lola like


Protein Interactions

Epigenetic inheritance to maintain the expression state of the genome is essential during development. In Drosophila, the cis regulatory elements, called the Polycomb Response Elements (PREs) function to mark the epigenetic cellular memory of the corresponding genomic region with the help of PcG and trxG proteins. While the PcG genes code for the repressor proteins, the trxG genes encode activator proteins. The observations that some proteins may function both as PcG and trxG members and that both these groups of proteins act upon common cis elements, indicates at least a partial functional overlap among these proteins. Trl-GAGA was initially identified as a trxG member but later was shown to be essential for PcG function on several PREs. In order to understand how Trl-GAGA functions in PcG context, the interactors of this protein were sought. lola like, aka batman, was identified as a strong interactor of GAGA factor in a yeast two-hybrid screen. lolal also interacts with polyhomeotic and, like Trl, both lolal and ph are needed for iab-7PRE mediated pairing dependent silencing of mini-white transgene. These observations suggest a possible mechanism for how Trl-GAGA plays a role in maintaining the repressed state of target genes involving lolal, which may function as a mediator to recruit PcG complexes (Mishra, 2003).

The BTB/POZ domains of several proteins have been reported to mediate homo- and hetero-dimerizations and can be functionally swapped between two proteins. It was reasoned that GAGA may carry out its diverse roles by recruiting different proteins to the target DNA sequences and the BTB domain may mediate this. A yeast two-hybrid screen using the BTB domain of GAGA, amino acids 1-245, was used as bait. The 0-16 h embryo cDNA library was screened to identify potential partners for Trl-GAGA protein. Out of the 46 adenine auxotrophs, 23 strongly activated the second reporter gene, ß-galactosidase. All the 23 positives interacted specifically with the bait upon retransformation and 14 turned out to be one single gene lola like (lolal). All the clones had the complete coding region of the gene. LOLAL also interacts with the full length GAGA protein. Quantitative assays show that the interaction of LOLAL with BTB and GAGA full-length protein is comparable. Indeed, in an independent screen using full length GAGA protein, LOLAL appeared eight times. Other BTB domain containing proteins were isolated multiple times and one already reported interactor of Trl-GAGA, Sin3A. GAGA factor itself was not isolated in these screens, which may suggest that BTB domain of this protein might not form a homodimer. When tested in a directed two hybrid assay, though, BTB-BTB interaction was seen. It has been reported that Trl-GAGA exists in several isoforms. All the experiments reported here have been done with the major isoform, GAGA 519. It is not known if other isoforms can interact differently. Furthermore, Pipsqueak, which binds to GAGA sequences and contains BTB domain has also been shown to be essential for sequence specific targeting of PcG protein complex. It is unclear, however, how Trl-GAGA is placed in this multi-component recruitment system functioning at the GAGA motif of polycomb response elements. Further studies will be needed to answer these questions (Mishra, 2003).

lolal was originally identified in the Drosophila gene disruption project. Later on this mutation was also found to enhance the homeotic phenotype of polyhomeotic (ph), and given the alternative name batman (Faucheux, 2003). lolal encodes a protein of 127 amino acids that contains a BTB domain of about 90 amino acids, leaving only few residues at both ends of the protein for any other functional motif/domain. Another PcG protein, Esc, consists almost entirely of six WD40 repeat motifs. Unlike multi-domain proteins, the ones made of a single domain alone may function as adaptor modules to bring together two different molecules/complexes. Trl-GAGA is known to activate transcription of several genes. In this context, lolal may function to inhibit this activation role of Trl-GAGA, in a way similar to MyoD-Id interaction. Further studies will be required to differentiate between these mechanisms (Mishra, 2003).

One of the key steps in the PcG/trxG mediated maintenance is the recruitment of the multi-protein complex of correct composition onto the PRE. Recent studies have shown that more than one or perhaps several recruiting processes take place in concert. It is likely that different recruitment possibilities provide the necessary variation that is needed for the establishment and maintenance of varying transcriptional states at hundreds of different loci. These studies identify a new member in this process. Trl-GAGA bound to specific sites on the PREs recruits LOLAL, which in turn, through direct or indirect means, incorporates Polyhomeotic (Ph) into the complex. This raises a question whether the other recruiting agents like pho, zeste, and others, function in cooperation or competition with each other. Also, it is not clear if the complex is assembled de novo on the PREs, a pre-assembled complex is recruited or partly assembled sub-complexes are recruited. Since large complexes of PcG proteins can be isolated, it is concluded that such structures, once assembled are stable. It is not clear though if these complexes are stable during cell division or they assemble each time a cell divides (Mishra, 2003).

Since most of the PREs contain Trl-GAGA binding sites, it is likely that at least some PcG complexes are recruited by GAGA factor through its direct interaction with LOLAL. These findings establish a molecular link between Trl-GAGA and the PcG complex. Since Trl-GAGA functions in several other processes that do not seem to be directly linked to the PRE function, it is likely that there are several interactors of Trl-GAGA. In the study of such interactors, initial observations suggest that a large number of proteins can interact with Trl-GAGA with a potential to target a repressive or activator function to different loci. It is not clear though how the selection of an appropriate partner is made. Is it in the context in which Trl-GAGA is bound or different heterodimers preexist in the nucleus and these are then recruited to appropriate loci? New assays will have to be designed to appropriately answer these questions (Mishra, 2003).

Genetic interaction studies show that lolal interacts with a variety of PcG and trxG mutations. This underscores the important role of this protein in the regulation of developmental genes. Interestingly, lolal interactions with ph mutation leads to transformation of 2nd (and some times 3rd) leg to 1st leg, an apparent anteriorization type of homeotic transformation in thoracic segments. But in the abdominal region, the same combination leads to posteriorization type of homeotic transformation, pigmentation of A4 (A4-->A5) reduction in the size of A6 (A6-->A7). However, appearance of sex comb in 2nd and 3rd legs is also known to be due to derepression of Scr in posterior segments thereby explaining this phenotype as due to loss of the repression function of the PcG proteins. Furthermore, trxG and PcG mutations upon interaction with lolal can give a similar phenotype. In lolal context, Asx and trg both show partial A6-->A5 transformation in the abdominal region. Pc is involved in pairing dependent silencing complex recruited by iab-7PRE. ph is also involved in the PS function of iab-7PRE. While it was known that lolal enhances the homeotic phenotype of ph, it has been demonstrated that both ph and lolal are involved in establishing the repressive complex at the iab-7PRE. This indicates that lolal and ph function in coordination to set up a repressive complex. Taken together, these results suggest that lolal may be acting along with Trl-GAGA or with other partners in different complexes in a locus or stage specific manner. Depending on the context it could be an activator or repressor function. Since not only 'ON' or 'OFF' but also several 'levels of expression states' for a given hox gene or indeed other regulated loci are maintained, it is likely that a unique combination of trxG and PcG proteins may be needed for each varying level of expression state of a given locus (Mishra, 2003).

Affinity pull-down experiments show that GAGA, Lolal, Ph and Pc proteins coexist in a complex. This is also in agreement with the genetic interaction studies, where interaction was found of lolal with ph and Pc. Genetic and biochemical studies also suggest that, like Trl, lolal could not specify the kind of complex to be assembled. It is likely, therefore, that specificity of the GAGA partner, PcG or trxG member, does not come from lolal. It is even possible that lolal could also be a multifunctional adapter of Trl-GAGA in assembling multi protein complexes. The specificity may come from yet another factor or even from the transcriptional activity around the locus. Recent observations that the transcription process itself may contribute to the cellular memory may support this view. This might bring together the ability of GAGA factor to support transcription and recruitment of multi-protein complexes and nucleosome remodeling activity in one mechanistic context (Mishra, 2003).

Trl-GAGA has been suggested to be involved in creating a nucleosome free region. The first step in establishing a PcG complex may be this Trl-GAGA mediated nucleosome remodeling of the chromatin on the PRE region to create a more accessible DNase I hypersensitive site. The recruitment of a protein complex to the accessible region may take place through GAGA factor or by other factors that can anchor the complex onto DNA. As the next step Lolal could mediate recruitment of initial complex, for example, Esc-E(z) protein complex, which modifies nearby histone tails to covalently mark the region for the recruitment of another complex, like PRC1. Consistent histone modifications and remodeling may be needed to maintain the chromatin conformation. These studies would place Lolal as the factor binding to the DNA bound GAGA even when the rest of the complex is not recruited and therefore serves to help in subsequent recruitment steps. In this context, the exact function of proteins like Lolal becomes very important. Further studies will be required to clarify these issues (Mishra, 2003).

Since Batman binds to many sites that are also targets of Ph and Trl on polytene chromosomes, this binding was further characterized in vitro on a defined PRE. The 70-bp MHS-70 fragment from the bxd PRE in Ubx was chosen. MHS-70 is required in vivo for the maintenance of embryonic silencing of a Ubx enhancer and binds in vitro to both Trl and Ph proteins partially purified from Kc cell nuclear extracts (Faucheux, 2003).

When tested in an EMSA (electrophoretic migration shift assay) in the presence of a nuclear extract from wild-type embryos, the radiolabeled MHS-70 probe gives rise to the formation of several retarded nucleoprotein complexes. The two slower-migrating complexes are specifically competed against by an excess of unlabeled MHS-70 probe. Batman is involved in the formation of these two complexes, since they are specifically supershifted in the presence of the batC11 antibody but not in the presence of a control anti-FLAG antibody. These complexes are formed with a much higher efficiency when MHS-70 is used as a probe in the presence of a nuclear extract from embryos expressing Batman-FLAG in an otherwise ban+ context. They are completely supershifted in the presence of the batC11 antibody, and partially supershifted in the presence of the anti-FLAG antibody, indicating that both endogenous Batman and Batman-FLAG participate in their formation. Together, these results demonstrate that Batman, as well as Batman-FLAG, binds to MHS-70 (Faucheux, 2003).

Since Batman does not contain much more than a single BTB/POZ protein-protein interaction domain, it was reasoned that its binding to DNA most likely requires an interaction with a DNA binding partner. Trl binds to GAGA sequence motifs found in MHS-70. In addition, Trl's own BTB/POZ domain provides a putative interface for dimerization with Batman. Thus, Trl may be the DNA binding partner mediating the binding of Batman to MHS-70. In order to test this hypothesis, it was first determined whether Trl target sequences are required for the binding of Batman to MHS-70. The formation of the Batman-containing complexes in the presence of a nuclear extract from Da:Gal4; UBF (UBF is the UAS:BAN-FLAG construct) transgenic larvae is specifically competed against by a 100-fold molar excess of the double-stranded GAGA oligonucleotide containing eight d(GA)3 motifs. In contrast, no competition is observed in the presence of a 100-fold molar excess of the MHS-70-derived fragment LS-1/9 in which two terminal d(GA)3 sequences have been mutated. These results indicate that the Batman-containing complexes bind to GAGA repeats in MHS-70, suggesting that their formation involves a GAGA binding factor such as Trl. Indeed, the two Batman-containing DNA-binding complexes are specifically supershifted in the presence of anti-Trl antibody, indicating that they contain Trl (Faucheux, 2003).

The preceding experiments suggest that the binding of Batman to a bxd PRE fragment involves its interaction with Trl or a Trl-containing complex. In order to test whether this interaction occurs independently from binding to a DNA target, protein immunoprecipitation experiments were performed, taking advantage of the tagged Batman-FLAG protein. Anti-FLAG antibody specifically precipitates the Batman-FLAG protein from UBF/+; da:Gal4/+ third instar larvae nuclear extracts. Under these conditions, the Trl protein efficiently coprecipitates with Batman-FLAG from larval nuclear extracts, as well as from embryonic extracts. These results provide further evidence that Batman and Trl are found in the same complexes in vivo, and this independently from binding to DNA (Faucheux, 2003).

The BTB/POZ domain has been shown to function as a hetero- or homo-dimerization interface. Therefore a test was performed to see whether an interaction between Batman and GAF is mediated by the BTB/POZ domains present in both proteins. In a yeast two hybrid assay, the Batman protein interacts with itself as well as with both the 519- and the 581-aa Trl isoforms. In contrast, Batman does not interact with the Trl519Delta[1-103] or Trl581Delta[1-103] variant proteins in which the BTB/POZ domain is deleted. In addition, Batman is unable to interact with the Trl BTB/POZ domain alone (Trl[1-121]). Therefore, it is concluded that the BTB/POZ domain of Trl is necessary but not sufficient to mediate the interaction of the two major Trl isoforms with the Batman BTB/POZ domain (Faucheux, 2003).



An antiserum was raised against the only region of Lolal/Batman that is not conserved among known proteins. Upon using this antiserum in Western blot experiments, a ~14-kDa band was detected in wild-type larvae. Both the 14-kDa band and a strong band with slightly lower mobility are detected in da:Gal4/UBF larvae. The latter band is detected by the anti-FLAG antibody, indicating that it corresponds to the Batman polypeptide flanked by the 8-aa FLAG epitope. The intensity of the 14-kDa band was reduced in larvae heterozygous for the Df(2R)PC4 deficiency uncovering ban, and the 14-kDa band was undetectable in homozygous banl(2)k02512 escaper third-instar larvae, indicating that the banl(2)k02512 allele is a strong hypomorphic allele. Immunostaining experiments using the batC11 antiserum indicate that the Batman protein is ubiquitously expressed in embryos as well as in larvae, a result that is in good agreement with in situ hybridization experiments using a ban cDNA probe. Since ban transcripts are detected in presyncytial embryos, a maternal contribution of ban may be sufficient to allow embryonic development to proceed until hatching of first-instar larvae (Faucheux, 2003).

The Batman protein, as detected by indirect fluorescence using the batC11 antibody, accumulates in the nucleus. During early embryonic development, Batman is found associated to condensed chromosomes during mitosis. On polytene chromosome spreads of third-instar salivary glands, a discrete banding pattern of more than 300 sites is observed for the Batman protein. A similar pattern on polytene chromosomes is seen using the anti-FLAG antibody in transgenic larvae expressing a Batman-FLAG tagged protein. These results indicate that Batman is a chromatin-associated protein (Faucheux, 2003).

The distribution of Batman was compared to that of the PcG protein, Ph, and to that of the trxG protein, Trl, both encoded by genes that show genetic interactions with ban mutations. Since the available antibodies against Batman, Trl, and Ph were obtained in rabbit, double-labeling experiments were performed on transgenic larvae expressing the Ph or the Batman proteins fused to the FLAG epitope, which was revealed using a mouse monoclonal antibody. The FLAG-Ph protein is functional. The Batman-FLAG protein is also functional since it fully rescues both the lethality and the E(ph) phenotype of the loss of function allele banl(2)k02512. In transgenic larvae expressing Ph-FLAG, 49 binding sites are shared by Batman and Ph-FLAG. Similar results were obtained in a symmetrical experiment in which Batman-FLAG sites were compared to endogenous Ph binding sites. Together, these results indicate that Batman codistributes with Ph on about half of Ph binding sites. The distribution of Batman-FLAG was compared to that of endogenous Trl in transgenic larvae expressing the Batman-FLAG protein. In contrast to the partial overlap observed between Batman and Ph, a perfect codistribution of Batman and Trl is found on polytene chromosomes. Consistently, Ph-FLAG shares about half of its binding sites with Trl, as was found for Batman. In addition, the position of the strongest binding sites for each of the two factors overlap on polytene chromosomes, suggesting that the stoichiometry of Batman and Trl is constant (Faucheux, 2003).

Taken together, these experiments indicate that the chromatin-associated protein Batman codistributes with both Trl and Ph on polytene chromosomes. This result is consistent with the genetic interactions observed between ban, Trl, and ph and suggests that Batman may cooperate with both PcG and trxG proteins for the regulation of common target genes (Faucheux, 2003).

Effects of Mutation or Deletion

The lolal/ban gene was identified based on the enhancement of the extra sexcomb phenotype of ph410 [E(ph)] associated with several noncomplementing P-element mutations (Faucheux, 2001). Four ban alleles were available at the beginning of this study. Three were lethal P[LacW] insertions [l(2)k02512, l(2)k11212, and l(2)k07907] located in the first intron of the ban gene, whereas the fourth, Df(2R)311a, was a lethal P[MTW] insertion associated with a 14-kb flanking deletion that eliminates several genes as well as the ban 5'-most sequences ( Faucheux, 2001). Viable revertants of banl(2)k02512 and banl(2)k11212 were recovered after excision of P[LacW], indicating that the lethality of these alleles is due to the P-element insertion (Faucheux, 2003).

The ban transcription unit was delimited by the ability of the 5.4-kb pBB genomic fragment to rescue both the lethality and the E(ph) effect of the banl(2)k02512 mutation in pBB transgenic flies. A major transcript of ~1.4 kb was detected by Northern blot analysis of embryonic and larval total RNA using the full LD14505 cDNA from the BDGP cDNA library that is included in the pBB genomic fragment as a probe. In situ hybridization on whole embryos using the same cDNA as a probe revealed that ban is ubiquitously expressed at all stages of embryogenesis. Sequencing of the 1,308-bp LD14505 cDNA indicated that it includes an ORF of 127 codons (Faucheux, 2003).

In order to test whether ORF127 includes all ban functions, transgenic lines were established with constructs designed to express ORF127 fused to a FLAG epitope (UBF construct) or to the EGFP (UBG construct) at the C terminus. These constructs were placed under the control of the UAS/GAL4 expression system. Ubiquitous expression of either construct using the da:Gal4 driver rescued both the lethality and the E(ph) phenotype of banl(2)k02512, thereby indicating that the BTB/POZ domain followed by the 10 aa contained in ORF127 is sufficient to fulfill known ban functions. This makes the Batman protein unique among BTB/POZ proteins, since previously known BTB/POZ domains are usually found as part of larger multidomain zinc finger-containing or actin-binding proteins (Faucheux, 2003).

All three P[LacW] insertions in ban are homozygous lethal predominantly at the first-instar larval stage, with very few escapers (7.2%) surviving until adulthood. Since ban transcripts are detected in presyncytial embryos, a maternal contribution of ban may be sufficient to allow embryonic development to proceed until hatching of first-instar larvae. To generate embryos lacking a maternal contribution, advantage was taken of the absence of expression of the pUAST-derived constructs UBF and UBG in the female germ line. banl(2)k02512/banl(2)k02512; da:Gal4, UBG/+ rescued adults were generated, and it was observed in females that no EGFP-tagged Batman protein was indeed detected in oocytes, whereas follicular cell nuclei were labeled. These females were crossed to sibling males of identical genotype and embryos showing no fluorescent signal were identified as homozygous banl(2)k02512/banl(2)k02512 embryos, which were thus lacking both the maternal and the zygotic contribution of ban. These embryos did not hatch, and all displayed cuticular defects compared to the wild type. All embryos showed incomplete head involution, and most of them showed defects in dorsal closure as well. In contrast, embryos derived from banl(2)k02512/banl(2)k02512; da:Gal4, UBG/+ rescued females crossed to wild-type males hatched and did not display cuticular defects, thereby indicating that the zygotic expression of a wild-type allele inherited from the father is sufficient to rescue the maternal loss of ban function (Faucheux, 2003).

The four ban alleles tested [banDf(2R)311a, banl(2)k02512, banl(2)k11212 and banl(2)k07907] enhance the extra sexcomb phenotype of both ph410 and Pc16 (Faucheux, 2001). This phenotype represents a homeotic transformation of mesothoracic or metathoracic legs toward prothoracic legs due to the deregulation of Sex combs reduced. In contrast, one extra copy of ban (carried on a genetically inserted construct) in an otherwise wild-type context for ban suppresses the extra sexcomb phenotype of ph410 (Faucheux, 2003).

A Contrabithorax (Cbx) phenotype, which is a wing-to-halter transformation due to ectopic expression of Ubx in the posterior part of the wing disc, was also detected in ban mutants heterozygous for alleles banl(2)k02512, banl(2)k11212, and banl(2)k07907 when also heterozygous for ph410, Pc16, Pc1, Pc3, or Pc15. The Cbx phenotype is further enhanced in ban heteroallelic viable combinations [banl(2)k02512/banDf(2R)311a, banl(2k11212/banDf(2R)311a, and banl(2)k07907/banDf(2R)311a], when also heterozygous for any of these Pc or ph alleles (Faucheux, 2003).

New alleles of ban were generated by excision of the P-element from the l(2)k02512 and l(2)k11212 insertions. The new alleles were selected as lethal or subviable in combination with banl(2)k02512. For two of these (Delta11phi and Delta11epsilon') ectopic sex comb teeth were observed on meso- or meta-thoracic legs in combination with banl(2)k02512 in about one-fifth of the male escapers. Thus, ban not only enhances PcG mutant phenotypes but also displays the extra sexcomb phenotype characteristic of PcG mutations (Faucheux, 2003).

In summary, the analysis of adult ban mutant phenotypes, together with that of the interactions between ban and ph or Pc, demonstrates a dose-dependent function for ban in the repression of the homeotic genes Scr and Ubx (Faucheux, 2003).

The presence of a conserved BTB/POZ domain in both Batman and Trithorax-like prompted a test of genetic interactions between the ban and Trl genes. The hypomorphic allele Trl13c is semilethal. Flies of the banl(2)k0251/+; Trl13c/± genotype are viable and show no visible phenotype. Viability of Trl13c mutant flies was assayed in a banl(2)k02512 heterozygous background. Whereas 12% of +/+; Trl13c/Trl13c flies hatched, only 4% of banl(2)k0251/+; Trl13c/Trl13c flies were recovered. Since the increase in the lethality could be due to second site mutations in either stock, the possibility of rescuing the lethality specifically with Trl and ban transgenes was tested. Two major isoforms of 519 and 580 aa are encoded by Trl. Both the hsp83:GAGA519 and the hsp83:GAGA581 transgenes have been shown to rescue the lethality of Trl13c/TrlR67 mutant combinations. Adding at least one copy of either the hsp83:GAGA519 or the hsp83:GAGA581 transgenes allowed the recovery of a much higher frequency of homozygous Trl13c escapers that are also heterozygous for banl(2)k02512 (up to 80% with four copies of hsp83:GAGA519). Symmetrically, the addition of an extra copy of batman also rescues banl(2)k02512/+; Trl13c/Trl13c flies. Together these experiments indicate that the increase in the lethality of flies that are mutant for Trl in a heterozygous banl(2)k02512 context results from the synergistic effect of ban and Trl loss of function mutations (Faucheux, 2003).

The Trl13c allele is a dominant enhancer of the weak homeotic transformation of halter toward wing found in Ubx130 heterozygotes. Flies that are doubly heterozygous for banl(2)k02512 and Ubx130 do not show a significant increase in the expressivity of the Ubx phenotype. In contrast, banl(2)k02512/+; Trl13c/Ubx130 flies display a higher frequency (18%) of strong halter-to-wing transformations compared to their +/+; Trl13c/Ubx130 siblings (6.3%). The synergistic interaction between ban and Trl on the Ubx phenotype indicates that ban function is required together with that of Trl for the activation of the homeotic gene Ubx (Faucheux, 2003).

Trl dosage is important for the pairing-sensitive silencing that is induced by PREs when they are placed next to a white derivative reporter gene. Two independent constructs containing a PRE fragment (260.1 iab-7 PRE and 5F24 25,2 Fab-7 PRE) were tested for a modification of eye pigmentation in Trl and in ban heterozygous mutant backgrounds. For both PRE constructs, it was found that reducing the dose of ban using several ban loss of function alleles leads to derepression of the white reporter gene, an effect similar to that observed when reducing the dose of Trl. When both Trl and ban functions are reduced in double heterozygotes, the pairing-sensitive silencing is further suppressed, thereby indicating that ban and Trl genetically interact for silencing the iab-7 PRE (Faucheux, 2003).

Taken together, these results suggest that ban cooperates with Trl in order to maintain the activation or the repression of Trl target genes that are necessary for viability and/or normal development of the flies (Faucheux, 2003).

The pairing-dependent silencing or pairing sensitivity (PS), assay is widely used for checking the PRE nature of a given sequence and studies have shown that mutations in PcG genes relieve this pairing-dependent silencing. Transgenic lines carrying iab-7PRE next to mini-white reporter, PRE-mw, show pairing dependent silencing of the mini-white and like several PcG mutations, this silencing is affected by Trl mutations. These PRE-mw lines were used to test if lolal is required for the PS function. Since lolal was shown to interact with ph (Faucheux, 2003), ph was checked in this PS assay (Mishra, 2003).

In several transgenic lines, PRE-mw/PRE-mw flies show lighter eye color as compared to the PRE-mw/+ flies. All the lines tested showed clear requirement of both lolal and ph for the PS function, since the eye color of PRE-mw/PRE-mw flies shows clear enhancement when brought in lolal (banDf(2R)311a) or ph (ph-p) background. Similar results were obtained with a P element insertion allele of lolal (lolalk02512). In this case, although there is a clear enhancement of pigmentation in PRE-mw/PRE-mw flies when tested in lolal background, the red pigment of these mutant combinations was also tested to distinguish the increase in pigmentation from mere additive effect of the light eye color of P element of the lolal allele. The results show that value for PRE-mw/PRE-mw in lolal/CyO context is 50% more than added values of PRE-mw/PRE-mw and lolal/CyO flies (Mishra, 2003).

The sex comb phenotype of ph is known to be enhanced by lolal and, by this criterion, lolal can be classified as a PcG member. Often members of the PcG interact with one another and also with the trxG members. lolal was tested in different PcG and trxG mutant backgrounds. Since most of these mutations are recessive lethal, only the heterozygote combinations could be tested, except for ph which is homozygous viable. As expected, sex combs were found on the middle leg and often on the third leg in ph/y; lolal/+ males. Similar interactions of lolal were found with Pc and Pcl. Interestingly, ph in combination with lolal shows partial homeotic transformation of A4-->A5, A5-->A6 and A6-->A7. In contrast, Asx shows partial A6-->A5 transformation in the abdomen. Pc in combination with lolal also gives a wing phenotype. All lolal/+; Pc/+ flies had crumbled wings with defective margin. Among the trxG mutations tested, mor had a rough eye phenotype and trg showed homeotic transformation in the abdomen. While trg/+ flies have several bristles on the 6th sternite in males, with the combination of lolal, an additional segment forms, indicating partial anteriorization transformation of A7 to A6 (Mishra, 2003).

Contrary to the expectations based on the biochemical studies, no apparent interaction of lolal with Trl13C was seen in heterozygous condition. Similarly, among the mutations tested, osa, trx, ash1, Psc, Scm did not show any interaction with lolal. Interestingly, it has been shown recently that Trl also enhances ph phenotype in a fashion similar to what is seen in the case of lolal and ph. This raises the possibility that there may be a networking of Trl-lolal-ph and, perhaps, other PcG proteins in the PRE mediated repression mechanisms. These interactions of lolal with ph and Pc suggest that association of lolal may be the key step of PcG function of Trl-GAGA (Mishra, 2003).


Faucheux, M., et al. (2001). Advantages of a P-element construct containing MtnA sequences for the identification of patterning and cell determination genes in Drosophila melanogaster. Mol. Genet. Genomics 265: 14-22. 11370860

Faucheux, M., et al. (2003). batman interacts with Polycomb and trithorax group genes and encodes a BTB/POZ protein that is included in a complex containing GAGA factor. Molec. Cell. Bio. 23: 1181-1195. 12556479

Mishra, K., et al. (2003). Trl-GAGA directly interacts with lola like and both are part of the repressive complex of Polycomb group of genes. Mech. Dev. 120: 681-689. 12834867

lola like: Biological Overview | Regulation | Developmental Biology | Effects of Mutation

date revised: 20 December 2003

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