Targets of Activity

The GAGA, NTF-1, and Zeste proteins activate the Ultrabithorax (Ubx) promoter in vitro. Differently mutated Ubx-promoter constructs containing binding sites for none, one, or all three of these transcription factors have been introduced into Drosophila by P-element transformation. Binding sites for each factor activate dramatically different patterns of transcription. In zeste mutant embryos, the activation by Zeste protein-binding sites is essentially abolished. These genetic data, when considered with earlier biochemical experiments, demonstrate that zeste directly and potently activates Ubx transcription in vivo (Laney, 1992). However, it has also been demonstrated that in vivo zeste function is not required for the activation of Ubx.

Transactivation of iab-5,6,7 (distal enhancers of abd-A) requires and may be mediated by the region between distal iab-7 and Abd-B. iab-5,6,7 transvection is independent of the allelic state of zeste, a gene that influences several other cases of transvection. The long-range nature of interactions in trans between iab-5,6,7 and ABD-B suggests that similar interactions could operate effectively in organisms lacking extensive somatic pairing. Therefore, transvection may be of more general significance than previously suspected (Hopmann, 1995).

The homeotic genes of the Drosophila bithorax complex are controlled by a large cis-regulatory region that ensures their segmentally restricted pattern of expression. A deletion that removes the Frontabdominal-7 cis-regulatory region (Fab-7') dominantly transforms parasegment 11 into parasegment 12. This chromosomal region contains both a boundary element and a silencer. Previous studies have suggested that removal of a domain boundary element on the proximal side of Fab-7' is responsible for dominantly transforming gain-of-function phenotype. The Fab-7 boundary element maps to two nuclease hypersensitive sites, HS1 and HS2. This article demonstrates that the Fab-7' deletion also removes a silencer element, the iab-7 PRE, which maps to a different DNA segment (the HS3 site) and plays a different role in regulating parasegment-specific expression patterns of the Abdominal-B gene. The iab-7 PRE mediates pairing-sensitive silencing of mini-white, and can maintain the segmentally restricted expression pattern of a bxd, Ubx/lacZ reporter transgene. Both mini-white and Ubx/lacZ silencing activities depend upon Polycomb Group proteins. Pairing-sensitive silencing is relieved by removing the transvection protein Zeste, but is enhanced in a novel pairing-independent manner by the zeste' allele. The iab-7 PRE silencer is contained within a 0.8-kb fragment that spans the HS3 nuclease hypersensitive site, and silencing appears to depend on the chromatin remodeling protein, the GAGA factor. It is suggested that PRE-PRE cooperation, either in trans or in cis, may be an important feature of the silencing process within the BX-C and that boundaries may limit PRE-PRE cooperation (Hagstrom, 1997).

It is not clear how transcription factors bind at a distal enhancer, nor how proximal promoter sequences cooperate to stimulate transcription in vivo. To distinguish between different models for the action of enhancer elements, DNA binding of the Drosophila activator Zeste was measured by in vivo UV crosslinking. Experiments in Drosophila embryos show that binding of Zeste protein to either the proximal promoter of the Ultrabithorax gene or to a Ubx enhancer element does not require the presence of the other element. However, significant transcription is observed only when both elements are present and bound by Zeste. The results indicate that stimulation by an enhancer can occur by a mechanism other than increasing the occupancy of an activator to binding sites near the start site of transcription, and suggest that the enhancer acts not by recruiting RNA polymerase to the promoter, but instead by activating an already bound polymerase (Laney, 1997).

Even-skipped inhibits transcriptional activators present at the Ultrabithorax (Ubx) proximal promoter when bound up to 1.5 kb away from these activators. Three adjacent regions of eve binding contribute to silencing. Repression in vitro correlates with binding of EVE protein to two low-affinity sites in the Ubx proximal promoter. One of the low-affinity sites overlaps an activator element bound by the Zeste transcription factor. Binding of EVE protein is shown to exclude binding by Zeste protein (TenHarmsel, 1993).

Both in vitro and in vivo transcription assays have been used to delineate the promoter for the 6-kb E74 mRNA. In vitro transcription of a series of 3' deletions defined the 3' in vitro promoter boundary at position +43. Additional 5'-flanking sequences, between -181 and -83, are necessary for efficient transcription in transfected Kc tissue culture cells. Two transcription factors that interact with the E74 promoter, Zeste and GAGA, were studied in DNA-binding assays. Zeste binds to two sites within the E74 promoter. These sites overlap with three of the six GAGA-binding sites. The Zeste- and GAGA-binding sites lie within domains identified by deletion mapping as cis-acting transcriptional control elements (Thummel, 1989).

Homologous association of the Bithorax-Complex during embryogenesis: consequences for transvection in Drosophila melanogaster

Transvection is the phenomenon by which the expression of a gene can be controlled by its homologous counterpart in trans, presumably due to pairing of alleles in diploid interphase cells. Transvection or trans-sensing phenomena have been reported for several loci in Drosophila, the most thoroughly studied of which is the Bithorax-Complex (BX-C). It is not known how early trans-sensing occurs nor the extent or duration of the underlying physical interactions. The physical proximity of homologous genes of the BX-C during Drosophila embryogenesis has been investigated by applying fluorescent in situ hybridization techniques together with high-resolution confocal light microscopy and digital image processing. The association of homologous alleles of the BX-C starts in nuclear division cycle 13, reaches a plateau of 70% in postgastrulating embryos, and is not perturbed by the transcriptional state of the genes throughout embryogenesis. After gastrulation, the pairing frequency of the Ubx and Abd-B loci increased to 60-70%. Pairing frequencies never reach 100%, indicating that the homologous associations are in equilibrium with a dissociated state. The effects of translocations and a zeste protein null mutation, both of which strongly diminish transvection phenotypes, have been investigated as to the extent of diploid homolog pairing. Although translocating one allele of the BX-C from the right arm of chromosome 3 to the left arm of chromosome 3 or to the X chromosome abolishes trans-regulation of the Ultrabithorax gene, pairing of homologous alleles surprisingly is reduced only to 20-30%. A zeste protein null mutation neither delays the onset of pairing nor leads to unpairing of the homologous alleles (Gemkow, 1998).

The data support a model for trans-sensing based on physical proximity and association of the homologous alleles. The translocation mutants that were investigated demonstrate that pairing can occur between segments of non-homologous chromosomes, albeit with a reduced frequency compared to that for the sites on homologous chromosomes. It is concluded that the efficacy of the trans-sensing effect is dependent on the sensitivity of the phenotype to the transcriptional level of the gene in question and to the stability or affinity of the specific paired locus. What model might be consistent with these data and those of others? In particular, what mechanism is responsible for the stable pairing of chromosomes? The information that loci on extreme positions of the 3R chromosome (near the centromere, central to the chromosome arm and close to the telomere) show the same frequency and onset of pairing argues against a zipper mechanism by which a centromeric association leads to a linear pairing along the chromosome to the telomere. The equal pairing frequencies observed for the translocation to the X chromosome and the opposite arm of chromosome 3 also argue against the centromere having a dominant role in the pairing mechanism and furthermore require flexibility in the chromosomal arms with respect to each other. In addition, the pairing properties of chromosome 4 argue against an earlier association of centromeric heterochromatin than euchromatic loci or a dominant role of heterochromatin-binding proteins in the recognition process. The heterochromatin-binding protein, HP1, does not appear in embryos until nuclear cycle 10, increasing dramatically in cycle 14. Although HP1 has been implicated as a protein causing heterochromatin aggregation, the data do not support a model by which heterochromatin regions would preferentially interact and drive homologue recognition and pairing. The HP1- binding loci remain independent in diploid embryonic nuclei even as late as stage 12. Distinct centromeres can be discerned in many stage 14 diploid nuclei in which chromosome 4 lies outside the regions occupied by the other centromeres (Gemkow, 1998).

No preferential disposition of the unpaired BX-C is seen in the lateral dimension of the nucleus in cycle 14 embryos. In cycle 14 embryos, the largest interallelic distance measured for probes from the BX-C is close to the diameter of the nucleus but is not as large as the axial dimension of the nucleus. This is most easily explained by the fact that, following the rapid early division cycles, the chromosomes are all still oriented with their centromeres at the apical surface, although no chromocenter exists. Decondensation of the chromosomes in this orientation predetermines a preferred axial disposition of the locus. That this interpretation is probably correct is supported by results for chromosome 4, which always lies on the apical surface in blastoderm embryos. These observations are compatible with a multipoint recognition of sequences dispersed along the chromosome, resulting in globally stable interactions despite relatively unstable (short lifetime) individual associations. The combination of numerous such associations would lead to a sudden increase in the overall stability of the paired chromosome, i.e. to a highly cooperative pairing along the whole chromosome once a threshold number of interactions was established. If the pairing is driven by associations of the chromosomes through protein-protein interactions then each individual paired site will have a finite binding constant and be in equilibrium with its unpaired state. Thus, if any individual locus (in this case, the genes of the BX-C) is probed there will be a probability (or frequency) of dissociation (in this case about 30%-35%). The fact that the paired state predominates and that the distance between unpaired loci becomes greatly reduced at later times during embryogenesis is consistent with this model. The paired alleles on the chromosome may be likened to the buttons on a shirt or blouse. Any one button may become unfastened yet the whole garment will not open. In addition, the probability of the unpaired region to pair again will be much higher than in the original recognition step since the local concentration, i.e. the total volume available to the homologous loci is reduced by some orders of magnitude due to the associations of sites flanking the test locus (Gemkow, 1998).

Several conclusions can be derived from these data: (1) the homology search occurs with approximately the same kinetics for the BX-C as for chromosome 4, as well as other sites on chromosome 3 as soon as the cell cycle is lengthened beyond successive S and M phases, (2) the frequency of pairing in postmitotic embryos reflects the affinity of the recognition elements scaled by the size of the chromosomal target locus, and (3) pairing or association of the chromosomes may be mediated through protein-protein interactions. To address this latter point, a protein was investigated that influences transvection of some genes (in particular white, yellow, Ubx and decapentaplegic), shows self-association, and binds at several hundred loci on polytene chromosomes: the protein Zeste. It has been argued that the self-association tendency of this protein promotes associations of Zeste-binding sites in both cis and trans. In addition, there are a number of clustered Zeste-binding sites in the upstream control region for the Ubx gene, the locus that was investigated in these experiments. The effect of a zeste null mutation on pairing at the Ubx locus was of interest, since this mutant changes the Ubx phenotypes in transvection-sensitive experiments. A higher variation of the pairing frequencies was observed but a similar mean was found for both the onset and extent of pairing, when compared to wild type; that is, mutant embryos were detected with pairing frequencies as low as 50%. Local decondensation of the fluorescent signals was detected in some of the chromosomes at the BX-C in zeste mutants. It is possible that the Polycomb group protein complexes themselves, which assemble at the PREs in the BX-C, are a stronger determinant of the local pairing affinity at this locus. Such a possibility is supported by the observation of the trans interactions of PREs. The present hypothesis is that many specific protein-protein interactions are responsible for recognition and pairing along the chromosomes and that they comprise proteins that have other functions such as enhancers or repressors. There is clear indication from data on mitotic recombination, that the length of time between mitoses sets the lower limit on the frequency of pairing. According to the model presented here, sites with strong protein-protein interactions would act as nucleating, sites but stable homolog associations along the length of the chromatids would only occur by multipoint recognition and interaction (Gemkow, 1998).

Zeste maintains repression of Ubx transgenes: support for a new model of Polycomb repression

During late embryogenesis, the expression domains of homeotic genes are maintained by two groups of ubiquitously expressed regulators: the Polycomb repressors and the Trithorax activators. It is not known how the activities of the two maintenance systems are initially targeted to the correct genes. Zeste and GAGA are sequence-specific DNA-binding proteins that are Trithorax group activators of the homeotic gene Ultrabithorax. Zeste and GAGA DNA-binding sites at the proximal promoter are also required to maintain, but not to initiate, repression of Ubx. Furthermore, the repression mediated by Zeste DNA-binding site is abolished in zeste null embryos. These data imply that Zeste and probably GAGA mediate Polycomb repression. A model is presented in which the dual transcriptional activities of Zeste and GAGA are an essential component of the mechanism that chooses which maintenance system is to be targeted to a given promoter (Hur, 2002).

Zeste, GAGA and a third transcription factor, NTF-1 (Grainy head), activate promoter constructs of the Ubx gene in embryos via an intermingled cluster of sites between nucleotides -200 to -31. However, the constructs that were used in these experiments contain only a small subset of the Ubx cis regulatory region, and while they reproduce many features of Ubx expression, they do not respond to Polycomb repression when inserted at many chromosomal locations. Consequently, they have not permitted a rigorous analysis of the role of the proximal promoter factors in maintaining repression. To address this question, larger constructs have been used that contain the 22 kb of DNA upstream of the Ubx mRNA start site. These constructs do not suffer from significant position effect variation; they more closely approximate the expression pattern of the endogenous Ubx gene than the shorter constructs; they maintain efficient repression in late embryos as shown by the lack of ß-galactosidase reporter gene expression in more anterior and posterior regions, and they are genetically under the control of PcG genes (Hur, 2002).

Deletion of nucleotides -200 to -31 essentially abolishes transcription from the large Ubx promoter constructs, indicating a crucial role for factors binding to the proximal promoter. To determine the role of each factor separately, three constructs were prepared, each containing binding sites for either Zeste, GAGA or NTF-1 inserted between the deletion end points of the above construct. Importantly, biochemical, in vivo u.v. crosslinking, and genetic experiments strongly suggest that the DNA-binding sites used in these constructs are recognized only by their cognate factor, and not by any other sequence-specific DNA-binding activities. Binding sites for each factor separately activate transcription of the large constructs during late embryogenesis. Strikingly, constructs containing only GAGA- or Zeste-binding sites at the proximal promoter are not expressed in the anterior or posterior of the embryo, whereas constructs bearing only NTF-1 sites are strongly transcribed in these terminal regions (Hur, 2002).

Ectopic expression of Ubx in anterior and posterior regions is generally caused by a failure of the initiating repressors or the Polycomb maintenance system. One interpretation of this result is that Zeste and GAGA are required for at least one form of repression, while NTF-1 is not. It is also possible, however, that Zeste and GAGA are not repressors. Instead, it may be that they are unable to activate expression in anterior or posterior regions, even though they are expressed at similar levels throughout the embryo. To distinguish between these two possibilities, constructs were examined that contained either Zeste and NTF-1 sites or GAGA and NTF-1 sites. These constructs are expressed in the central region of the embryo; but, importantly, they are not significantly expressed in anterior or posterior regions. Since NTF-1 can activate Ubx transcription in these terminal regions, the absence of terminal expression is consistent with GAGA and Zeste directly repressing transcription in addition to their activation function (Hur, 2002).

To establish decisively if Zeste and GAGA are repressors, it was desirable to use a genetic test. Unfortunately, GAGA is a lethal gene and a broadly acting regulator required for expression of transcription factors that regulate Ubx in early embryos. Thus, it has not been possible to determine genetically whether GAGA is a direct repressor of Ubx. By contrast, zeste is a largely redundant gene. zeste null embryos and flies are essentially wild type, and the endogenous Ubx gene is expressed normally in these animals; but because the 22UZ transgenes lack the cis regulatory elements through which factors that redundantly share the function of zeste act, these transgenes should be regulated by zeste (Hur, 2002).

Consistent with this idea, transgenes containing only Zeste sites at the proximal promoter fail to express in zeste mutant embryos, whereas constructs containing only GAGA or NTF-1 binding sites are expressed in this same genetic background. Thus, this genetic experiment confirms that Zeste bound at the proximal promoter is required to activate transcription of the 22UZ constructs in the normal domain of Ubx expression. To test the role of Zeste in repression, constructs containing binding sites for both Zeste and NTF-1 at the proximal promoter were compared in wild type and zeste mutant embryos. In the normal domain of Ubx expression, these constructs are expressed at similar levels in mutant and wild-type embryos. Importantly, these constructs are derepressed in anterior and posterior regions of embryos lacking zeste. Thus, Zeste actively represses transcription in terminal regions of the embryo via binding sites at the proximal promoter (Hur, 2002).

To distinguish if Zeste is required for the initiation or the maintenance of repression, expression of the 22UZ ZESTE/NTF-1 construct was examined at an earlier stage. In embryos that lack zeste, the 22UZ ZESTE/NTF-1 transgene is almost fully repressed in anterior and posterior regions at this earlier stage. Only weak derepression is observed in a few isolated cells. Thus, the transiently expressed factors that initiate repression in the early embryo must be active, and the extensive derepression observed later must be due to a failure in the maintenance system (Hur, 2002).

The PcG genes are an essential part of system that maintains repression of the endogenous Ubx gene. To confirm that these genes also act on these transgenes, the 22UZ Zeste and 22UZ GAGA constructs were crossed into PcG mutant embryos. Both transgenes are derepressed in late stage embryos lacking the Polycomb gene. Similar results were obtained in embryos lacking another PcG gene, extra sex combs. Thus, Zeste -- and probably also GAGA -- act together with the Polycomb system to maintain repression of Ubx (Hur, 2002).

It is suspected that GAGA and Zeste have redundant, overlapping functions in maintaining repression because the 22UZ Native construct, which contains Zeste, GAGA and NTF-1 sites, is not derepressed in zeste mutant embryos, which contrasts with the behavior of the 22UZ ZESTE/NTF-1 construct. Such redundancy in repression would parallel the known redundancy between these two transcription factors in activating Ubx in the central portions of the animal, and helps explain the previous lack of evidence that Zeste and GAGA are repressors (Hur, 2002).

The data presented in this paper are consistent with the earlier genetic data that suggested that some trxG and PcG proteins may have dual activities. Further support for this idea comes from recent biochemical experiments that have shown that GAGA is complexed with two PcG proteins in Drosophila nuclear extracts and Zeste is part of a multisubunit complex that contains Polycomb. In addition, PcG proteins are frequently associated in vivo with promoter regions that include Zeste or GAGA DNA recognition sites, including the Ubx proximal promoter examined in this paper. Most PcG proteins do not recognize specific DNA sequences; thus, the interaction with Zeste and GAGA may serve to recruit PcG proteins to promoters (Hur, 2002).

But is it essential that some proteins, such as Zeste and GAGA, participate in both repression and activation, or is it mere coincidence? This joint participation may be essential. At the transition between the initiating repressors and the Polycomb system, one possibility is it that Polycomb proteins are recruited to or activated on only those genes that are bound by initiating repressors; the initiating repressors may physically bind to PcG proteins to recruit them. However, Polycomb repression can be established on Ubx promoter constructs that lack initiating repressors elements, provided that initiating enhancer elements are also absent. In other words, at the transition between the establishment and maintenance of the Ubx expression pattern, the Polycomb systems reads the absence of activation, rather than the presence of repression or repressors (Hur, 2002).

Endogenous Zeste protein binds to Ubx promoter constructs in vivo whether they are transcribed or not. It is suggested that in the early embryo in the cells in which Ubx is activated, Zeste is complexed, directly or indirectly, with initiating activators on the Ubx promoter. These complexes mask surfaces on Zeste that would otherwise be bound by components of the Polycomb system. By contrast, in those cells where Ubx is not activated, Zeste is still bound to the promoter but is not a part of an activating complex. Surfaces on Zeste protein would then be exposed and could serve as the signal that the Polycomb system reads to initiate the maintenance phase of repression. The dual activities of Zeste and GAGA could be a key to understanding this fascinating regulatory mechanism (Hur, 2002).

Regulation of Polycomb group complexes by the sequence-specific DNA binding proteins Zeste and GAGA

Repression and activation of the expression of homeotic genes are maintained by proteins encoded by the Polycomb group (PcG) and trithorax group (trxG) genes. Complexes formed by these proteins are targeted by PcG or trxG response elements (PREs/TREs), which share binding sites for several of the same factors. The repressive class II PcG complex PRC1 has more than 30 protein subunits, including 5 that have been genetically defined as PcG proteins: Polycomb (Pc), Posterior sex combs (PSC), Polyhomeotic (Ph), dRING1, and, at substoichiometric levels, sex combs on midlegs (SCM). GAGA factor and Zeste bind specifically to PREs/TREs and have been shown to act as both activators and repressors. Purified proteins and complexes have been reconstituted from recombinant subunits to characterize the effects of GAGA and Zeste proteins on PcG function using a defined in vitro system. Zeste directly associates with the PRC1 core complex (PCC) and enhances the inhibitory activity of this complex on all templates, with a preference for templates with Zeste binding sites. GAGA does not stably associate with PCC, but nucleosomal templates bound by GAGA are more efficiently bound and more efficiently inhibited by PCC. Thus Zeste and GAGA factor use distinct means to increase repression mediated by PRC1 (Mulholland, 2003).

To demonstrate directly that GAGA enhances template recognition by PCC, a recruitment assay was developed. Ubx5S DNA, consisting of a portion of the Ubx promoter with known high-affinity Zeste binding sites, was biotinylated, assembled into chromatin, and immobilized on streptavidin-coated magnetic beads. Nucleosomal arrays were incubated with either buffer only or GAGA for 15 min at 30°C prior to addition of PCC. Following a 20-min binding period, array-bound beads and all material bound to them were separated magnetically from unbound protein. Western blot analysis demonstrates that PCC and GAGA components bound to the nucleosomal array-bead complex and that PCC association is increased on arrays bound by GAGA. GAGA and PCC bind only minimally to unconjugated beads. Increasing amounts of competitor nucleosomal array result in a loss of PCC association with the array-bead complex, but do not affect PCC association with the GAGA array-bead complex. These results demonstrate that PCC has a higher affinity for nucleosomal templates bound by the GAGA factor. Prebinding the GAGA{Delta}POZ protein does not lead to increased recruitment of PCC (Mulholland, 2003).

These experiments show that a template prebound by GAGA factor is more efficiently bound and repressed by PCC than an unbound template. GAGA factor might recruit or stabilize PCC binding by directly interacting with its subunits, or GAGA might alter the template in a manner that favors PCC binding. GAGA factor oligomers have been shown to be able to bind multiple templates simultaneously, bringing them together. Binding by GAGA factor might create a network of templates that is more efficiently bound and recognized by PCC than an individual template might be (Mulholland, 2003).

In the defined in vitro system used in this study, both Zeste and GAGA factor can enhance the activity of a PRC1 core complex to repress remodeling of a nucleosomal template. Zeste binds directly to these PcG proteins to generally increase their repressive function, and prebinding GAGA factor to the template recruits the PRC1 core to that template. Previous genetic and mechanistic studies have suggested that regulation of PRC1 repression is a complicated process involving targeting by sequence-specific DNA binding proteins, covalent modification of histone tails, and perhaps targeting by siRNAs. The differences in function of GAGA and Zeste suggest that their role in PcG repression is more complex than previously suspected. One hypothesis for how sequence-specific factors establish PcG repression is that they create a binding surface with greater affinity for PRC1. Although these experiments with GAGA factor are consistent with this hypothesis, the experiments with Zeste suggest that additional mechanisms contribute to targeting by sequence-specific factors. Zeste binds tightly to the core components of PRC1 and enhances their activity even when templates do not contain targeting sequences. This might be important in facilitating the ability of PRC1 repression to spread away from PRE elements, and thus may facilitate the repression of large domains by PRC1 (Mulholland, 2003).

Although originally identified as an activator, Zeste can also function in vivo as a repressor. For example, in zeste mutant flies, a transgene containing the Ubx promoter modified to contain only Zeste binding sites is derepressed in the anterior and posterior segments of the embryo. Two distinct substitution mutants of zeste express proteins that repress rather than activate the white gene but retain activator function required for transvection, suggesting that Zeste has both inherent activation and repression activities that can be separated. It is possible that, when incorporated into PRC1, Zeste is configured so as to only display surfaces responsible for repression (Mulholland, 2003).

It is widely believed that PcG activity is targeted and maintained throughout the course of development by multiple systems. For instance, ESC/E(z) can methylate H3 K27, and PC can bind to this modification, suggesting that a methylation mark might also play a key role in targeting PRC1 and/or in regulating the spread of PRC1 activity. The combined effects of factors such as GAGA that target PRC1 activity, factors such as Zeste that augment PRC1 activity, and other systems such as those for covalent modification of histones might be necessary for faithful maintenance of PRC1 association with a template. It is likely that further mechanisms, such as RNAi, also contribute (Mulholland, 2003).

These multiple mechanisms might be additive or synergistic. Additionally, redundancy between them would provide a fail-safe scheme for maintenance of repression. For instance, if methylation at H3 K27 and increased function by Zeste each were sufficient to establish repression by PRC1, then repression could be established even if one or the other were to fail. Consistent with this hypothesis of redundant function, experiments were performed in which both Zeste and GAGA were present; no significant additive or synergistic effects on PCC function was seen. The establishment of defined in vitro systems, such as used here, will aid in unraveling the connections between the different mechanisms that contribute to regulation of PcG function (Mulholland, 2003).

GAGA facilitates binding of Pleiohomeotic to a chromatinized Polycomb response element: Requirement for zeste

Polycomb response elements (PREs) are chromosomal elements, typically comprising thousands of base pairs of poorly defined sequences that confer the maintenance of gene expression patterns by Polycomb group (PcG) repressors and trithorax group (trxG) activators. Genetic studies have indicated a synergistic requirement for the trxG protein GAGA and the PcG protein Pleiohomeotic (PHO) in silencing at several PREs. However, the molecular basis of this cooperation remains unknown. Using DNaseI footprinting analysis, a high-resolution map is provided of sites for the sequence-specific DNA-binding PcG protein PHO, trxG proteins GAGA and Zeste and the gap protein Hunchback (HB) on the 1.6 kb Ultrabithorax (Ubx) PRE. Although these binding elements are present throughout the PRE, they display clear patterns of clustering, suggestive of functional collaboration at the level of PRE binding. While GAGA can efficiently bind to a chromatinized PRE, PHO alone is incapable of binding to chromatin. However, PHO binding to chromatin, but not naked DNA, is strongly facilitated by GAGA, indicating interdependence between GAGA and PHO already at the level of PRE binding. These results provide a biochemical explanation for the in vivo cooperation between GAGA and PHO and suggest that PRE function involves the integrated activities of genetically antagonistic trxG and PcG proteins (Mahmoudi, 2003).

This study has determined the precise distribution within the Ubx PRE of the recognition elements for four sequence-specific DNA-binding proteins that have all been implicated in Ubx regulation in vivo: PcG protein PHO, gap protein HB and trxG proteins GAGA and Zeste. The results indicate that, rather than a random collection, the binding site distribution within the Ubx PRE reflects a functional arrangement, allowing cooperation between distinct PRE binding proteins. Of particular interest is the observation that chromatin binding by the PcG protein PHO is strongly facilitated by the trxG protein GAGA. This finding provides a molecular mechanism for the requirement for both factors during PRE-directed silencing in vivo, and suggests that PHO and GAGA elements together may form a functional module (Mahmoudi, 2003).

Several independent genetic studies have pointed to a concurrent requirement for GAGA and PHO during gene silencing directed by distinct PREs. The PcG-dependent silencing conferred by a 230 bp fragment of the iab-7 PRE is dependent on both GAGA and PHO binding. Similarly, a 138 bp fragment of the MCP silencer, which was found to be sufficient for maintenance of embryonic silencing, contains PHO and GAGA sites. Mutations in either PHO or GAGA sites compromised silencing and revealed cooperation between both proteins. Particularly relevant for the current study are results that support a critical role in PcG silencing for GAGA and PHO sites within the Ubx PRE (Mahmoudi, 2003).

Functional dissection of the Ubx PRE has revealed that a Pc-dependent PRE silencer is contained in the central 567 bp fragment from position 577 to 1143, which includes all PHO and the highest density of GAGA sites. Another study showed that an oligomerized subfragment, corresponding to positions 890-1079 within PRE C, harboring two PHO and five GAGA elements, is able to confer PcG silencing in vivo. Finally, deletion of a 160 bp region corresponding to positions 851-1011 within PRE C impairs maintenance of silencing. The large extent of overlap between the DNA fragments identified in these independent studies strongly suggests that the common region within PRE C represents the critical core of the Ubx PRE. The most noticeable feature of this region is the many alternating GAGA and PHO binding elements. Moreover, it is of interest to note that footprinting analysis revealed the presence of Zeste as well as HB sites within this region, which may also contribute to the in vivo maintenance of repression (Mahmoudi, 2003).

The identification of Zeste as a component of the PRC1 PcG complex, suggests that it may play a direct role in PcG complex recruitment to the Ubx PRE. Further evidence for the involvement of Zeste in the maintenance of Ubx repression as well as activation has been provided by transgene experiments. Finally, the presence of HB sites within the Ubx PRE suggests a potential role for HB, not only during the initiation of Ubx repression, but also during the transition from establishment to maintenance. One attractive possibility is that this transition involves dMi-2 recruitment by HB. It should be noted that in the absence of initiating activation and repression elements, HB-independent PcG repression of the Ubx promoter has been documented (Mahmoudi, 2003).

Although there is substantial evidence for the notion that the proteins discussed above are involved in PcG silencing of homeotic genes, it remains unclear whether they can be sufficient for targeting or whether additional factors are required. One way to determine a minimal set of protein recognition sequences that can mediate PcG silencing will be the generation of synthetic PREs, which should be tested in vivo. The results suggest that, within such a PRE, PHO sites will need to be flanked by GAGA sites in order to facilitate chromatin binding. The proteins GAGA and Zeste may be particularly well adapted for such a purpose. Both GAGA and Zeste form large homo-oligomers that bind cooperatively to the multiple sites present in their natural response elements, such as the Ubx PRE and promoter. This cooperative mode of DNA-binding may allow these proteins to first bind an accessible site within a nucleosomal array and then progressively displace histones during binding to flanking sites. In addition, GAGA and Zeste have both been shown to recruit selective ATP-dependent chromatin remodeling factors. The process of targeting of remodelers to specific DNA elements may enable GAGA and Zeste to create nucleosome-free or remodeled areas, thus facilitating binding of other regulators. It is considerede likely that the remodeling complexes present in the chromatin preparations used in assays, are involved in the observed synergistic binding between PHO and either GAGA or Zeste (Mahmoudi, 2003).

GAGA oligomerization may also promote the communication between the Ubx PRE and promoter. Both elements, which are separated by ~24 kb of intervening DNA, contain a preponderance of binding sites for GAGA. GAGA oligomerization through its POZ domain allows it to form a protein bridge that directs long-range enhancer-promoter association. In fact, GAGA could even mediate enhancer function in trans by simultaneous binding of two separate DNA fragments. Thus, it is tempting to speculate that GAGA may link the Ubx PRE to the Ubx promoter. It should be noted that both the chromatin remodeling and long-range bridging functions of GAGA might accommodate PRE-mediated activation as well as repression (Mahmoudi, 2003).

The interdependence between proteins belonging to antagonistic genetic groups for efficient chromatin binding described it this study will have to be taken into account when interpreting mutational analysis of PRE function. Thus, removal of recognition sequences for the trxG protein GAGA may block its activation function but could also affect binding of the PcG protein PHO. Moreover, recent results suggest additional opportunities for cross-talk during recruitment of non-DNA-binding PcG complexes. Although a clear consensus between different studies is still lacking, there is experimental evidence for PcG complex recruitment by PHO, GAGA and Zeste. Because binding sites for either one of these proteins alone do not confer PRE function, it appears likely that they work in a combinatorial fashion. Depending on their context, the multitude of distinct binding elements that constitute a PRE might be redundant, cooperative or antagonistic to each other. Furthermore, distinct PREs may require different sets of PRE-binding proteins, and additional recruiters may be involved in PcG-silencing. Attractive candidates are GAGA-related factors batman and the PHO-related factor PHO-like (Mahmoudi, 2003).

In conclusion, current evidence suggests that PRE-directed maintenance of gene activation or repression is not achieved by a simple binary switch set by competing trxG and PcG proteins. Although their relative ratios vary considerably and correlate with transcription levels, they coexist at PREs during gene activation as well as repression. Likewise, genetic suppressor studies indicated extensive cross-talk between PcG and trxG proteins. This study has shown that, already at the level of PRE binding, there is strong interdependence between trxG protein GAGA and PcG protein PHO. The results demonstrate a direct biochemical mechanism for the cooperation between PcG and trxG proteins during PRE binding (Mahmoudi, 2003).

Transcription of Drosophila troponin I gene is regulated by two conserved, functionally identical, synergistic elements

The Drosophila wings-up A gene encodes Troponin I. Two regions, located upstream of the transcription initiation site (upstream regulatory element) and in the first intron (intron regulatory element), regulate gene expression in specific developmental and muscle type domains. Based on LacZ reporter expression in transgenic lines, upstream regulatory element and intron regulatory element yield identical expression patterns. Both elements are required for full expression levels in vivo as indicated by quantitative reverse transcription-polymerase chain reaction assays. Three myocyte enhancer factor-2 binding sites have been functionally characterized in each regulatory element. Using exon specific probes, it was shown that transvection is based on transcriptional changes in the homologous chromosome and that Zeste and Suppressor of Zeste 3 gene products act as repressors for wings-up A. Critical regions for transvection and for Zeste effects are defined near the transcription initiation site. After in silico analysis in insects (Anopheles and Drosophila pseudoobscura) and vertebrates (Ratus and Coturnix), the regulatory organization of Drosophila seems to be conserved. Troponin I (TnI) is expressed before muscle progenitors begin to fuse, and sarcomere morphogenesis is affected by TnI depletion as Z discs fail to form, revealing a novel developmental role for the protein or its transcripts. Also, abnormal stoichiometry among TnI isoforms, rather than their absolute levels, seems to cause the functional muscle defects (Marin, 2004).

This in vivo study reveals two regulatory regions, URE and IRE, located immediately upstream and downstream to the transcription initiation site and defined by a characteristic array of binding sites for the transcription factors Mef2, Biniou, and Tinman. The regions are qualitatively identical in their effects, but both are required for proper levels of transcription. Given the span of the genomic fragments tested for the reporter expression, it seems that the full set of positive regulatory elements has been identified. Putative repressor sites, however, remain to be identified. Finally, the transvection experiments suggest that, in addition to the cis-requirements, transcription is also dependent on trans-effects occurring probably at a small critical region close to the putative promoter (Marin, 2004).

The genomic fragments analyzed in LacZ reported transgenes allow identifying regions that contain positive regulatory elements that direct expression to specific tissues and developmental stages. These regulatory modules are revealed as overlapping stretches of DNA rather than separate and mutually exclusive units. For example, the modules for somatic and visceral muscles share ∼1 kb of sequences. None of the smaller fragments tested that subdivide this 1 kb, however, could reproduce the original somatic or visceral expression patterns. The case has precedents in other genes such as mef2, tubulin, or tropomyosin 2, and illustrate the intimate relationship between specific sequences (i.e., enhancers or repressors) and the topology of the surrounding chromatin in the context of proper gene expression (Marin, 2004 and references therein).

The location of the two regulatory regions could sustain a particular chromatin structure, perhaps of a hair-pin type, for normal transcription. The spacing requirement and the linear order of interference effects shown by the three rearrangements support this speculation. In females, normal transcription requires correct pairing between both IRE + URE complements, and the spacing becomes less critical as long as it is the same in both chromosomes. The transvection effect that takes place when two homologous copies of the gene are present clearly implies enhanced transcription from the trans-homologue, as demonstrated by QRT-PCR assays in genotypes that allow to discriminate the chromosomal origin of some transcripts (Marin, 2004).

The initiation of transcription is clearly dependent on Mef2. Maintenance, however, does not seem to rely exclusively on this transcription factor. The difference between these two types of transcription has been recently documented, and the present case may indicate that it is a general phenomenon. Because the analyzed regions contain canonical binding sites for Tinman and Biniou, it seems puzzling why these factors are not able to drive gene expression in a mef2 null background. One possibility is that Mef2, in addition to its direct DNA binding activity on wupA and its role as a transcription factor, acts also as a trancriptional cofactor for Tinman and Biniou. Also, the role of other transcription factors such as the one encoded in Dmeso18E remains to be integrated into this scenario. Purification and analysis of the corresponding protein complexes would be required to test these speculations (Marin, 2004).

Concerning the Trithorax group of transcriptional cofactors assayed, the data demonstrate that zeste acts as a repressor for wupA. In addition, Su(z)3 is also a repressor and it behaves similarly to zeste with respect to transvection effects. Additional data on a third gene, Trithorax-like, which encodes the GAGA factor yielded similar effects. They represent cis- and trans-requirements for normal transcription. There seems to be, however, a critical domain near the promoter where the effects of these repressors become evident. Based on the immunity of PG31 heterozygotes to Trithorax group mutant backgrounds, this critical region could be defined by the-249 position as the upstream limit. In addition, the perfect transvection in PL87/PG31 heterozygotes suggest also a critical region of pairing for transcription. It is plausible that both critical regions are coincident. Presumably, pairing of these two rearrangements will be facilitated by the common sequences and size of the inserts. Their different site of insertion and the different transgenes, however, most likely will distort pairing to some extent. Thus, the critical region for transvection might be as small as 30 base pairs upstream of the initiation site. Extensive studies in the gene yellow have reached the same conclusion where the critical region for transvection seems to be the TATA box and an initiator element located in cis. The case of wupA, which does not contain TATA box, suggests that the critical region is the promoter per se, independently of its type. These observations should help to direct future in vitro studies with chromatin fragments (Marin, 2004).

It may seem counterintuitive the observation that z and Su(z)3 mutant backgrounds result in an increase of transcription at wupA, whereas the phenotypic effect shows a loss of transvection. Because the transcriptional change has been consistently observed in all genotypes assayed, including the flies sorted by wing position, it is evident that the phenotype does not correlate with the absolute levels of TnI transcripts. Furthermore, in spite of the very low levels of transcription in 23437/hdp3 females, they are viable and muscles are functional, except those involved in flight. The most plausible interpretation of these observations is that the deleterious effect on muscle structure results from changes in the stoichiometry of certain TnI isoforms rather than in their absolute levels. It is likely that, as in TnI isoform replacement experiments with the vertebrate homologues, the unbalance of certain TnI isoforms lead to unsuitable thin filaments in vivo. If this is also the case in humans, certain muscular diseases, particularly those revealed under intense exercise, may result from mutations in regulatory regions, and thus may have escaped detection under standard sequencing procedures. In this context, the conserved gene regulatory array should be useful to guide future mutant screenings in humans (Marin, 2004).

Z discs are thought to be the anchoring points where thin filaments exert force and contract the sarcomere during muscle activity. It is somewhat unexpected that reduced levels or structural modifications of TnI can result in defective Z discs. The aspect and spacing of these Z disk-like structures suggest that a Z disk results from the lining of independent substructures deposited on the thin filaments and latter organized in register. It is worth noting, however, that a thin filament does not necessarily anchor at a Z disk. The quasi-normal sarcomeres from double mutants in troponin I, hdp2, and myosin or tropomyosin, Su(hdp2), show frequent cases of thin filaments extending more than one sarcomere in length. All these abnormal features of Z discs observed in mutants that involve TnI may indicate additional developmental functions of this protein beyond the well known regulatory role in sarcomere mechanics. Alternatively, these features may result from depletion of bona fide Z disk components whose expression is downregulated because of TnI mutations. This possibility would require a coordinated regulation of gene expression among thin filament components (Marin, 2004).

Vertebrate TnI encoding genes are not yet amenable to the in vivo analysis that Drosophila allows. Nevertheless, previous studies on the quail fast and slow TnI genes show functional evidences of a very similar regulatory structure to that described here for wupA. Equivalent regions to IRE and URE can be revealed by sequence analysis in other TnI members with the exception of the cardiac gene. The array of regulatory regions and their characteristic features seem fairly well conserved in TnI encoding genes of insects and vertebrates. This observation will be relevant toward the design of tools that aim to mimic the native gene expression in otherwise pathological conditions. Beyond this utilitarian use, the conservation of the TnI regulatory landscape in other genes that encode thin filament components might be indicative of common trends that would ensure proper quantitative expression of these components and, eventually, could help to translate gene regulation into physiology (Marin, 2004).

Requirements for gene activation by Zeste

The zeste gene is involved in two chromosome pairing-dependent phenomena: transvection and the suppression of white gene expression. Both require the ability of Zeste protein to multimerize, dependent on three interlaced hydrophobic heptad repeats in the C-terminal domain. The first step is dimerization through a leucine zipper. Two other heptad repeats are then required to form higher multimers. A zeste mutation, which causes the pairing-dependent suppression of white, creates a new hydrophobic nucleus that allows the formation of a new and larger aggregate. One mutation suppresses even unpaired copies of white and makes even larger aggregates. The phenotypic suppression of white by a series of mutants is strictly correlated with hyperaggregation and the larger the hyperaggregates, the weaker the requirement for the pairing of white. (Chen, 1993a).

The Zeste protein forms multimeric species in vitro through its C-terminal leucine zipper domain. Multimerization is required for efficient binding to DNA containing multiple recognition sequences. Increasing the number of binding sites stimulates binding in a cooperative manner. Zeste protein binds to DNA in a highly cooperative manner that depends on its ability to form multimers able to interact simultaneously with multiple recognition sequences. DNA affinity is restored only if multimerization can occur. The DNA binding domain alone is not sufficient for gene stimulation, suggesting that another region of the protein is required for proper function (Chen, 1993b).

Mutants that can only form dimers still bind to a dimeric site, but with lower affinity. Mutations or progressive deletions from the C-terminal show that when even dimer formation is prevented, DNA-binding activity is lost. Surprisingly, binding activity is regained with larger deletions that leave only the DNA-binding domain. Additional protein sequences apparently inhibit DNA binding unless they permit multimerization (Chen, 1993b).

Mutations of zeste, particularly the null state, are strong recessive enhancers of position-effect variegation (PEV) for the white, roughest and Notch loci. Zeste's involvement with these diverse phenomena suggests that the normal Zeste product functions in the decondensation of chromatin. One model proposes that Zeste is important for opening and stabilizing domains of chromatin, a step in gene determination and the establishment of cell memory. It postulates that chromatin domains that have been structurally modified by chromosomal rearrangement or by insertion of transposable elements are particularly sensitive to the absence or modification of the Zeste protein. Such a view unifies the role of Zeste in transcription, transvection and PEV (Judd, 1995).

Genome-wide prediction of Polycomb/Trithorax response elements

Polycomb/Trithorax response elements (PRE/TREs) maintain transcriptional decisions to ensure correct cell identity during development and differentiation. There are thought to be over 100 PRE/TREs in the Drosophila genome, but only very few have been identified due to the lack of a defining consensus sequence. The definition of sequence criteria that distinguish PRE/TREs from non-PRE/TREs is reported in this study. Using this approach for genome-wide PRE/TRE prediction, 167 candidate PRE/TREs are reported, that map to genes involved in development and cell proliferation. Candidate PRE/TREs are shown to be bound and regulated by Polycomb proteins in vivo, thus demonstrating the validity of PRE/TRE prediction. Using the larger data set thus generated, three sequence motifs that are conserved in PRE/TRE sequences have been identified (Ringrose, 2003).

The detection of PRE/TREs by prediction generates a large data set that can be used to search for further common sequence features. To this end, the 30 highest scoring PRE/TRE hits were scanned for motifs that occur significantly more often in PRE/TREs than in randomly generated sequence. Five significant motifs were found. Not surprisingly, but reassuringly, two known motifs, the GAF and PHO binding sites were found. The Zeste binding motif was not found by this analysis, although it occurs as frequently as GAGA factor in the 30 sequences analyzed. This is probably due to the shortness and degeneracy of the Zeste motif, and suggests that other such short motifs will also be missed by this approach (Ringrose, 2003).

Nevertheless, three additional motifs were found. The first, called GTGT, is found several times in 14 of the sequences. The second motif, poly T, is found several times in almost all 30 PRE/TRE sequences analyzed. Some variants of this site match the binding consensus for the Hunchback protein, which has been shown to be an early regulator at some PRE/TREs. The third motif, TGC triplets, occurs several times in 13 of the PRE/TRE sequences. No binding factor for this sequence has yet been identified (Ringrose, 2003).

To further examine these three motifs, motif occurrence was evaluated in all 167 predicted PRE/TREs and in the promoter peaks described above. In contrast to the known GAF, Z, and PHO motifs, the three motifs each occur in only a subset of predicted and known PRE/TREs, and do not occur significantly together. These motifs may thus each define a subclass of PRE/TREs. Consistent with this idea, some of the lowest scoring known PRE/TRE sequences indeed contain one or more of the three motifs (Ringrose, 2003).

Although no correlation between particular sites and high scores was found, a negative correlation was found between numbers of GAF/Z and PHO sites (a correlation coefficient of -0.78, indicating that when many GAF/Z sites are present, there are few PHO sites, and vice versa). This suggests that each PRE/TRE may have a preferred ground state, in which it is either predisposed to silencing (many PHO sites) or to activation (many GAF/Z sites) (Ringrose, 2003).

In summary, this analysis identifies three motifs that occur significantly in association with known PRE/TRE motifs. Further functional characterization of these motifs and the proteins that bind them may contribute to a more complete definition of the sequence requirement for PRE/TRE function, and of subclasses of PRE/TREs (Ringrose, 2003).

This study offers four main contributions to the understanding of PRE/TRE function. First, a larger set of sequences have been defined that will facilitate the more complete definition of PRE/TRE sequence requirements. Three motifs have been identified that may contribute to this goal. The definition of the minimal requirement for PRE/TRE function will not be a trivial task. Analysis of motif composition and order in the 167 predicted PRE/TREs reveals that there is a great diversity of patterns, with no preferred linear order. It is possible that each different pattern of motifs reflects a subtly different function. However, the concept of a linear order of motifs may well be irrelevant, because these elements operate in the three-dimensional context of chromatin. The fact that such a diversity of PRE/TRE designs exist indicates that the vast majority of them would defy detection by conventional pattern-finding algorithms, and underlines the advantages of the approach described in this study (Ringrose, 2003).

Although no linear constraints on motif order were found, the fact that only motif pairs, and not single motifs, are able to identify PRE/TREs strongly suggests that this close spacing of sites has functional significance. Multiple sites may work in concert, to promote cooperative binding of similar proteins (e.g., repeated PHO sites) or to provoke competition between dissimilar proteins (e.g., closely spaced GAGA factor and PHO sites). In addition, in chromatin, only a subset of sites will be exposed and optimally available for binding at any one time, while others will be occluded by nucleosomes. The trxG includes nucleosome remodeling machines, raising the intriguing possibility that remodeling of PRE/TREs in chromatin may contribute to epigenetic switching by exposing different sets of protein binding sites (Ringrose, 2003).

Second, a PRE/TRE peak is observed at the promoter of all the genes examined. This strongly suggests that promoter binding is a general principle of PRE/TRE function. It has been reported that PcG proteins can interact with general transcription factors. It has hitherto been unclear whether the observed PcG/trxG binding at promoters of the genes they regulate is mediated indirectly via such an interaction, or whether the PcG and trxG bind directly to PRE/TREs at the promoters. The high scores observed at promoters favor the latter interpretation (Ringrose, 2003).

Third, it has been shown that in most cases, PRE/TREs do not occur in isolation, but are accompanied by one or more other peaks nearby. These grouped PRE/TREs may create multiple attachment sites for PcG and trxG proteins, which come together to build a fully operational complex at the promoter. Alternatively, grouped PRE/TREs may be individually regulated by tissue-specific enhancers as in the BX-C. Thus, each of the many PRE/TREs of the homothorax gene may interact with the promoter PRE/TRE in different tissues. This idea is consistent with the fact that Homothorax has specific roles in diverse developmental processes (Ringrose, 2003).

Finally, the current list of about ten PcG/trxG target genes has been expanded to over 150 genes, identifying candidates for epigenetic regulation. The genes thus identified encompass every stage of development, suggesting that the PcG/trxG are global regulators of cellular memory. Experiments to further investigate and compare this regulation for individual genes are currently underway (Ringrose, 2003).

A double-bromodomain protein, FSH-S, cooperates with zeste to activate the homeotic gene Ultrabithorax through a critical promoter-proximal region

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. However, its direct role in homeotic gene activation has been obscured by complex phenotypes in mutant embryos. 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 and the extra terminal (ET) domain at its C terminus, is identical to the N-terminal half of the large isoform FSH-L. 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 and references therein).

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. 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. However, subsequent studies failed to show this activity in the mouse counterpart, FSRG-1, despite more than 90% sequence identity. 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. Mouse BRD4 stimulates transcription by binding to positive transcription elongation factor b. 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).

Protein Interactions

Polycomb group genes are necessary for maintaining homeotic genes repressed in appropriate parts of the body plan. Some of these genes, for example Psc, Su(z)2 and E(z), are also modifiers of the zeste-white interaction. The products of Psc and Su(z)2 have been immunohistochemically detected at 80-90 sites on polytene chromosomes. The chromosomal binding sites of these two proteins were compared with those of Zeste protein and two other Polycomb group proteins, Polycomb and polyhomeotic. The five proteins co-localize at a large number of sites, suggesting that they frequently act together on target genes. In larvae carrying a temperature sensitive mutation in Enhancer of zeste ( E[z]), both the Su(z)2 and Psc products become dissociated from chromatin at non-permissive temperatures from most but not all sites, while the binding of the Zeste protein is unaffected. The polytene chromosomes in these mutant larvae acquire a decondensed appearance, frequently losing characteristic constrictions. These results suggest that the binding of at least some Polycomb group proteins requires interactions with other members of the group. Although Zeste can bind independently, its repressive effect on white expression involves the presence of at least some of the Polycomb group proteins (Rastelli, 1993).

A goal of modern biology is to identify the physical interactions that define 'functional modules' of proteins that govern biological processes. One essential regulatory process is the maintenance of master regulatory genes, such as homeotic genes, in an appropriate 'on' or 'off' state for the lifetime of an organism. The Polycomb group (PcG) of genes maintain a repressed transcriptional state, and PcG proteins form large multiprotein complexes2, but these complexes have not been described owing to inherent difficulties in purification. A major PcG complex, PRC1, has been purified to 20%-50% homogeneity from Drosophila embryos. Thirty proteins have been identified in these preparations, then the preparation has been further fractionated and Western analyses have been used to validate unanticipated connections. The known PcG proteins Polycomb, Posterior sex combs, Polyhomeotic and dRING1 exist in robust association with the sequence-specific DNA-binding factor Zeste and with numerous TBP (TATA-binding-protein)-associated factors that are components of general transcription factor TFIID (dTAFIIs). Thus, in fly embryos, there is a direct physical connection between proteins that bind to specific regulatory sequences, PcG proteins, and proteins of the general transcription machinery (Saurin, 2001).

The inheritance of established expression patterns of certain genes during multiple cell divisions is essential for the correct development of an animal. In Drosophila, the expression patterns of the homeotic genes that govern body segment identity are established early in embryogenesis by the products of the gap and pair-rule genes, but are maintained throughout the rest of development by proteins of the PcG and trithorax group (trxG). The trxG maintains the transcriptionally active state of the homeotic genes, whereas the PcG prevents ectopic expression by maintaining a repressive state. The PcG genes encode components of multiple complexes. One of these complexes, Polycomb repressive complex 1 (PRC1), contains the Polycomb (PC), Polyhomeotic (PH) and Posterior sex combs (PSC) proteins (Saurin, 2001).

To better understand the mechanisms of this cellular memory system, an epitope-tag strategy was used to purify PRC1 over 3,000-fold from Drosophila embryos. This complex has been extensively washed in 1 M salt and has a high specific activity in functional analyses; however, contaminating proteins remain associated. Extensive efforts to fractionate this complex to homogeneity in reasonable quantity were blocked by unacceptably low yields on a wide variety of subsequent purification steps. The advent of genome-wide sequence analysis provided an alternative route to identify the components of PRC1. Using mass spectrometry and the recently completed Drosophila genome, almost all of the proteins were identified in the highly fractionated material derived from the M2-affinity column. The sensitivity of Western analysis was used to validate of the association of proteins during subsequent chromatography steps (Saurin, 2001).

The presence of the previously identified PcG proteins PH, PC, and PSC was confirmed by mass spectrometry. In addition, a Drosophila homolog of the mammalian RING1 protein (dRING1) was identified; dRING1 has been found to colocalize with PC on polytene chromosome preparations and its mammalian counterparts have previously been shown to associate with mammalian PcG proteins. Thus, the PcG complement of PRC1 is made up from PH, PSC, PC, dRING1 and sub-stoichiometric amounts of Sex Combs on Midleg (SCM) (Saurin, 2001).

Of the remaining proteins identified by mass spectrometry, the presence of several dTAFII proteins and Zeste is particularly striking. Zeste is a sequence-specific DNA-binding factor, with binding sites in the promoter and regulatory regions of some homeotic genes. The dTAFII proteins were initially identified in the general transcription factor TFIID, a central component for transcriptional initiation, but are also found in histone acetyltransferase complexes. The dTAFII proteins identified by mass spectrometry and Zeste all appear approximately stoichiometric with the PcG complement in the M2 fraction, and maintain a quantitative association with PcG proteins on gel filtration and heparin agarose (Saurin, 2001).

PRC1 fractionates as an extremely large complex, and contains several other proteins in addition to the PcG, dTAFII and Zeste proteins described above. The sequence information provides speculative information on the identity of these proteins, but further work is needed to validate each of these associations. The constitutively expressed Heat shock cognate 3 and 4 (HSC3 and HSC4) proteins were found. The requirement for HSCs in PcG action during development has been demonstrated genetically in flies, where a mutant allele of HSC4 enhances the homeotic phenotype of PC-heterozygous flies. Proteins were found that have been linked to histone deacetylase complexes, including HDAC (RPD3), dMi-2, dSin3A, p55 and SMRTER, a functional homolog of the human SMRT/N-CoR corepressors. While dMi-2 has been linked genetically to PcG repression, and HDAC and p55 have been found present in the Esc/E(z) PcG complex, further studies are clearly needed to examine their association with PRC1. These proteins are present in low stoichiometry: cofractionation of these proteins with PcG on subsequent steps could not be accurately assessed owing to lack of signal, and PRC1 has low deacetylase activity when acetylated core histones and histone peptides are used as substrate (Saurin, 2001).

The most surprising connection revealed in this study is that between PcG proteins and several dTAFIIs. TAFII proteins have previously been found in TFIID and in histone acetyltransferase complexes, and in both contexts have been linked to transcriptional activation. This study suggests that they may also function in PRC1-mediated PcG repression. PcG complexes are targeted to specific genes by sequences called Polycomb response elements (PREs), but are also known to associate at promoters. The presence of dTAFII proteins in PRC1 provides a direct physical connection between PcG proteins and components of the general transcription machinery that bind at promoters. In addition, several of these dTAFIIs have similarities with core histone proteins (dTAFIIs 62, 42 and 30beta) and have been biochemically and structurally demonstrated to associate with each other in a histone octamer-like substructure. Although quite speculative, the structural similarities between the dTAFII42/62 heterotetramer with the histone H3/H4 heterotetramer might indicate a direct role in interacting with nucleosomes and/or DNA to help maintain a stable association of PcG proteins across the numerous rapid cell divisions of the embryo (Saurin, 2001).

The presence of Zeste in PRC1 may serve to assist in the targeting of PcG proteins to repressed loci. Indeed, Zeste can be found localized with PcG proteins at some PcG-repressed loci and recent data demonstrate that Zeste is directly involved in the maintenance of the repressed state of some of these loci. Zeste binds to both PRE and promoter sequences, and thus may serve to bridge the connection of the PcG proteins to these elements. Zeste has also been shown to interact directly with the BRM complex of the trxG. Zeste thus appears be involved in both PcG function and trxG function, consistent with previous genetic studies implying a role in activation and repression (Saurin, 2001).

zeste: Biological Overview | Developmental Biology | Effects of Mutation | References

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