two pathways is different between GCN4 and Tdf. The DNA-binding domain of Drosophila FTZ-F1 carries a basic region homologous to those in bZIP proteins and binds Mbf1 through this region. However, loss of mbf1 showed no effect on FTZ-F1-dependent transcription in vivo, suggesting that the activation by FTZ-F1 relied solely on the pathway through its transactivation domain. In an in vitro transcription system, the transactivation domain does not seem to be functional because FTZ622 polypeptide bearing only the DNA-binding domain of FTZ-F1 shows the same transcriptional activity as the intact FTZ-F1. This may explain the difference in the MBF1 requirement between FTZ-F1-dependent transcription in vivo and in vitro (Liu, 2003).

It is possible that the role of Mbf1 becomes more critical under certain circumstances, when rapid induction of gene expression is demanded by environmental conditions. The expression of the TDS-lacZ reporter in mbf1- background varies considerably from embryo to embryo, suggesting that certain conditions that are uncontrolled in these experiments may render transcription particularly dependent on the Mbf1-mediated pathway. In the natural environment, there are many stimuli that alter the gene expression profile: UV radiation, poison agents, nutrient starvation and so on. Therefore, direct recruitment of TBP by Mbf1 may become essential for rapid activation of transcription under such conditions. In agreement with this idea, the yeast mbf1 disruptant is viable under normal culture conditions, but sensitive to amino acid starvation (Liu, 2003).

Studies on MBF1 homologs also support the idea that MBF1 may function when gene expression is required in response to developmental or environmental signals. Rat MBF1 has been isolated as a calmodulin-associated peptide 19 (CAP-19) and human MBF1 has been identified as endothelial differentiation-related factor 1 (EDF1). EDF1/MBF1 is downregulated when endothelial cells are induced to differentiate. Interestingly, EDF1/MBF1 binds to calmodulin in the cytoplasm under low Ca2+ conditions but the two proteins dissociate when intracellular Ca2+ is high. The released EDF1/MBF1 is then phosphorylated and shuttled into the nucleus, where it binds TBP. Nuclear translocation of MBF1 has also been observed at a specific stage of molting in the silkworm B. mori. Considering the Ca2+ elevation upon exposure to the molting hormone ecdysteroid , these data raise an intriguing possibility that MBF1 is involved in Ca2+-induced gene activation. Although in this study the developmental roles of MBF1 were studied only in association with Tdf function, Drosophila Mbf1 may also be involved in other biological processes, such as stress response, homeostasis and longevity (Liu, 2003).

Several lines of evidence suggest that Drosophila Mbf1 has partners other than Tdf. Mbf1 is expressed in a wide spatiotemporal pattern, including tissues and stages where Tdf is absent. Although Tdf is not expressed in the salivary gland, immunolocalization of Mbf1 on salivary gland chromosome revealed a large number of loci associated with Mbf1. Furthermore, FLAG-tagged Mbf1 pulled down many proteins besides Tdf. Although mbf1-null mutants are viable under laboratory conditions, tdf becomes haploinsufficient in mbf1- genetic background, clearly indicating the importance of Mbf1 in the expression of the genomic information. This finding opens a way to identify new partners of Mbf1 through genetic screening for loci that exhibit dominant phenotypes in the absence of Mbf1. Characterization of Mbf1 partners will contribute to the knowledge of how co-activators mediate specific biological events (Liu, 2003).

Multiprotein bridging factor 1 (MBF1) is an evolutionarily conserved transcriptional coactivator that connects a regulatory factor and TATA element-binding protein

One Drosophila Tbp interaction with transcription factors involves a coactivator of the transcription factor FTZ-F1. The coactivator, Multiprotein bridging factor 1 (MBF1), makes possible a connection, or bridges, the TATA box-binding protein (Tbp) and the nuclear hormone receptor FTZ-F1. MBF1 is functional in interactions with Tbp and a positive cofactor MBF2. MBF1 makes a direct contact with FTZ-F1 through the C-terminal region of the FTZ-F1 DNA-binding domain and stimulates the binding of FTZ-F1 to its recognition site. The central region of MBF1 (residues 35-113) is essential for the binding of FTZ-F1, MBF2, and Tbp. MBF1, in the presence of MBF2, and FTZ622 bearing the FTZ-F1 DNA-binding domain, support selective transcriptional activation of the fushi tarazu gene. Mutations that disrupt the binding of FTZ622 to DNA or MBF1, or an MBF2 mutation that disrupts the binding to MBF1, all abolish the selective activation of transcription. These results suggest that tethering the positive cofactor MBF2 to a FTZ-F1-binding site through FTZ-F1 and MBF1 is essential for the binding site-dependent activation of transcription (Takemaru, 1997).


Search PubMed for articles about Drosophila Mbf1

Grima, D. P., Sullivan, M., Zabolotskaya, M. V., Browne, C., Seago, J., Wan, K. C., Okada, Y. and Newbury, S. F. (2008). The 5'-3' exoribonuclease pacman is required for epithelial sheet sealing in Drosophila and genetically interacts with the phosphatase puckered. Biol Cell 100(12): 687-701. PubMed ID: 18547166

Jindra, M., Gaziova, I., Uhlirova, M., Okabe, M., Hiromi, Y. and Hirose, S. (2004). Coactivator MBF1 preserves the redox-dependent AP-1 activity during oxidative stress in Drosophila. EMBO J 23(17): 3538-3547. PubMed ID: 15306851

Jones, C. I., Zabolotskaya, M. V. and Newbury, S. F. (2012). The 5' --> 3' exoribonuclease XRN1/Pacman and its functions in cellular processes and development. Wiley Interdiscip Rev RNA 3(4): 455-468. PubMed ID: 22383165

Jones, C. I., Pashler, A. L., Towler, B. P., Robinson, S. R. and Newbury, S. F. (2016). RNA-seq reveals post-transcriptional regulation of Drosophila insulin-like peptide dilp8 and the neuropeptide-like precursor Nplp2 by the exoribonuclease Pacman/XRN1. Nucleic Acids Res 44(1): 267-280. PubMed ID: 26656493

Li, F. Q., Ueda, H. and Hirose, S. (1994). Mediators of activation of fushi tarazu gene transcription by BmFTZ-F1. Mol Cell Biol 14(5): 3013-3021. PubMed ID: 8164657

Lim, A. K., Tao, L. and Kai, T. (2009). piRNAs mediate posttranscriptional retroelement silencing and localization to pi-bodies in the Drosophila germline. J Cell Biol 186(3): 333-342. PubMed ID: 19651888

Liu, Q. X., Jindra, M., Ueda, H., Hiromi, Y. and Hirose, S. (2003). Drosophila MBF1 is a co-activator for Tracheae Defective and contributes to the formation of tracheal and nervous systems. Development 130(4): 719-728. PubMed ID: 12506002

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Biological Overview

date revised: 22 October 2018

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