InteractiveFly: GeneBrief

female sterile (1) homeotic: Biological Overview | References

More than a dozen trithorax group (trxG) proteins are involved in activation of Drosophila HOX genes. How they act coordinately to integrate signals from distantly located enhancers is not fully understood. The female sterile (1) homeotic [fs(1)h] gene is one of the trxG genes that is most critical for Ultrabithorax (Ubx) activation. One of the two double-bromodomain proteins encoded by fs(1)h acts as an essential factor in the Ubx proximal promoter. Three aspects are noted: (1) overexpression of the small isoform FSH-S, but not the larger one, can induce ectopic expression of HOX genes and cause body malformation; (3) FSH-S can stimulate Ubx promoter in cultured cells through a critical proximal region in a bromodomain-dependent manner; (3) purified FSH-S can bind specifically to a motif within this region that was previously known as the ZESTE site. The physiological relevance of FSH-S is ascertained using transgenic embryos containing a modified Ubx proximal promoter and chromatin immunoprecipitation. In addition, FSH-S is involved in phosphorylation of itself and other regulatory factors. It is suggested that FSH-S acts as a critical component of a regulatory circuitry mediating long-range effects of distant enhancers (Chang, 2007).

Drosophila HOX genes control development of body segments via highly restricted expression domains. These domains are first established by transiently expressed segmentation genes in early embryos and then maintained in an epigenetically heritable manner by the Polycomb group (PcG) of repressors, and the trithorax group (trxG) of activators. Like mammalian promoters that are regulated by distant elements, transcriptional regulation of HOX genes also requires coordinated long-range interactions between the basal transcription machinery assembled around the initiation sites and factors recruited at distant regulatory elements. How the epigenetic inheritance imposed by PcG and trxG is integrated into the general framework of such long-range interactions remains unclear. Its elucidation should provide an important model for understanding the regulatory mechanisms of genes under strict developmental control (Chang, 2007).

PcG repressors form at least two types of multimeric complexes that are targeted by sequence-specific binding proteins to a core PcG response element located ~25 kb upstream of the homeotic gene Ultrabithorax (Ubx). These complexes may block the access of the regulatory elements or modify chromatin by associated histone deacetylase and histone methyltransferase activities. In contrast to the highly targeted activities of PcG repressors, trxG activators appear to employ diverse mechanisms for chromatin remodeling and long-range interactions. For example, trithorax (trx) and absent, small or homeotic discs 1 (ash1) encode histone methyltransferases that are targeted to PcG response elements, promoters, and transcribed regions. In addition to these targeted activities, brahma, moira, and osa encode subunits of an ATP-dependent chromatin remodeling complex that can modulate the nucleosome fluidity to provide an open access of regulatory sequences. Moreover, kohtalo and skuld encode subunits of the Mediator coactivator complex that can facilitate interactions between distal factors and basal transcription machinery (Chang, 2007).

How signals provided by distal elements are integrated at the Ubx basal promoter remains unclear. The Ubx proximal region has several unique features. Instead of the consensus TATA box in the −30 region, Ubx contains the initiator around +1 and the downstream promoter element around +30, which are frequently found in genes lacking the TATA box in Drosophila and mammals. The ability of these elements to support Ubx transcription in vitro and in vivo indicates that they represent an authentic basal promoter. However, this basal promoter fails to integrate regulatory signals from distant elements without a proximal region from −200 to −32, revealing a critical requirement for this region in mediating long-range interactions (Chang, 2007).

Interestingly, this critical proximal region (CPR) contains multiple binding sites for Zeste and Trithorax-like (Trl) proteins. The Zeste sites appear to be particularly important, since CPR activity can be substantially replaced by tandem Zeste sites. Consistent with the transactivating role, zeste was initially identified as required for Ubx expression through transvection, a pairing-dependent effect believed to facilitate the transutilization of the regulatory elements on one chromosome by the promoter on homologous chromosome. Zeste protein can stimulate Ubx transcription in vitro and is necessary for the expression of Ubx transgenes containing subsets of regulatory sequences. Paradoxically, zeste is not essential for normal development or for expression of the endogenous Ubx promoter or a Ubx transgene with more complete regulatory sequences. The role of zeste is further complicated by the finding that zeste may be involved in Ubx repression. Clearly, other factors must be required for the activating effect of the Zeste sites in the CPR (Chang, 2007).

The maternal-effect gene female sterile (1) homeotic [fs(1)h] was identified as a transactivator of Ubx by its strong genetic interactions with Ubx, trx, and ash1 mutations (Digan, 1986; Gans, 1980; Shearn, 1989). However, its direct role in homeotic gene activation has been obscured by complex phenotypes in mutant embryos (Huang, 1990). Sequence analysis indicates that fs(1)h encodes two putative proteins of approximately 120 and 210 kDa. The small isoform FSH-S, containing two widely spaced bromodomains (Haynes, 1992; Tamkun, 1992) and the extra terminal (ET) domain at its C terminus (Lygerou, 1994), is identical to the N-terminal half of the large isoform FSH-L (Haynes, 1989). Bromodomains can bind acetylated lysine or histones and are frequently found in transcription or chromatin modification factors, whereas ET domains are found in a small family of double-bromodomain proteins (BET proteins) with no designated function. Several interesting properties have been shown for mammalian BET proteins. For example, human RING3 (or BRD2) is a growth-stimulated nuclear kinase acting on serine and threonine. Mouse BRD2-like protein can be copurified with the Mediator transcriptional coactivator complex. Recently, mouse BRD4 has been shown to be involved in the recruitment of positive transcription elongation factor b (Chang, 2007).

This report provides several lines of evidence to support a direct role of fs(1)h in homeotic gene activation and the idea that FSH-S is primarily responsible for this function. Furthermore, it is shown that FSH-S acts directly on the Zeste site of the CPR. These results support a critical role for FSH-S in integrating signals from distal factors (Chang, 2007).

While many trxG mutations were identified by their suppressing effects on specific homeotic phenotypes caused by PcG mutations, their contributions to regulation of individual HOX genes have not been systematically examined. To address this issue, the effect of mutations of 18 trxG genes was examined on homeotic phenotypes caused by reduced Ubx expression, i.e., transformation of the third to second thoracic segment in adult flies. Interestingly, only fs(1)h [i.e., Df(1)C128], trx, and ash1 mutations showed strong enhancement on Ubx¹³⁰ phenotypes (increased from <1% to ~10%). Other trxG mutations showed weak or no effects on Ubx¹³⁰ mutation, despite that many could suppress PcG phenotypes as strongly as trx mutations. Thus, Ubx activation appeared to be highly sensitive to the dosages of fs(1)h, trx, and ash1. This selective effect was further supported by genetic interactions between fs(1)h and other trxG mutations. Again, fs(1)h showed strong synergistic effects with trx (>20%) and ash1 mutations (~10%) on Ubx phenotypes. By contrast, it showed weaker or no interactions with other trxG mutations. These results strongly suggest that fs(1)h, trx, and ash1 share some common role in certain critical steps of Ubx activation (Chang, 2007).

Loss of fs(1)h function results in complex defects in early embryos, leading to severe body distortion and lethality (Forquignon, 1981; Huang, 1990). These defects hamper the analysis of the role of fs(1)h in Ubx activation. To circumvent these problems, fs(1)h function was inactivated by shifting heat-sensitive fs(1)h¹ mutant embryos from the permissive temperature (21°C) to the restrictive temperature (29°C) during the onset of gastrulation. In wild-type embryos, high levels of Ubx transcripts can be detected in the ventral nerve cord (VNC) in a domain encompassing parasegments (PS) 5 to 12. In mutant embryos, a marked reduction of Ubx transcripts was seen. By contrast, no change was observed for caudal (cad), a HOX gene controlling the development of most posterior segments. Thus, fs(1)h appeared to be required for a subset of HOX genes (Chang, 2007).

fs(1)h encodes two double-bromodomain proteins, FSH-S and FSH-L. To define their roles in HOX activation, the Gal4/UAS binary system was used to induce high levels of FSH-S or FSH-L and examine their effects on HOX expression. UAS transgenes containing epitope-tagged FSH-S or FSH-L were driven by dpp-Gal4 in small subsets of imaginal cells. Targeted expression of FSH-S caused striking defects in the adult. Frequently, adults heads lacked maxillary palpi, and their aristae were transformed into distal legs with claws. Severe defects were also found in thoracic legs, including bifurcation of tibial segments and deletion of tarsal segments. Surprisingly, no discernible defect was seen in adults with targeted FSH-L expression, suggesting that FSH-L and FSH-S act differently (Chang, 2007).

Antenna-to-leg transformations can be induced by ectopic expression of the HOX gene Antennapedia (Antp) in antennal discs. To determine whether extra legs induced by FSH-S might be related to ectopic Antp expression, eye-antennal discs from third instar larvae were stained with an anti-ANTP antibody. Whereas ANTP is normally not expressed in these discs, strong ANTP signals were seen in antennal discs of transgenic animals. Using an anti-Flag antibody to mark tagged FSH-S, extensive overlaps were found between FSH-S and ANTP signals, suggesting that FSH-S is directly involved in ANTP induction. By contrast, no ectopic ANTP was induced by FSH-L, which was consistent with the normal appearance of adult flies. These effects further distinguished the role of FSH-S and FSH-L in HOX activation. Curiously, very little ANTP expression was induced by FSH-S in eye discs, despite its comparable levels in antennal and eye discs. The nature of this tissue-dependent response is unclear. Furthermore, no ectopic Ubx signal was found in eye-antennal and other discs. To avoid problems caused by induction timing or tissue dependence, an en-Gal4 line was used to drive FSH-S expression. Under such conditions, most larvae died before the third instar, while rare adult escapers (less than 1%) showed partial deletion of thoracic segments. In second instar larvae, ectopic Ubx signals could be detected in ventral ganglions. In addition to the transverse rows normally found within PS5 to PS12, Ubx signals appeared in small clusters of cells near the lateral margins of PS4, PS3, and PS2 at anteriorly diminishing frequencies. Occasionally, ectopic Ubx was found in PS4 extending to PS2 on both sides of the ganglion. These results strongly suggested that FSH-S can induce HOX genes (Chang, 2007).

Previous analysis predicted that fs(1)h protein products might be membrane associated, implicating a role in signal transduction. To further characterize FSH-S, antibodies were raised against three regions (S1, S2, and S3) common to both FSH-S and FSH-L. Using affinity-purified antibodies, two common bands were detected in embryonic extracts. The sizes of these two bands were consistent with predicted sizes of FSH proteins (~210 and ~120 kDa). In addition, an antibody specific for FSH-L (i.e., L3) reacted only with the larger protein. The authenticity of these proteins was further confirmed by the analysis of a larval-lethal mutant, fs(1)h¹⁷, which results from an insertion of a copia element in the intron following the FSH-S coding sequences. Unlike many other fs(1)h mutations, this mutation did not cause homeotic effects. Interestingly, the larger protein was severely diminished in mutant larvae at third instar, while the small one was unaffected. These results indicate that these proteins represent the two specific FSH isoforms and, more importantly, that FSH-L is not essential for the homeotic effect (Chang, 2007).

The developmental profiles and subcellular localization of FSH proteins in embryos were analyzed by immunostaining with affinity-purified S1 antibody. Muclear staining was clearly seen in syncytial embryos. Although S1 antibody reacted with both FSH-S and FSH-L, this nuclear staining was attributed to FSH-S, since FSH-L is primarily a centrosomal protein at this stage. In addition, tagged FSH-S was localized in the nuclei in both transgenic lines. The staining intensity appeared to be uniform throughout all developmental stages except in those cells located near invaginating furrows or in VNC. To further confirm the distribution pattern of FSH-S, whole-mount in situ hybridization was performed using a probe from the 3' UTR of FSH-S mRNA, which is absent in FSH-L mRNA. Again, a ubiquitous distribution of FSH-S transcripts was observed (Chang, 2007).

The genetic interactions, induction of HOX gene expression, nuclear localization, and the presence of a double bromodomain raised a strong possibility that FSH-S might directly affect HOX promoters. To test this, the ability of FSH-S to stimulate reporter constructs containing various promoters was tested by cotransfection experiments in a Drosophila haploid cell line which was shown to recapitulate Ubx regulation by trx and Pc. Reporter activities from constructs containing the P1 or P2 promoters of Antp or promoters of Ubx and the Heat shock protein 70 (Hsp70) were assayed following cotransfection of an Act5C-FSH-S effector or an Act5C control vector. The activities of the Antp-P2 and Ubx promoters were stimulated approximately 10-fold and 20-fold, respectively, while the Antp-P1 and Hsp70 promoters were only weakly affected. Thus, the effect of FSH-S appeared to be highly selective (Chang, 2007).

Several trxG genes have been shown to act on regulatory sequences located about 20 kb upstream of the initiation site. Since the UC construct used in this study only contained sequences from −3142 to +360, FSH-S appeared to act via distinct sequences near the basal promoter. To identify the FSH-S response elements (FRE), the effect of FSH-S on a series of Ubx deletion mutants was analyzed. Sequential deletion of 5' sequences from −3142 to −1762 (5Δ1), −628 (5Δ2), or −226 (5Δ3) did not alter the ability of the Ubx promoter to respond to cotransfected FSH-S. Deletion from +360 to +161 (3Δ1) resulted in a general reduction of the promoter activity by about twofold, regardless of the presence or absence of cotransfected FSH-S. Since the stimulatory effect of FSH-S was not affected, this downstream region most likely contains a positive element that is unrelated to FRE. No further effect was observed when sequences from +161 to +36 (3Δ2) were deleted. These results indicated that the FRE is not present in the regions upstream of −226 or downstream of +36. Consistently, a construct containing sequences from −226 to + 36 (3Δ22) was sufficient to respond to FSH-S. Conversely, an internal deletion of sequence from −200 to −32 (InΔ1; In is initiator) almost completely abolished the promoter activity. Since the initiator (ACATTC from −2 to +4) and downstream promoter elements (GGATA from +23 to +27) were intact in InΔ1 construct, the inactivation of the Ubx promoter should reflect the removal of regulatory elements. These results led to the conclusion that the FRE is located between −200 and −32, which corresponds to the CPR determined previously. Further refinement of the boundaries of the FRE was unsuccessful, since deletions from −226 to −127 (3Δ23) or from −127 to −32 inactivated the promoter (Chang, 2007).

Whether any specific domain of FSH-S is required for transactivation was examined. Mutant constructs carrying deletions of the N-terminal half of the first bromodomain (Δ1), the entire second bromodomain (Δ2) and its flanking sequences (Δ3), or both bromodomains (Δ12) or the C-terminal sequences including the ET domain (Δ4-6) were tested in transfection assays. It appeared that deletion of the first bromodomain results in a complete inactivation of FSH-S, suggesting a critical requirement of this domain. However, the full activity of FSH-S was also dependent on the second bromodomain and ET domain, since deletion of these domains resulted in partial inactivation. Interestingly, although FSH-L contains the entire FSH-S sequence, it appeared to be much less active than FSH-S. These results are consistent with the observation that FSH-L could not induce HOX genes in imaginal tissues and support further that FSH-S is primarily, if not exclusively, responsible for the transactivation function of fs(1)h (Chang, 2007).

Next, whether FSH-S could bind any specific sequences in the CPR was examined. An inducible S2 cell line containing the metallothionein promoter-driven Flag-tagged FSH-S was established. Tagged FSH-S was purified by immunoaffinity chromatography from whole-cell extracts after (NH₄)₂SO₄ enrichment. In addition to the major band corresponding to FSH-S, several less abundant proteins were also copurified. Although fs(1)h mutant showed strong genetic interactions with trx or ash1 mutants, FSH-S was not copurified with these proteins or Osa. In addition, FSH-S was not associated with Zeste protein, which was shown to bind the CPR. The ability of purified FSH-S to bind specific sequences of the CPR was demonstrated by EMSAs. Upon addition of increasing amounts of FSH-S to labeled Ubx-5 probe, a slower-migrating band appeared near the top of 3.5% native polyacrylamide gels, indicating the formation of protein-DNA complexes. The exceedingly slow mobility of this band suggested that a multisubunit protein complex is involved. FSH-S is a constituent of this putative complex, since a small but significant supershift was observed when an antibody against FSH-S was briefly incubated with FSH-S protein. A supershift was not observed when an antibody to FSH-L was used instead. Furthermore, this binding was sequence specific, since it could be completely blocked by the addition of excess amounts of unlabeled Ubx-5 or Ubx-6 but not by a random DNA fragment. Similar results were also obtained when Ubx-6 was used as the probe. To further narrow the binding region, four smaller probes from the CPR were used for EMSA. Specific binding was observed with probes Ubx-5b (−167 to ~−94) and Ubx-6a (−104 to ~−35) but not Ubx-5a (−226 to ~−146) or Ubx-6b (−55 to ~+36), indicating that the FRE is located between −167 and −35 (Chang, 2007).

The CPR contains clusters of binding sites for Zeste, Trl (also known as GAGA factor), and NTF-1. To determine whether any of these sites might correspond to FRE, competition assays were performed with DNA fragments containing tandem repeats of Zeste, Trl, or NTF-1 binding sites. Interestingly, only Zeste repeats effectively blocked binding activity. To exclude the possibility that fortuitous binding sites might be generated by multimerizaton of these repeats, an oligonucleotide containing one consensus Zeste site (CGAGTG) was tested with different flanking sequences. This oligonucleotide also blocked the binding activity of FSH-S. Thus, the Zeste site should represent the core FRE (Chang, 2007).

For further analyses of DNA binding properties, FSH-S and recombinant Zeste proteins were compared by an in-gel chemical footprinting technique. The DNA-cleaving ions OP-Cu used in this study gain more access to unprotected sequences than DNase I and are thus capable of revealing detailed differences in binding properties. Similar to studies with DNase I, three sites (Z1 to Z3) were protected by Zeste or FSH-S proteins in the Ubx-6a fragment. Despite an overall similarity, several important differences between these patterns were noticed. For example, the regions unprotected by Zeste produced bands with intensities comparable to those from free probes. However, fainter intervening bands were produced by FSH-S, suggesting weak protection on flanking sequences. Two additional differences were found over the Z1 site. FSH-S appeared to protect more 5′ sequences than Zeste. However, Zeste produced several bands more intense than the control, suggesting DNA distortion in this region. The lack of detectable Zeste protein and the distinct DNA binding properties exhibited by FSH-S clearly support the involvement of a novel binding factor (Chang, 2007).

If the FRE indeed corresponds to the Zeste site, the function of the Zeste site might be inactivated by fs(1)h mutations. Therefore, the effects were examined of fs(1)h mutation on expression of Ubx-lacZ transgenes containing two distal regulatory domains (BXD and ABX) and ~3 kb of immediate upstream sequences in addition to a wild-type CPR (Uβ) or tandem Zeste sites (Uβ-Z). In the wild-type background, strong lacZ signals were observed from PS5 to more posterior parts of the VNC in Uβ embryos. In addition, there was weaker misexpression in anterior parts of the VNC. The misexpression was more pronounced in Uβ-Z embryos. More importantly, lacZ transcripts were severely reduced throughout the entire VNC in both Uβ and Uβ-Z embryos upon inactivation of fs(1)h, indicating a strict requirement of fs(1)h. These results strongly support the physiological relevance of FSH-S to the Zeste site (Chang, 2007).

To further demonstrate that FSH-S is indeed associated with CPR of the endogenous promoter in vivo, chromatin immunoprecipitation assays were performed with formaldehyde-fixed chromatin prepared from male fs(1)h¹⁷ mutant larvae, which contain normal levels of FSH-S but diminishing amounts of FSH-L. Using five pairs of primers to cover sequences of more than 2 kb around Ubx start sites, it was found that FSH-S is preferentially associated with a CPR-containing DNA fragment. Although the antibody used here could cross-react with FSH-L, the contribution of FSH-L to the binding is excluded, because only a minute amount of FSH-L was present in fs(1)h¹⁷ mutant larvae, and, more importantly, no FSH-L signal was detectable in the Ubx promoter. In addition, this association appeared to be promoter specific, since only background signal was detected in the cad promoter (Chang, 2007).

Human RING3 protein, a FSH-S-like protein, has been shown to be a novel nuclear Ser/Thr kinase with scrambled subdomains (Denis, 1996). However, subsequent studies failed to show this activity in the mouse counterpart, FSRG-1, despite more than 90% sequence identity (Rhee, 1998). To determine whether FSH-S could act as a kinase, the kinase activity in FSH-S preparations was examined. Addition of [γ-³²P]ATP resulted in substantial phosphorylation of FSH-S and an additional protein of ~56 kDa. Because this smaller protein was consistently copurified, it will be referred to as FAP56 (FSH-associated protein of 56 kDa). Phosphoamino-acid analysis of in vitro phosphorylated proteins revealed that FSH-S was phosphorylated at the serine residue, while FAP56 was phosphorylated at both serine and threonine residues. Although FSH-S phosphorylation was readily detected by radioactive labeling, no mass increase was found upon incubation with 0.1 mM ATP. However, when treated with calf intestine phosphatase, the mass of FSH-S appeared to decrease slightly, indicating a limited phosphorylation of FSH-S (Chang, 2007).

The kinase activity of RING3 kinase could be restored by renaturation on nitrocellular filter after SDS-PAGE. Using this procedure, no FSH-S phosphorylation was detected in parallel experiments. However, it was reasoned that if FSH-S is a kinase, it must be able to bind ATP. An ATP analog, FSBA, has been used for affinity labeling of ATP binding proteins including kinases. Therefore, the reactivity of FSH-S toward FSBA was examined. Using an FSBA-specific antibody, it was found that FSH-S could indeed be covalently linked to FSBA. More importantly, the degree of cross-linking was substantially reduced by excessive ATP, indicating that FSH-S can bind ATP specifically (Chang, 2007).

Addition of FSH-S to cell extracts in which endogenous kinases were heat inactivated resulted in phosphorylation of many proteins, suggesting the presence of many kinase substrates. The clustering of multiple binding sites for FSH-S (or Zeste) and Trl in the CPR suggests that they might be spatially juxtaposed upon binding to the CPR, raising the possibility that Trl might be a potential kinase substrate. Using in vitro kinase assays, it was found that addition of FSH-S to purified recombinant Trl indeed resulted in its phosphorylation (Chang, 2007).

This report has provided several lines of evidence to support a direct role of FSH-S in HOX gene activation. Unlike other trxG proteins, FSH-S acts directly on the CPR of the Ubx promoter. The revelation of several interesting properties of FSH-S offers important mechanistic insights into the Ubx regulatory circuitry. Lack of functional fs(1)h is known to cause complex developmental defects including homeotic transformation and early embryonic lethality. The contribution of two different fs(1)h products to these effects had not been determined. Based on the following observations, it is suggested that FSH-S is primarily involved in HOX regulation. First, it was shown that FSH-S, but not FSH-L, can effectively activate homeotic promoters in imaginal discs and cultured cells. Second, FSH-S is a nuclear protein, while FSH-L is mainly found in centrosomes and is involved in organization of mitotic spindles in early embryos. Third, FSH-S can bind and function both in vitro and in vivo through a specific motif in the CPR. Lastly, no homeotic phenotype has been observed in an fs(1)h¹⁷ mutant lacking FSH-L. Thus, FSH-S is directly responsible for the homeotic effect of fs(1)h. Although FSH-L contains the entire sequence of FSH-S, these results clearly indicate that it does not play any significant role in the homeotic effect. The complex developmental functions of fs(1)h are very likely to be divided between different isoforms (Chang, 2007).

The abilities of FSH-S to bind a specific motif and to affect promoter activity through the CPR indicate that FSH-S plays an important role at the CPR for activation of the Ubx promoter. Among 18 trxG genes examined, fs(1)h, trx, and ash1 form a small but interesting subgroup that is most critical for Ubx activation and is known to act through specific regulatory sequences. Previous studies have shown that TRX and ASH1 act primarily through distal sequences that are essential for domain-specific Ubx expression. Recently, they have also been implicated in transcriptional elongation by their association with promoter and transcribed sequences. FSH-S is the only factor that functions primarily, if not entirely, on the CPR. Given the critical role of the CPR in promoter activity, FSH-S is very likely to play a key role in integration of activating signals from distal elements and factors. The strong synergistic effects reported in this study for fs(1)h, trx, and ash1 mutations indicate that they are involved in a critical step of Ubx promoter activation and that intimate functional relationships probably exist between these factors. Although they appear to exist in distinct protein complexes, it is highly likely that they interact directly or through associated factors. Such interactions may facilitate the action of TRX and ASH1 in the promoter and more downstream regions. An alternative (but not mutually exclusive) possibility is that FSH-S might be involved in attenuation of the repressing activity of PcG proteins. Since the distal response elements for PcG proteins and TRX/ASH1 are largely overlapping and their histone modification activities are functionally antagonistic, destabilization of PcG complexes could result in more efficient occupancy and/or more potent chromatin modification by TRX and ASH1. In either case, the activities of these distal factors might also be modulated by the kinase activity associated with FSH-S (Chang, 2007).

The stimulatory effects of FSH-S on the Ubx basal promoter also suggest that FSH-S may directly affect the basal transcription machinery. A closely related Saccharomyces cerevisiae protein, BDF1, has been shown to be a TFIID-associated factor, acting potentially as a functional substitute for TAF1 in higher organisms (Matangkasombut, 2000). Mouse BRD4 stimulates transcription by binding to positive transcription elongation factor b (Jang, 2005; Yang, 2005). Although it is unclear whether FSH-S possesses similar activities, the presence of structurally similar domains suggests that it may interact with these basal transcription factors. Therefore, it is speculated that FSH-S provides a dual interface for interactions with distal factors and basal transcriptional machinery for optimal Ubx transcription (Chang, 2007).

The sharing of the same target sequences between FSH-S and Zeste may help clarify a long-standing enigma about the role of Zeste in Ubx regulation. The function of zeste was revealed by a pairing-dependent phenomenon called transvection in which Ubx alleles with defective promoters can partially complement alleles with impaired regulatory sequences. Thus, zeste can facilitate the transutilization of the regulatory sequences on one chromosome by the Ubx promoter on a paired homologous chromosome. However, zeste is not required for expression of an intact endogenous Ubx gene or expression of a Ubx transgene containing more complete regulatory sequences (i.e., 35-kb sequences in 35UZ transgene), despite the fact that Zeste binds to the CPR and is required for expression of Ubx transgenes containing partial regulatory sequences. Moreover, zeste is dispensable for viability. These findings indicate that zeste is not essential for Ubx expression under normal genetic contexts. In contrast, FSH-S is indispensable for Ubx regulation and for development. The ability of FSH-S to bind the same target sequences indicates that FSH-S represents a critical component of a regulatory circuitry that utilizes regulatory signals present on the same chromosome to insure proper transcription of intact Ubx promoter. It is interesting that zeste-independent transvection has also been found that appears to employ the mechanisms that normally operate between the distal elements and the proximal promoter. It is speculated that FSH-S is also very likely to play a role in zeste-independent transvection (Chang, 2007).

The finding of DNA binding activity in FSH-S is surprising, since the double-bromodomain and the C-terminal ET domain, two prominent domains required for the function of FSH-S, are not known for DNA binding activity. It is possible that FSH-S may possess a novel DNA binding domain. Alternatively, the binding activity might be contributed by a factor that is associated with FSH-S, since several proteins were copurified with FSH-S and since recombinant FSH-S did not show the same activity. Further characterization of FSH-S and associated factors is necessary to resolve this question (Chang, 2007).

Another interesting feature of FSH-S is the kinase activity. The structural similarities to the RING3 nuclear kinase, the detection of a similar kinase activity, and the ATP binding activity are consistent with the notion that FSH-S contains a Ser/Thr kinase activity. However, the lack of kinase activity in bacterially expressed FSH-S suggests that posttranslational modification or an additional factor(s) is required for such an activity. It is interesting that, in addition to FSH-S and FAP56, many other proteins including Trl can be phosphorylated by FSH-S in vitro, suggesting a broad substrate specificity. Thus, it seems plausible that FSH-S may modulate the activities of factors that are brought into its proximity. Once it occupies the CPR, it is speculated that FSH-S may affect multiple factors that are in close contact with CPR by either short- or long-range interactions (Chang, 2007).

Search PubMed for articles about Drosophila fs(1)h

Chang, Y.-L. et al. (2007). A double-bromodomain protein, FSH-S, activates the homeotic gene Ultrabithorax through a critical promoter-proximal region. Mol. Cell. Biol. 27(15): 5486-5498. Medline abstract: 17526731

Denis, G. V., and Green, M. R. (1996(. A novel, mitogen-activated nuclear kinase is related to a Drosophila developmental regulator. Genes Dev. 10: 261-271. Medline abstract: 8595877

Digan, M. E., et al. (1986). Genetic and molecular analysis of fs(1)h, a maternal effect homeotic gene in Drosophila. Dev. Biol. 114: 161-169. Medline abstract: 3007240

Forquignon, F. (1981). A maternal effect mutation leading to deficiencies of organs and homeotic transformations in the adults of Drosophila. Wilhelm Roux's Arch. 190: 132-138

Gans, M., Forquignon, F. and Masson, M. (1980). The role of dosage of the region 7D1-7D5-6 of the X chromosome in the production of homeotic transformation in Drosophila melanogaster. Genetics 96: 887-902. Medline abstract: 6790337

Haynes, S. R., et al. (1989). The Drosophila fsh locus, a maternal effect homeotic gene, encodes apparent membrane proteins. Dev. Biol. 134: 246-257. Medline abstract: 2567251

Haynes, S. R., et al. (1992). The bromodomain: a conserved sequence found in human, Drosophila and yeast proteins. Nucleic Acids Res. 20: 2603. Medline abstract: 1350857

Huang, D.-H. and Dawid, I. B. (1990). The maternal-effect gene fsh is essential for the specification of the central region of the Drosophila embryo. New Biol. 2: 163-170. Medline abstract: 1982070

Jang, M. K., et al. (2005). The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol. Cell 19: 523-534. Medline abstract: 16109376

Lygerou, Z., et al. (1994). The yeast BDF1 gene encodes a transcription factor involved in the expression of a broad class of genes including snRNAs. Nucleic Acids Res. 22: 5332-5340. Medline abstract: 7816623

Matangkasombut, O., et al. (2000). Bromodomain factor 1 corresponds to a missing piece of yeast TFIID. Genes Dev. 14: 951-962. Medline abstract: 10783167

Rhee, K., et al. (1998). Expression and potential role of Fsrg1, a murine bromodomain-containing homologue of the Drosophila gene female sterile homeotic. J. Cell Sci. 111: 3541-3550. Medline abstract: 9811568

Shearn, A. (1989). The ash-1, ash-2 and trithorax genes of Drosophila melanogaster are functionally related. Genetics 121: 517-525. Medline abstract: 2497049

Tamkun, J. W., et al. (1992). brahma: a regulator of Drosophila homeotic genes structurally related to the yeast transcriptional activator SNF2/SWI2. Cell 68: 561-572. Medline abstract: 1346755

Yang, Z., et al. (2005). Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol. Cell 19: 535-545. Medline abstract: 16109377

date revised: 15 December 2007

Home page: The Interactive Fly © 2007 Thomas Brody, Ph.D.