pho mutants have a strong maternal component (Breen, 1986 and Girton, 1994). Consistent with this, PHO mRNA is present ubiquitously in early embryos (Brown, 1998).

The effects of mutations of the pleiohomeotic (pho) locus (formerly called l(4)29) on embryonic and adult development of Drosophila were investigated. The normal function of pho is vital for pattern formation during embryonic and adult development. The hypomorphic, adult-viable phocv allele produces maternal-effect embryonic lethality: the lethal embryos show homeotic transformations of head, thoracic, and abdominal segments and abnormal development of the central and peripheral nervous systems. Hypomorphic and amorphic pho alleles are recessive lethals with the lethal individuals showing partial homeotic transformations of antennae, legs, abdominal segments, and internal structures. pho mutations product pattern abnormalities in the legs and a novel sixth tarsal segment phenotype. The pho adult phenotypic effects are restricted to discrete spatial regions of the imaginal discs. In leg discs these effects are localized in the upper medial quarter of the discs and show a striking correlation with the organization of positional information as proposed by the polar coordinate model (Girton, 1994).

DNA from flies heterozygous for pho¹ or pho^c was analyzed by long PCR using primers throughout the pho cDNA. The pho^c mutant chromosome has a 7.5 kb insertion within an 86 bp intron in the 5' noncoding region of the transcript. The pho¹ mutant chromosome has a 4.5 kb insertion into the coding region, upstream of the zinc finger region of the protein. Northern analysis showed that the amount of wild-type pho mRNA is reduced about 50% in pho^c and pho¹ heterozygous adults, suggesting that the insertion elements disrupt the production of the wild-type transcript in these two mutants. In pho¹ mutants, a larger mRNA of about 7 kb was also seen. This RNA is most likely a hybrid transcript between PHO mRNA and the insertion element present on this chromosome. RNA was examined from pho^cv, a homozygous viable female sterile derivative of pho^c, which has the same insertional polymorphism as pho^c. In homozygous pho^cv adults, no PHO mRNA is detected, suggesting that the mRNA seen in pho^c heterozygotes is due to that contributed by the wild-type chromosome present in this stock. This result also suggests that the pho RNA seen in adults was largely due to that deposited in the developing oocytes. It is suggested that the absence of this transcript in pho^cv adults leads to their female sterility. It is not understood why pho^cv and pho^c have the same apparent molecular lesion, yet one is homozygous lethal and one is homozygous viable but sterile. The simplest hypothesis is that an additional mutation has occurred on the pho^cv chromosome that leads to the production of a PHO transcript zygotically, and this rescues the lethality (Brown, 1998).

Like some other PcG mutants, homozygous mutant pho individuals from heterozygous mothers die as pharate adults, showing phenotypes associated with homeotic gene derepression, including posteriorly directed homeotic transformations of the antenna and legs (Gehring, 1970 and Girton, 1994). pho mutants have a strong maternal component (Breen, 1986 and Girton, 1994). Consistent with this, PHO mRNA is present ubiquitously in early embryos (Brown, 1998).

It has been proposed that certain PcG genes are required for the maintenance of the expression domains of knirps and giant, through a mechanism similar to the regulation of homeotic genes. The regionalization of the Drosophila embryo depends on the maternally supplied products of bicoid (bcd), hunchback (hb), and nanos (nos). Nos represses the translation of the maternal HB mRNA in the posterior embryonic region. This permits the expression of the zygotic gap genes knirps (kni) and giant (gt), which specify posterior identities. These genes would otherwise be repressed by Hb. Embryos from nos/nos mothers form no abdominal segments, but this phenotype can be rescued by a total lack of hb in the maternal germline. It can also be dominantly rescued by the mutation of maternally supplied regulator molecules that normally repress kni and gt in the zygote. Pelegri and Lehmann (1994) have shown that certain mutant products of the PcG genes E(z), Psc, and pleiohomeotic can partially rescue nos by such a maternal effect. To determine if mutation of multi sex combs (mxc) also affects this regulation, the cuticles of embryos were examined from mxc/+;hb nos/nos mothers that were heterozygous for different mxc mutations. This genetic background was used because a decrease in the amount of maternal hb product can partially rescue the nos phenotype in F1 embryos. Such embryos can differentiate a few abdominal denticle belts and form an adequate background to evaluate increased rescue of nos. Thus loss-of-function PcG mutations should have a strong effect on rescue, and the embryos from hb nos/nos mothers that have two PcG mutations in their genetic background should permit increased rescue of the nos phenotype (Saget, 1998).

Any of three E(z)^son (suppressor of nanos) alleles or a hypomorphic pleiohomeotic allele partially rescue the phenotypes of hb nos/nos progeny by a maternal effect; deficiencies covering E(z) or the Psc/Su(z)2 complex also allow some maternal rescue of hb nos/nos progeny, yet the strongest effect is observed with the gain-of-function E(z)^son alleles. The EMS-induced allele mxcG48 rescues the hb nos/nos progeny phenotype, whereas a deficiency of mxc does not. Some rescue with the Psc/Su(z)2 complex deletion Df(2)vgB is also observed and strong rescue (consistently >50%) is observed with an EMS-induced pleiohomeotic allele phob, described as amorphic. This suggests that phob and mxcG48 are probably not amorphic alleles, and that maternal rescue of hb nos/nos progeny by a PcG gene is most efficient with a non-null mutation (Saget, 1998).

Segmentation of embryos from transheterozygous mothers was also examined. Because neither a reduction of wild-type PcG product nor two PcG mutations in trans in the hb nos/nos mothers increases nos rescue, these data strongly suggest that, whatever the mechanism of gap gene regulation by these PcG mutations may be, it does not function like the PcG-mediated maintenance of homeotic gene expression in embryos and in imaginal discs. The strong rescue provided by several non-null EMS-induced mutations, which may produce mutant proteins, leads to a proposal that modified PcG proteins are poisoning a normal process. How this process depends on wild-type regulation by PcG products has yet to be established (Saget, 1998).

Polycomb group proteins (PcG) repress homeotic genes in cells where these genes must remain inactive during Drosophila and vertebrate development. This repression depends on cis-acting silencer sequences, called Polycomb group response elements (PREs). Pleiohomeotic (Pho), the only known sequence-specific DNA-binding PcG protein, binds to PREs, but pho mutants show only mild phenotypes compared with other PcG mutants. pho-like, a gene encoding a protein with high similarity to Pho, has been characterized. Pho-like binds to Pho-binding sites in vitro and pho-like; pho double mutants show more severe misexpression of homeotic genes than do the single mutants. These results suggest that Pho and Pho-like act redundantly to repress homeotic genes. The distribution of five PcG proteins on polytene chromosomes was examined in pho-like, pho double mutants. Pc, Psc, Scm, E(z) and Ph remain bound to polytene chromosomes at most sites in the absence of Pho and Pho-like. At a few chromosomal locations, however, some of the PcG proteins are no longer present in the absence of Pho and Pho-like, suggesting that Pho-like and Pho may anchor PcG protein complexes to only a subset of PREs. Alternatively, Pho-like and Pho may not participate in the anchoring of PcG complexes, but may be necessary for transcriptional repression mediated through PREs. In contrast to Pho and Pho-like, removal of Trithorax-like/GAGA factor or Zeste, two other DNA-binding proteins implicated in PRE function, do not cause misexpression of homeotic genes or reporter genes in imaginal discs (Brown, 2003).

phol is located on chromosome 3 in polytene subdivision 67B and is designated by the Drosoophila genome project as CG3445. This sequence is predicted to encode a protein of 669 amino acids with four zinc fingers that share 80% sequence identity with the four zinc fingers of Pho. Although this is less conservation than between Pho and human YY1, which have 96% sequence identity over the zinc-finger region, all amino acids involved in making important DNA contacts are conserved in Phol. In addition, a short region of the spacer, conserved between Pho and YY1, is also conserved in Phol, although to a lesser extent. No other regions of conservation between Pho and Phol or between Phol and YY1 were detected (Brown, 2003).

Since the amino acids contacting the DNA are identical in Pho and Phol, Phol was expected to have the same DNA-binding specificity as Pho. Gel shift assays with the Phol zinc-finger domain showed that this protein specifically binds an oligonucleotide containing a Pho-binding site. Binding was not competed by an oligonucleotide containing a mutated Pho-binding site. Thus Pho and Phol can bind to the same DNA sequence with the same apparent binding specificity (Brown, 2003).

A Drosoophila strain (EP0559) containing a P element insertion in the untranslated leader region of the phol transcription unit was obtained from the Drosoophila genome project. Flies that are homozygous or hemizygous for this P-element insertion are viable and fertile. Two phol deletion alleles were generated by imprecise excision of the P-element. In the phol^81A null mutation, part of the P-element was deleted along with the entire phol-coding region. In phol^106C, the EP0559 element was completely deleted along with 1389 bp of the phol transcription unit, but leaving the phol promoter and the zinc-finger region intact. Therefore, it is possible that phol^106C encodes a truncated Phol protein. Importantly, the flanking transcription unit, CG3448, is left intact in both alleles. In phol^81A, the deletion ends 846 bp upstream of the CG3448 mRNA. phol^106C and phol^81A are both homozygous and hemizygous viable; males are fertile but females are sterile. The female sterility of both mutant alleles is rescued by a phol transgene. Homozygotes for either phol allele look phenotypically normal and the mutants show no obvious homeotic phenotypes. Eggs laid by mothers that are homozygous for either phol allele look normal, are fertilized, but do not develop. Embryos derived from germline clones from heterozygous phol^106C and phol^81A mothers have the same phenotype showing that phol is required in the germ cells. The requirement for phol in the germline did not allow for the generation of embryos that lack Phol protein and therefore the role of phol in regulation of homeotic genes in embryos could not be examined (Brown, 2003).

pho homozygotes die as pharate adults with weak homeotic transformations, while phol homozygotes survive and are phenotypically normal adults. By contrast, phol; pho double mutants die as third instar larvae and fail to pupate. Examination of phol, pho larvae shows that the brain is smaller than normal, the discs are misshapen and smaller than wild-type discs, and the salivary gland polytene chromosomes are enlarged. The larger salivary gland polytene chromosomes may be due to additional rounds of endoreplication in the double mutants. To test whether phol functions in PcG repression, Ubx and Abd-B expression was examined in wing imaginal discs from single and double mutants of phol and pho. As expected, no Ubx or Abd-B expression was observed in wild-type or phol mutant wing discs. pho mutants showed misexpression of Ubx in a few cells in the wing pouch, but did not misexpress Abd-B. By contrast, phol; pho double mutants strongly misexpress Ubx and Abd-B in the wing disc. This suggests that Phol and Pho redundantly repress homeotic genes in imaginal discs and can partially substitute for each other. Ubx misexpression is confined to the wing pouch in phol; pho double mutants; the lack of Ubx misexpression in more peripheral areas of the disc possibly reflects downregulation by Abd-B, which is strongly misexpressed in these regions of the disc (Brown, 2003).

Whether removal of phol during larval development would also cause derepression of Ubx and Abd-B was tested by generating clones of phol mutant cells in imaginal wing discs of pho mutant larvae. In these experiments, phol mutant cells were identified by the absence of a GFP marker. Strong misexpression of Ubx and Abd-B was observed in double mutant cells in the wing pouch similar to the misexpression observed in wing discs from the phol; pho double mutant larvae. These observations suggest that either Phol or Pho is required throughout development to keep homeotic genes repressed (Brown, 2003).

It has been suggested that pho may not play a role in PRE function in embryos. This suggestion is surprising given reports showing misexpression of engrailed, abd-A and Abd-B in pho mutant embryos. In addition, the severe defects observed in embryos lacking maternal pho function suggests a strong requirement for Pho during oogenesis and/or embryogenesis. The role of pho in embryos has been reexamined by testing the requirement for pho and Pho binding sites in the regulation of PRE-containing reporter genes (Brown, 2003).

No additional mis-expression of Ubx or AdbB is seen in phol; pho double mutant embryos over that seen in pho single mutants. Thus, an embryonic experiment was carried out in pho single mutants. lacZ expression from a Pbx-Bxd-Ubx-lacZ (BP01) reporter gene was examined. This reporter is derepressed in Pc mutant embryos. Similarly, it was found to be derepressed in a pho mutant. This shows that Pho protein is required for the silencing of this reporter gene in the embryo. Next examined was whether mutation of the Pho-binding sites within a PRE disrupts silencing. A construct containing PRE_D, a 567 bp fragment from the Ubx gene, was used. Mutation of Pho-binding sites in PRE_D inactivates its silencing capability in imaginal discs. Mutation of Pho-binding sites has been shown not to cause a loss of PRE_D silencing in embryos. However, different results were obtained using the same lines. Expression was examined from three wild-type PRE_D lines and five PRE_DPhomut. All wild-type PRE_D lines gave the expression pattern. Two out of five PRE_DPhomut lines gave similar expression. A third PRE_DPhomut line also showed unrestricted expression in embryos but the levels were lower compared with the other lines. A fourth line showed no silencing in the embryonic epidermis and in discs, but maintained restricted expression in the embryonic CNS. A fifth line showed restricted expression similar to the wild-type PRE_D control lines. These results show that Pho protein and Pho-binding sites do play a role in repression during embryogenesis (Brown, 2003).

These experiments suggest that the DNA-binding proteins Pho and Phol play important and redundant roles in PcG repression. One possible role of these two proteins may be to anchor other PcG proteins to PREs. To test this hypothesis, binding of five different PcG proteins to polytene chromosomes was analyzed in phol; pho double mutants (Brown, 2003).

First, the localization of Pho proteins was examined on polytene chromosomes of wild-type larvae. Pho has been reported to bind to about 35 chromosomal sites. Using a new Pho antiserum, combined with immunofluorescent techniques, Pho binding to about 100 sites on polytene chromosomes was detected. Psc colocalizes with Pho at about 65% of these sites. Psc has also been reported to bind to 65% of the Pc sites (Brown, 2003).

Next, the distribution of the PcG proteins Pc, Psc, Polyhomeotic (Ph), Sex combs on midleg (Scm) and Enhancer of zeste [E(z)] on polytene chromosomes was examined. Pc, Ph and Psc are all core components of the PcG protein complex called PRC1. Scm has also been reported to co-purify with PRC1. Scm and Ph may also be present in protein complexes other than PRC1. E(z) is a component of the Esc-E(z) complex, which is distinct from PRC1. The analysis focused on PcG protein binding sites on the X chromosome and on the right arm of chromosome 3, which includes the bithorax and Antennapedia gene complexes (BXC and ANTC) (Brown, 2003).

Pho, Pc, Psc, Ph and Scm all bind the same three sites in wild-type chromosomes. As expected, in phol; pho double mutants, no Pho protein is detected. Binding of Pc, Psc and Scm is lost at polytene subdivision 2D in phol; pho double mutants; binding of these proteins to all other sites on the X chromosome is unaffected. Binding of Ph is completely unaffected in phol; pho double mutants. In particular, the Ph signal at 2D is present, suggesting that Ph can bind at this site even if other PcG proteins are removed. Pc binding to 2D is not lost in either pho or phol single mutants, suggesting that the presence of either of these two proteins is sufficient for Pc to bind to this site (Brown, 2003).

It was of particular interest to know whether E(z) protein distribution would change in the double mutants because the vertebrate homologues of Pho and Esc interact in in vitro binding experiments and Pho co-immunoprecipates with Esc in early embryos. One attractive hypothesis is that Pho might be required for the binding of E(z)/Esc protein complexes to chromatin. However, no changes were detected in any E(z) chromosomal sites on either the X chromosome or on 3R in phol, pho double mutants. It has been reported that E(z) binds to chromosomal subdivision 2D; however, E(z) was detected at this site on only about 20% of the wild-type chromosomes. Although E(z) was never at 2D on phol; phol double mutant chromosomes, it cannot definitely be concluded there is a difference between this and wild type (Brown, 2003).

The patterns of binding of Psc, Ph, Scm and E(z) proteins on chromosome arm 3R were indistinguishable in wild type and phol; pho double mutants. In particular, these PcG proteins still bind to regions that include the BXC and ANTC loci in phol; pho double mutants. The binding of Pc to the BXC and ANTC, and most other loci was also unaltered in the double mutant, but binding to two specific chromosomal sites was lost. Interestingly, Psc, Scm and E(z) were not detected at these sites on wild-type chromosomes, suggesting that Pc binds independently of these proteins at these sites. Ph was present at one of these two sites, but its binding was not altered in phol; pho double mutants (Brown, 2003).

Taken together, the immunolocalization data suggest that binding of PcG proteins to most sites is unaltered in the absence of Pho and Phol protein, but that these two proteins are redundantly required for PcG protein binding at a few specific sites. Intriguingly, it appears that all PcG proteins tested in this study are still associated with the BXC and ANTC loci. Nevertheless, the BXC genes Ubx and Abd-B are derepressed in phol; pho double mutant wing discs. Several different explanations for this paradox are proposed. (1) Derepression of homeotic genes and binding of PcG proteins were not assayed in the same tissues. It was not possible to detect derepression of Ubx in salivary gland cells of phol, pho double mutants. (2) Pho and Phol may only be required for anchoring PcG proteins at some PREs in the BXC. Different DNA-binding proteins may provide this function at other PREs. This is supported by the finding that binding of PcG proteins is lost at some sites in phol; pho double mutants. Moreover, several different PREs have been identified in the Ubx gene. The resolution of antibody signals on polytene chromosomes is not refined enough to resolve distinct PREs in a single gene and, hence, loss of only a fraction of PcG protein complexes may not be detectable. Finally, Phol and Pho may not be necessary for the anchoring of PcG protein complexes to the DNA, but may confer the actual transcriptional repression mediated by PREs in imaginal discs, while the PcG protein complexes might function in the propagation and memory of the repression. Thus, PcG protein complexes might serve to recruit Phol and Pho or their co-repressors to the DNA (Brown, 2003).

The GAGA factor protein is encoded by the Trithorax-like (Trl) gene. A hypomorphic Trl allele was originally isolated due to mutant phenotypes that suggested a requirement for activation of homeotic gene expression. The proposal that Trl functions in PcG repression was based on the observations that Trl protein binds to PRE sequences, that it co-immunoprecipitated with Pc, and that mutation of Trl-binding sites cause a loss of mini-white silencing and PRE function in reporter genes. These conflicting data prompted an analysis of the role of Trl in homeotic gene regulation by generating clones of Trl mutant cells in imaginal discs. In these experiments, Trl^R85, a null allele was used, and the mutant cells were again marked by the absence of a GFP marker protein (Brown, 2003).

Trl mutant clones were analysed in the wing disc for misexpression of Ubx and Abd-B and no evidence was found for such misexpression. Since PREs often contain Pho- and Trl-binding sites in close proximity, and these is a weak genetic interaction between pho and Trl heterozygous mutants, whether removal of Trl in pho mutant wing discs would exacerbate the misexpression of Ubx observed in pho mutants was tested. This was not the case. pho mutant wing discs with clones of Trl homozygous cells show no additional misexpression of Ubx compared with pho single mutants. Thus, no evidence is found for a genetic interaction between Trl and pho (Brown, 2003).

The effects of removing Trl on the silencing capabilities of two different PRE-containing Ubx-LacZ reporter transgenes was examined: PRE_1.6 contains a PRE from the Ubx gene and MCP725 contains a PRE from the Abd-B gene. In wild-type flies, expression of both transgenes is confined to the posterior compartments of the haltere and third leg discs, and both transgenes are misexpressed in a variety of PcG mutants. By contrast, no misexpression was found of either transgene in Trl mutant clones in wing imaginal discs (Brown, 2003).

Whether mutation of Trl protein binding sites (i.e. GAGAG sequences) in a PRE from the Ubx gene would compromise its silencing capability was examined. For this experiment a previously described reporter gene, PRE_D, was used that is stably silenced in the wing imaginal disc due to the presence of the 567 bp long PRE core fragment. Mutation of Pho protein-binding sites within PRE_D abolish repression of this reporter transgene in wing imaginal discs. By contrast, mutation of all five GAGAG motifs in PRE_D causes no misexpression of this reporter transgene. Sixteen lines were obtained: five produced expression caused by positional effects and could not be analysed. The other eleven all showed silencing in the wing disc (Brown, 2003).

Finally, the requirement for Trl in maintaining expression of Ubx and Abd-B in their normal expression domains was examined. Intriguingly, no obvious reduction of Ubx or Abd-B expression was observed in Trl mutant clones in the haltere and third leg disc (Ubx) or in the genital disc (Abd-B). By contrast, clones of trithorax (trx) mutant cells show a dramatic reduction in Ubx protein levels (Brown, 2003).

These results fail to support a role for Trl in PcG repression in imaginal discs. However, the possibility that Trl is playing a role in the establishment of PcG repression in the embryo cannot be excluded. The requirement for Trl function in the germline and the early embryo does not allow an analysis of embryos lacking Trl protein. Similarly, zeste is not redundant with Pho for repression of homeotic genes in imaginal discs (Brown, 2003).

These results show a strong requirement for the DNA-binding proteins Pho and Pho-like in homeotic gene silencing in imaginal discs. In fact, the strong misexpression of homeotic genes observed in phol; pho double mutant imaginal cells is comparable with that seen in imaginal disc clones mutant for Pc, Scm, Sce or Pcl. The loss of PcG protein binding at only a small number of sites in phol, pho polytene chromosomes is consistent with the idea that Phol and Pho are required to recruit PcG protein complexes at only a subset of PREs. Alternatively, Phol and Pho may be required for transcriptional repression mediated by PREs, but not for anchoring of PcG protein complexes (Brown, 2003).