Gene name - polyhomeotic proximal and polyhomeotic distal
Cytological map position - 2D3-4
Function - transcription factor
Keywords - Polycomb group
Symbol - ph-p and ph-d
Genetic map position - 1-0.5
Classification - zinc finger
Cellular location - nuclear
|Recent literature||Schuster, K.J. and Smith-Bolton, R.K. (2015). Taranis protects regenerating tissue from fate changes induced by the wound response in Drosophila. Dev Cell 34(1):119-28. PubMed ID: 26096735
Regenerating tissue must replace lost structures with cells of the proper identity and function. How regenerating tissue establishes or maintains correct cell fates during regrowth is an open question. This study identified a gene, taranis, that is essential for maintaining proper cell fate in damaged and regenerating Drosophila wing imaginal discs but that is dispensable for these fates in normal wing development. In regenerating tissue with reduced levels of Taranis, expression of the posterior selector gene engrailed is silenced through an autoregulatory silencing mechanism that requires the PRC1 component polyhomeotic, resulting in a transformation of posterior tissue into anterior tissue late in regeneration. An essential component of the wound response, JNK signaling, induces this misregulation of engrailed expression. Taranis can suppress these JNK-induced cell fate changes without interfering with JNK signaling activity. Thus, taranis protects regenerating tissue from deleterious side effects of wound healing and regeneration.
|Wani, A. H., Boettiger, A. N., Schorderet, P., Ergun, A., Munger, C., Sadreyev, R. I., Zhuang, X., Kingston, R. E. and Francis, N. J. (2016). Chromatin topology is coupled to Polycomb group protein subnuclear organization. Nat Commun 7: 10291. PubMed ID: 26759081
The genomes of metazoa are organized at multiple scales. Many proteins that regulate genome architecture, including Polycomb group (PcG) proteins, form subnuclear structures. Deciphering mechanistic links between protein organization and chromatin architecture requires precise description and mechanistic perturbations of both. Using super-resolution microscopy, this study shows that PcG proteins are organized into hundreds of nanoscale protein clusters. PcG clusters were manipulated by disrupting the polymerization activity of the sterile alpha motif (SAM) of the PcG protein Polyhomeotic (Ph) or by increasing Ph levels. Ph with mutant SAM disrupts clustering of endogenous PcG complexes and chromatin interactions while elevating Ph level increases cluster number and chromatin interactions. These effects can be captured by molecular simulations based on a previously described chromatin polymer model. Both perturbations also alter gene expression. Organization of PcG proteins into small, abundant clusters on chromatin through Ph SAM polymerization activity may shape genome architecture through chromatin interactions.
|Cheutin, T. and Cavalli, G. (2018). Loss of PRC1 induces higher-order opening of Hox loci independently of transcription during Drosophila embryogenesis. Nat Commun 9(1): 3898. PubMed ID: 30254245
Polycomb-group proteins are conserved chromatin factors that maintain the silencing of key developmental genes, notably the Hox gene clusters, outside of their expression domains. Depletion of Polycomb repressive complex 1 (PRC1) proteins typically results in chromatin unfolding, as well as ectopic transcription. To disentangle these two phenomena, this study analyzed the temporal function of two PRC1 proteins, Polyhomeotic (Ph) and Polycomb (Pc), on Hox gene clusters during Drosophila embryogenesis. The absence of Ph or Pc affects the higher-order chromatin folding of Hox clusters prior to ectopic Hox gene transcription, demonstrating that PRC1 primary function during early embryogenesis is to compact its target chromatin. Moreover, the differential effects of Ph and Pc on Hox cluster folding match the differences in ectopic Hox gene expression observed in these two mutants. These data suggest that PRC1 maintains gene silencing by folding chromatin domains and impose architectural layer to gene regulation.
|Medina, I., Calleja, M. and Morata, G. (2021). Tumorigenesis and cell competition in Drosophila in the absence of polyhomeotic function. Proc Natl Acad Sci U S A 118(45). PubMed ID: 34702735
Cell competition is a homeostatic process that eliminates by apoptosis unfit or undesirable cells from animal tissues, including tumor cells that appear during the life of the organism. In Drosophila there is evidence that many types of oncogenic cells are eliminated by cell competition. One exception is cells mutant for polyhomeotic (ph), a member of the Polycomb family of genes; most of the isolated mutant ph clones survive and develop tumorous overgrowths in imaginal discs. To characterize the tumorigenic effect of the lack of ph, the growth was studied of different regions of the wing disc deficient in ph activity; the effect was restricted to the proximal appendage. Moreover, it was found that ph-deficient tissue is partially refractory to apoptosis. Then, the behavior of clones lacking ph function was studied; many suffered cell competition but were not completely eliminated. Unexpectedly, it was found that nonmutant cells also undergo cell competition when surrounded by ph-deficient cells, indicating that within the same tissue cell competition may operate in opposite directions. Two reasons are suggested for the incompleteness of cell competition inph mutant cells: 1) These cells are partially refractory to apoptosis, and 2) the loss of ph function alters the identity of imaginal cells and subsequently their cell affinities. It compromises the winner/loser interaction, a prerequisite for cell competition.
|Kapur, I., Boulier, E. L. and Francis, N. J. (2022). Regulation of Polyhomeotic Condensates by Intrinsically Disordered Sequences That Affect Chromatin Binding. Epigenomes 6(4). PubMed ID: 36412795
The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph) forms phase-separated condensates with DNA or chromatin in vitro, suggesting that PcG bodies may form through SAM-driven phase separation. Three distinct low-complexity regions were identified in Ph based on sequence composition. The role of each of these sequences was tested in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affected Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occurred when the central, glutamine-rich intrinsically disordered region (IDR) was removed, which resulted in large Ph condensates. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. Chromatin fractionation experiments indicated that the removal of the glutamine-rich IDR reduced chromatin binding and that the removal of either of the other IDRs increased chromatin binding. These data suggest that all three IDRs, and functional interactions among them, regulate Ph condensate size and number. These results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation. These observations highlight the complexity of regulation of biological condensates housed in single proteins.
|Kapur, I., Boulier, E. L. and Francis, N. J. (2022). Regulation of Polyhomeotic Condensates by Intrinsically Disordered Sequences That Affect Chromatin Binding. Epigenomes 6(4). PubMed ID: 36412795
The Polycomb group (PcG) complex PRC1 localizes in the nucleus in condensed structures called Polycomb bodies. The PRC1 subunit Polyhomeotic (Ph) contains an oligomerizing sterile alpha motif (SAM) that is implicated in both PcG body formation and chromatin organization in Drosophila and mammalian cells. A truncated version of Ph containing the SAM (mini-Ph) forms phase-separated condensates with DNA or chromatin in vitro, suggesting that PcG bodies may form through SAM-driven phase separation. In cells, Ph forms multiple small condensates, while mini-Ph typically forms a single large nuclear condensate. It is therefore hypothesized that sequences outside of mini-Ph, which are predicted to be intrinsically disordered, are required for proper condensate formation. In this study identified three distinct low-complexity regions in Ph based on sequence composition. The role of each of these sequences was systematically tested in Ph condensates using live imaging of transfected Drosophila S2 cells. Each sequence uniquely affected Ph SAM-dependent condensate size, number, and morphology, but the most dramatic effects occurred when the central, glutamine-rich intrinsically disordered region (IDR) was removed, which resulted in large Ph condensates. Like mini-Ph condensates, condensates lacking the glutamine-rich IDR excluded chromatin. Chromatin fractionation experiments indicated that the removal of the glutamine-rich IDR reduced chromatin binding and that the removal of either of the other IDRs increased chromatin binding. These data suggest that all three IDRs, and functional interactions among them, regulate Ph condensate size and number. The results can be explained by a model in which tight chromatin binding by Ph IDRs antagonizes Ph SAM-driven phase separation. These observations highlight the complexity of regulation of biological condensates housed in single proteins.
Early ph expression is activated by Bicoid and Engrailed and repressed by Oskar. polyhomeotic is involved in a negative autoregulatory loop. As the number of polyhomeotic copies in the genome increases, the level of transcription of each decreases (Fauvarque, 1995).
Of particular interest is the activation of polyhomeotic by Engrailed. When examined at stage 10, the expression domains of en and ph are found to overlap. At this stage ph is expressed in 14 evenly spaced bands in the anterior compartment of each parasegment, coincident with engrailed expression domains. Engrailed binding sites exist in two regions between ph-p and ph-d and in the one promoter site proximal to ph-d.
What function could engrailed activation of ph serve? At some time after the initial ph activation, ph expression becomes independent of EN. There is a transition period when maintenance of en expression is actually dependent on ph. During the larval stage, EN activates ph expression during wing morphogenesis, even as it represses ph expression in the hindgut. It would appear that EN can act as either an activator or as a repressor of ph expression. It appears that engrailed, a gene whose activity is essential in the correct formation of segment polarity, has coopted PH as its own private Polycomb-group protein, adding a segment polarity element to gene silencing (Serrano, 1995).
Transheterozygous adult flies, mutant for both engrailed and polyhomeotic, show a gap in the fourth vein. In the corresponding larval imaginal discs, a polyhomeotic-lacZ enhancer trap is not normally activated in anterior cells adjacent to the anterior-posterior boundary. This intermediary region corresponds to the domain of low engrailed expression that appears in the anterior compartment, during L3. This en expression depends on the putative serine-threonine kinase protein fused and on the level of hh expression in the posterior cells abutting the A/P boundary, and so depends indirectly on en expression in the posterior compartment. The exact role of this late L3 anterior compartment en expression is still not understood (Maschat, 1998).
Several arguments show that engrailed is responsible for the induction of polyhomeotic in these cells. The role of polyhomeotic in this intermediary region is apparently to maintain the repression of hedgehog in the anterior cells abutting the anterior-posterior boundary, since these cells ectopically express hedgehog when polyhomeotic is not activated. Analysis of the expression patterns of different genes of the Hh signaling pathway that are normally expressed in this intermediary region showed that the segmentation gene patched is highly affected in ph/en mutant discs. The gap in the fourth vein can therefore be correlated with a misregulation of patched in the posterior compartment. Interestingly, this ectopic ptc expression appears not only in the cells where ph is affected, but also in neighboring posterior cells. This ectopic expression of ptc progressively invades the posterior compartment during the third instar to fill the whole compartment in mature larvae. Genetic data indicate that the level of hh expression is involved in this phenomenon, suggesting that the progressive invasion of the posterior compartment by Ptc is due to an increased secretion of Hh by the cells of the anterior intermediary region, towards cells localized more posteriorly. As a consequence of this ptc misregulation, cubitus-interruptus (ci) and decapentaplegic (dpp) are activated in the posterior compartment, suggesting that the intermediary region, where dpp expression is normally confined, expands posteriorly. As a result of the absence of ph activation by En in cells abutting the A/P boundary, this boundary is not maintained at its normal position, but is progressively shifted posteriorly, while cells lose their posterior identity. Thus posterior cells express a new set of genes that are normally characteristic of anterior cells, suggesting a change in the cell identity. Altogether, these data indicate that engrailed and polyhomeotic interactions are required to maintain the anterior-posterior boundary and the posterior cell fate, just prior to the evagination of the wing (Maschat, 1998).
Considering that hh is responsible for the changes appearing in the posterior compartment of ph/en flies implies that posterior cells might become competent to respond to the Hh signal. Such competence could be attributed to the presence of a low level of posterior compartment ci, which is present ectopically in a [ph-; en-/+] background. Indeed, ph has been shown to be a repressor of ci in the posterior compartment and now it seems both en and ph are likely to be responsible for ci repression in the posterior compartment. Transcriptional repression of ci in the posterior compartment could be initiated by en and maintained by ph, the ph expression depending itself on en expression. Indeed, posterior heterozygous en/+ cells do not show any phenotype unless they are also mutant for ph. One could hypothesize a feedback loop involving en and ph to maintain the level of en expression and ci repression in the posterior compartment. If the basic level of en expression in the posterior compartment depends on both en and ph, en could be maintained at a lower level in a [ph-; en-/+] background. These cells might now produce enough Ci and Ptc to become competent to receive the Hh signal. If posterior cells are not competent to receive an Hh signal, higher amounts of Hh would not affect the posterior cells. Such a feedback loop mechanism between en and ph, maintaining the level of en expression, could also explain the lack of hypomorphic en mutants, since such mutants would be detectable only when ph is affected (Maschat, 1998).
The opposing actions of polycomb (PcG) and trithorax group (trxG) gene products maintain essential gene expression patterns during Drosophila development. PcG proteins are thought to establish repressive chromatin structures, but the mechanisms by which this occurs are not known. Polycomb repressive complex 1 (PRC1) contains PcG proteins Polyhomeotic (Ph), Polycomb (Pc), Posterior sexcombs (Psc) and Ring and inhibits chromatin remodeling by trxG-related SWI/SNF complexes. A functional core of PRC1 has been defined by reconstituting a stable complex using four recombinant PcG proteins. One subunit, Psc, can also inhibit chromatin remodeling on its own. These PcG proteins create a chromatin structure that has normal nucleosome organization and is accessible to nucleases but excludes hSWI/SNF (Francis, 2001).
To assemble an active recombinant complex of PcG proteins that could be purified in large amounts and used for detailed functional analyses, Sf9 cells were coinfected with Flag-Ph, Pc, Psc, and Ring. Flag-Ph and associated proteins were purified by affinity chromatography. Pc, Psc, and Ring were purified quantitatively with Flag-Ph. If Sf9 cells are coinfected with Psc, Ph, and Pc, with either Psc or Ph carrying the Flag epitope, the three proteins copurify but the eluted fractions contain a significant excess of the epitope-tagged subunit; this suggests Ring is important for stable complex formation. Purification of Flag-Ph from cells expressing Flag-Ph, Ring, and Pc did not result in purification of a complex, but mainly of Flag-Ph. Thus, Pc, Ph, Psc, and Ring form the most stable complex, which will be referred to as PCC (PRC1 core complex) for simplicity. PCC fractions invariably contain a fifth, 70 kDa protein; this protein may be an HSC70 homolog from Sf9 cells since it is immunoreactive with antibodies to HSP70. HSC70 may interact specifically with PcG proteins since HSC proteins copurify with PcG proteins in PRC1 and mutations in HSC4 enhance Pc phenotypes; alternatively, it may associate with the complex simply because the PcG proteins are overexpressed (Francis, 2001).
To determine whether PCC shares the ability of PRC1 to block chromatin remodeling by hSWI/SNF, PCC was tested in the plasmid supercoiling assay originally used to define PRC1 activity. When incubated with a nucleosomal plasmid array and topoisomerase I, hSWI/SNF induces an ATP-dependent decrease in negative supercoiling, which can be identified on agarose gels as a change in the distribution of plasmid topoisomers. PRC1 blocks the ability of hSWI/SNF to alter plasmid topology when preincubated with the template, but not when added simultaneously with hSWI/SNF. Preparations of PCC inhibit remodeling in this assay in a preincubation-dependent manner that mimics the activity of PRC1 (Francis, 2001).
Each component of PCC was purified to determine whether any individual protein shared functional characteristics with PCC or PRC1. One subunit, Psc, also inhibits chromatin remodeling in the plasmid supercoiling assay in a manner that is strictly dependent on preincubation. Preparations of Pc, Ph, and Ring were less active than Psc in this assay. It is concluded that PCC and at least one of its subunits share the ability of PRC1 to inhibit chromatin remodeling (Francis, 2001).
To compare the relative efficiency of PRC1, PCC, and Psc, a quantifiable assay for inhibition was developed based on restriction enzyme accessibility. Assembly of DNA into chromatin blocks the ability of restriction enzymes to digest DNA at nucleosomal sites, but this decrease in accessibility can be counteracted by ATP-dependent chromatin remodeling factors such as hSWI/SNF. Nucleosomal arrays were assembled by the use of templates consisting of two sets of five 5S nucleosome positioning sequences that flank DNA sufficient to assemble two nucleosomes, one of which is predicted to overlap a unique HhaI site. The extent of digestion of the HhaI site in a chromatin template exposed to hSWI/SNF was used to quantify inhibition of chromatin remodeling by PcG proteins (Francis, 2001).
PRC1 blocks the ATP-dependent stimulation of restriction enzyme digestion by hSWI/SNF when preincubated with the template at a concentration of approximately 1 nM. Under the same conditions, approximately 2-4 nM PCC also inhibits chromatin remodeling, suggesting the core complex is up to 50% as efficient as PRC1. Psc inhibits chromatin remodeling at higher concentrations than PCC. Thus, both Psc and PCC inhibit chromatin remodeling and are nearly as efficient as PRC1 (Francis, 2001).
PcG preparations could inhibit remodeling through interactions with the nucleosomal array, by directly inhibiting hSWI/SNF function, or by a combination of these mechanisms. PRC1 does not efficiently inhibit remodeling of mononucleosomes. This suggestes that the complex might require a nucleosomal array substrate. Because large amounts of concentrated PCC and Psc could be purified, tests were performed to see whether these proteins inhibit remodeling of mononucleosomes over a wide range of protein concentrations in excess of nucleosomes. PCC and Psc were titrated into a restriction enzyme accessibility assay for hSWI/SNF remodeling of mononucleosomes assembled on a 155 bp DNA fragment. PCC and Psc have no effect on remodeling of mononucleosomes at concentrations greater than those required to completely inhibit remodeling of arrays, and Psc inhibits remodeling only when present at greater than 20-fold excess over template. These results suggest that PCC or Psc inhibit chromatin remodeling by interacting with the substrate and that these proteins have specific substrate requirements not present in mononucleosomes, such as linker DNA or multiple contiguous nucleosomes. These experiments also confirm that concentrations of PCC and Psc that inhibit remodeling of nucleosomal arrays do not directly inhibit hSWI/SNF activity (Francis, 2001).
These experiments demonstrate that the stable PcG complex PCC and the single subunit Psc share the ability of PRC1 to inhibit remodeling. The definition of this minimal system and the ability to obtain large quantities of highly purified PCC and Psc allows a detailed characterization of the ability of PcG proteins to inhibit remodeling. The ability of PCC and Psc to bind DNA and to interact with nucleosomal arrays to prevent remodeling was examined (Francis, 2001).
Although one mammalian Psc homolog, MEL-18, has been demonstrated to bind DNA, this has not been shown to be the case with the PcG proteins present in PCC. The ability of PCC and Psc to inhibit remodeling on arrays but not on mononucleosomes has suggested that they might recognize free DNA. PCC and Psc were tested for DNA binding activity using filter binding with a 155 bp probe; both PCC and Psc bind DNA with high affinity. The measured KDs of PCC and Psc for DNA were similar. Filter binding assays were used to determine what fraction of molecules in each of the protein preparations were active for DNA binding. A high fraction of the molecules in PCC and Psc preparations were active for DNA binding (PCC 30%-60%; Psc 20%-50%), assuming binding as monomers (Francis, 2001).
It was of interest to determine whether PcG proteins generally block access of all enzymes to chromatin or, more specifically, prevent chromatin remodeling. Considerable attention has been given to the ability of remodeling factors to increase accessibility of DNA sites, such as is measured in the restriction enzyme accessibility assay. However, nucleosome movement stimulated by remodelers can also cause previously exposed sites to become occluded. A SacI site in the 5S array template is predicted to reside between nucleosomes; consistent with this prediction, the SacI site is accessible in 40%-60% of the nucleosomal arrays in the absence of hSWI/SNF. If the template is remodeled with hSWI/SNF, the accessibility of this site is dramatically reduced. The SacI site therefore allows the dissociation of effects of PCC and Psc on chromatin remodeling from direct effects on the restriction enzyme or on template accessibility, since in this case inhibition of remodeling would be expected to increase SacI digestion after incubation with hSWI/SNF. When the template was remodeled by SWI/SNF for 30 min and subsequently digested with SacI for 5 min, a clear decrease in restriction enzyme accessibility was observed, consistent with repositioning of nucleosomes over this site on some templates. This hSWI/SNF-dependent decrease in accessibility is blocked by preincubation of the template with PCC or Psc so that digestion levels were similar to those in the absence of hSWI/SNF (40%-60% digestion). It is concluded that Psc and PCC block ATP-dependent remodeling without blocking access of restriction enzymes to the template (Francis, 2001).
To examine whether inhibition of remodeling by hSWI/SNF is accompanied by changes in nucleosome organization, the effect of PRC1, PCC, and Psc on MNase digestion of nucleosomal arrays was examined. MNase preferentially digests the linker DNA between nucleosomes. On arrays of nucleosomes that are positioned by 5S sequences, digestion with MNase produces a distinct ladder, which can be visualized by probing Southern blots of the digested array with the 5S sequence. Incubation of the array with hSWI/SNF and ATP causes a smearing of the banding pattern, reflecting randomization of nucleosome positioning. PRC1, PCC, and Psc block disruption of the MNase ladder by hSWI/SNF. Significantly, none of these PcG proteins or complexes alter the MNase digestion pattern of the array, implying that MNase is still able to access the array and that nucleosome organization is not grossly altered by the presence of these PcG proteins. These results suggest that complexing these PcG proteins with the template prevents hSWI/SNF-mediated nucleosome movement but not nuclease access to the template (Francis, 2001).
In these MNase experiments, it was possible that PCC or Psc altered nucleosome positions but nonetheless maintained a regular organization of nucleosomes on the template. To determine whether nucleosome positions were altered, indirect end labeling was carried out after digestion with MNase or DNaseI and nucleosome position was examined in the presence or absence of PCC or Psc. Neither Psc nor PCC altered nucleosome position as assayed by either MNase or DNaseI digestion, both of which yield nucleosomal ladders due to the tight positioning of the nucleosomes on the 5S array. Similar results were obtained in MNase experiments with PRC1. Interestingly, however, arrays incubated with PCC or Psc were approximately 5- to 10-fold less sensitive to digestion by MNase, but about 10-fold more sensitive to digestion by DNase I. Although as yet there is no explanation for this observation, it is consistent with PCC or Psc altering the structure of the chromatin template and perhaps the orientation of the linker DNA (Francis, 2001).
Psc and PCC could prevent remodeling by preventing hSWI/SNF from binding to the template, by interfering with the remodeling of chromatin carried out by bound hSWI/SNF, or by a combination of both mechanisms. To determine whether binding of Psc or PCC to the template interferes with the binding of hSWI/SNF, in vitro chromatin immunoprecipitations (ChIPs) were carried out using antisera to BRG1, the major ATPase subunit and remodeling protein of hSWI/SNF. The amount of DNA precipitated by anti-BRG1 was compared on templates incubated with or without Psc or PCC and these results were correlated with inhibition of remodeling in the same reactions. When arrays were preincubated with concentrations of Psc or PCC that inhibit chromatin remodeling, the amount of BRG1 bound to the template was decreased by at least 10-fold. Titrations of Psc and PCC indicate that exclusion of BRG1 from the template correlates with inhibition of remodeling. Thus, these experiments suggest that Psc or PCC can exclude hSWI/SNF from a nucleosomal array, and are consistent with exclusion accounting, at least in part, for inhibition of remodeling (Francis, 2001).
Psc is active as an isolated polypeptide, raising the possibility that this protein is an important component of PCC activity and could function in the absence of the other PCC subunits. Indeed, recent evidence suggests Psc is an essential component of the silencing mechanism and may function independently of the other PCC subunits in certain circumstances. (1) Loss of Psc and its homolog suppressor of zeste 2 [Su(Z)2] or Ph, but not of other PcG proteins, has been shown to result in rapid upregulation of homeotic genes. Repression can be restored by resupply of Psc [and Su(Z)2] provided only a few cell generations has passed. These results are consistent with an essential role for Psc in the actual silencing mechanism. (2) Immunocytochemistry in Drosophila embryos demonstrates that the majority of Ph, Pc, and Psc dissociate from chromosomes during mitosis; Psc reassociates with the chromosomes before Pc and Ph, suggesting Psc may function independently of these subunits in vivo during reestablishment of repression (Francis, 2001).
In vivo, PcG proteins are targeted to appropriate genes by PREs, but PCC does not require PRE sequences to prevent chromatin remodeling. Preliminary results with templates that include PRE sequences from the Ubx gene suggest that PREs may not target PCC and Psc in vitro. This raises the possibility that the basic mechanisms by which PcG proteins influence chromatin can be mechanistically separated from those which target repression to appropriate genes. It seems likely that PcG proteins interact, perhaps directly, with DNA binding factors that target them to PREs. The characterization of PCC activity provides a system in which targeting of PcG proteins can be analyzed in vitro (Francis, 2001).
Polycomb group (PcG) proteins are conserved epigenetic regulators that are linked to cancer in humans. However, little is known about how they control cell proliferation. This study reports that mutant clones of the PcG gene polyhomeotic (ph) form unique single-cell-layer cavities that secrete three JAK/STAT pathway ligands, which in turn act redundantly to stimulate overproliferation of surrounding wild-type cells. Notably, different ph alleles cause different phenotypes at the cellular level. Although the ph-null allele induces non-autonomous overgrowth, an allele encoding truncated Ph induces both autonomous and non-autonomous overgrowth. It is proposed that PcG misregulation promotes tumorigenesis through several cellular mechanisms (Feng, 2011). <>In summary, mosaic clones homozygous for the ph-null allele induce overproliferation of surrounding wild-type cells through Notch-Upd-JAK/STAT signalling, whereas mosaic clones homozygous for a ph hypomorphic allele that encodes truncated Ph proteins induce both autonomous and non-autonomous cell overproliferation. These results highlight an important but largely overlooked phenomenon: different mutations in the same gene might induce tumours and cancers through distinct cellular mechanisms, depending on the nature of the mutations and/or genetic backgrounds. This fact adds another layer of complexity to cancer pathology (Feng, 2011).
Drosophila polyhomeotic (ph) is one of the important polycomb group genes that is linked to human cancer. In the mosaic eye imaginal discs, while phdel, a null allele, causes only non-autonomous overgrowth, ph505, a hypomorphic allele, causes both autonomous and non-autonomous overgrowth. These allele-specific phenotypes stem from the different sensitivities of ph mutant cells to the Upd homologs that they secrete (Feng, 2012).
Different ph alleles cause tissue overgrowth in different ways. While a ph null allele, phdel , causes only non-autonomous cell over-proliferation, a ph hypomorphic allele, ph505 , causes both autonomous and non-autonomous cell overproliferation. In mosaic tissues, overproliferation of mutant cells was defined as autonomous, whereas over-proliferation of genotypically wild type cells induced by mutant cells was defined as non-autonomous. The signaling pathway involved in phdel induced non-autonomous cell over-proliferation. In summary, elevated Notch activity in ph cells up-regulates the expression of JAK/STAT pathway ligands Upd homologs, which in turn activate the JAK/STAT pathway in neighboring wild type cells and cause their over-proliferation. This study addressed why a ph null allele and a ph hypomorphic allele both cause tumors but in such different ways (Feng, 2012).
First whether the same signaling pathway underlay non-autonomous overproliferation induced by both phdel and ph505 was tested. The functions of Notch and Upd homologs in the ph505 mosaic eyes were examined with the same strategy used for phdel. A ph505 -Notch double mutant line was generated, and eyes mosaic for this line were essentially of the same size as wild type eyes. The mosaic eye discs had normal size and normal cell proliferation level, as shown by PH3 staining, which marks mitotic cells. Moreover, the size of ph505 -Notch clones was significantly reduced when compared to that of ph505 clones. These results indicated that Notch was required for both autonomous and non-autonomous overproliferation induced by ph505 (Feng, 2012).
Next ph505 was recombined with updΔ1-3, a deficiency line that lacks all three upd homologs in the Drosophila genome Mosaic analyses were then performed using this double mutant line. ph505 -updΔ1-3 mosaic eyes were significantly smaller than ph505 mosaic eyes and were comparable to wild type eyes, indicating that tissue overgrowth was largely suppressed. PH3 staining of the double mutant mosaic eye discs showed that these discs had relatively normal size and cell proliferation level. Importantly, ph505 -updd1-3 clones were also drastically reduced in size compared to ph505 clones. These results indicated that Upd homologs are required for not only non-autonomous but also autonomous cell over-proliferations induced by ph505 (Feng, 2012).
It is not surprising that the same signaling pathway is responsible for non-autonomous over-proliferation induced by both phdel and ph505 , and it is not completely unexpected that Notch is also required for ph505 induced autonomous over-proliferation, as Notch is a transcription factor that has been shown to autonomously regulate cell proliferation. However, the three Upd proteins are secreted and are not expected to have any direct effect on autonomous cell proliferation. To interpret these observations, it was hypothesized that ph505 cells still respond to Upd ligands secreted by themselves in an autocrine or paracrine manner, and therefore over-proliferate. However, phdel cells were thought to be no longer responsive to Upd ligands (Feng, 2012).
To functionally test this hypothesis, the double mutant strategy was applied, taking advantage of the fact that the genes domeless (dome, encoding the only transmembrane receptor of the Drosophila JAK/STAT pathway) and hopscotch (hop, encoding the only Drosophila JAK kinase) are also on X chromosome as is ph. First ph505 was recombined with two dome alleles to generate ph505 -dome double mutant lines. Eye discs mosaic for these lines were still significantly larger than wild type, but the size of double mutant clones was dramatically reduced, so that only a tiny portion of the disc was composed of mutant cells. PH3 staining indicated that non-autonomous proliferation level was still high, but autonomous proliferation largely disappeared. The adult eyes mosaic for such double mutant lines were further examinedm and these eyes were found to be still much larger than wild type and similar to ph505 mosaic eyes in size, but they generally were not folded as seen in ph505 mosaic eyes (Feng, 2012).
Next a ph505 -hop double mutant line was generated. Autonomous proliferation was found in mosaic eye discs of this double mutant that was also significantly suppressed, with mutant cells only accounted for a small portion of the whole disc. In contrast, non-autonomous cell over-proliferation was not affected and the overall size of these discs was still significantly larger than wild type. Adult eyes mosaic for this double mutant showed similar phenotypes as those of ph505 -dome mosaic eyes. These eyes were still significantly larger than wild type but they were generally not folded. Therefore, the removal of either dome or hop from ph505 cells only suppressed autonomous over-proliferation but did not affect non-autonomous overproliferation, making such double mutant mosaic discs phenotypically similar to phdel mosaic discs (Feng, 2012).
As controls, phdel -dome and phdel -hop double mutant lines were also generated using the same dome and hop alleles. Mosaic analyses on eye discs showed that the removal of dome or hop from phdel cells did not affect non-autonomous cell over proliferation. It did, however, mildly reduce the mutant clone size, suggesting that phdel cells might still have a weak response to Upd ligands. Adult eyes mosaic for these double mutant lines were phenotypically indistinguishable from phdel mosaic eyes, consistent with the above observations in mosaic eye discs (Feng, 2012).
Finally it was asked why phdel and ph505 cells responds differently to the Upd ligands secreted by themselves. It was hypothesized that some of the JAK/STAT pathway modulators might be differentially expressed in phdel and ph505 cells. To test this hypothesis, TU-Tagging, a technique that enables the purification of RNA from mutant cells without having to physically isolate such cells, was chosen. Briefly, Drosophila is unable to synthesize uridine from uracil due to the lack of phosphoribosyltransferase (UPRT). When exogenous UPRT is expressed in mutant cells by MARCM, such cells would acquire the ability to utilize uracil. If these larvae are fed with 4-thiouracil (4-TU), a uracil derivative that contains a thio group, only mutant cells would be able to use 4-TU and eventually incorporate thio- containing uridine into newly synthesized RNA. This treatment has little toxicity, and the thio-labeled RNA can be purified from total RNA using conventional biochemical methods (Feng, 2012).
TU-tagging was performed to isolate RNA from phdel cells and ph505 cells, and qRTPCR was used to examine candidate gene expression. The expression of the JAK/STAT pathway receptor dome was significantly higher in ph505 cells than in phdel cells. A higher receptor expression might sensitize ph505 cells to the Upd ligands. The levels of enok and socs42a, both negative regulators of the JAK/STAT pathway, were also significantly higher in ph505 cells compared to phdel cells. This might represent feedback loops that negatively regulate the pathway activity. In fact, several such negative feedback loops, in which elevated pathway activity upregulates a negative pathway regulator, have been reported in JAK/STAT pathway (Feng, 2012).
Together, it is concluded that phdel and ph505 both cause autonomous over-expression of Upd homologs in mutant cells, which represents the only driving force of cell overproliferation in phdel and ph505 mosaic tissues and in essence acts non-autonomously to activate JAK/STAT pathway. The different phenotypes of these two types of mosaics are due to different sensitivity of mutant cells to Upd homologs. ph505 mutant cells robustly respond to Upd ligands that they secreted. Therefore, Upd ligands secreted by ph505 cells simultaneously induce over-proliferation in both mutant and wild type cells. In contrast, phdel cells are largely insensitive to Upd ligands, so that Upd ligands secreted by phdel cells only induce over-proliferation in wild type but not mutant cells. Furthermore, differential expression of the JAK/STAT pathway receptor dome might underlie the different sensitivity of phdel and ph505 cells to Upd ligands (Feng, 2012).
The polyhomeotic locus of Drosophila is a complex locus essential for the maintenance of segmental identity. Genetic analysis suggests that two independent units contribute to ph function. Comparison of genomic sequence shows that the ph locus has been duplicated, and that it contains proximal and distal transcription units. The proximal transcription unit encodes two embryonic mRNAs of 6.4 and 6.1 kb; the distal unit encodes a 6.4-kb embryonic mRNA. The distal protein is very similar to the proximal product, except for the absence of an amino terminal region, and a small region near the carboxy terminus. The long open reading frame in the distal cDNA does not begin with an ATG codon; an internal ATG is used for a start codon. The proximal protein occurs in two forms that are developmentally regulated, and that probably arise from use of two different initiator methionine codons (Hodgson, 1997).
The PH-P protein has 4 glutamine rich regions, a C4 zinc finger domain, a C-terminal alpha helix motif and a serine and threonine rich domain. Comparison to the mammalian ph homolog rae28 reveals two more regions of similarity, indicated as H1 and H2. Drosophila Sex comb on midleg protein, another PcG protein, contains a domain homologous to Ph corresponding to the H2 region. Further analysis has revealed two other motifs, a GXXXXGK consensus GTP binding region near the amino terminus, and a DEPPKKKATMQ nuclear localization sequence near the H1 homology region (De Camillis, 1992 and Hodgson, 1997)).
Single copies of an approximately 65-70 residue domain are present in the sequences of 14 eukaryotic proteins, including yeast byr2, STE11, ste4, and STE50, which are essential participants in sexual differentiation. This domain, named SAM (sterile alpha motif), appears to participate in other developmental processes because it is also present in Drosophila polyhomeotic gene product and related homologs. Its appearance in byr2 and STE11, which are MEK kinases, and in proteins containing pleckstein homology, src homology 3, and discs-large homologous region domains, suggests possible participation in signal transduction pathways (Ponting, 1995).
The Sex combs on midleg and Polyhomeotic proteins share homology with regard to the SPM domain. This domain is 38% identical between the two proteins, over a length of 65 amino acids. Each protein has an SPM domain located at its respective C termini. This domain is predicted to be largely alpha-helical. Besides these proteins, there are numerous proteins that contain a related domain with much lower overall identity). These more distantly related proteins include members of the Ets family of transcription factors and yeast proteins required for mating. The high-homology domain subgroup that includes the Scm and Ph versions are referred to as the SPM domain, and the extended domain family is referred to as the SAM domain (Ponting, 1995). One of the more well-characterized SAM domains is present in the human TEL oncoprotein, an Ets class transcription factor, where the SAM has been referred to as a helix-loop-helix (HLH) domain. Recent studies have shown that this domain mediates self-binding and oligomerization of TEL protein and of TEL fusion protein derivatives (Peterson, 1997).
date revised: 2 January 2023
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