InteractiveFly: GeneBrief

Xrp1: Biological Overview | References

BIOLOGICAL OVERVIEW

The elimination of unfit cells from a tissue is a process known in Drosophila and mammals as cell competition. In a well-studied paradigm 'loser' cells that are heterozygous mutant for a haploinsufficient ribosomal protein gene are eliminated from developing tissues via apoptosis when surrounded by fitter wild-type cells, referred to as 'winner' cells. However, the mechanisms underlying the induction of this phenomenon are not fully understood. This paper reports that a CCAAT-Enhancer-Binding Protein (C/EBP), Xrp1, which is known to help maintaining genomic stability after genotoxic stress, is necessary for the elimination of loser clones in cell competition. In loser cells, Xrp1 is transcriptionally upregulated by an autoregulatory loop and is able to trigger apoptosis - driving cell elimination. Xrp1 acts in the nucleus to regulate the transcription of several genes that have been previously involved in cell competition. It is therefore speculated that Xrp1 might play a fundamental role as a molecular caretaker of the genomic integrity of tissues (Baillon, 2018).

Tissues are composed by genetically heterogeneous cells as a result of the accumulation of different mutations over time. Unfit and potentially detrimental cells are eliminated from tissues via apoptosis triggered by a process known in both insects and mammals as cell competition. The eliminated cells, referred to as 'loser' cells, are normally viable and capable of growing, but are eliminated when surrounded by fitter, 'winner' cells. In Drosophila melanogaster, the majority of ribosomal protein genes (RPGs) are haploinsufficient (hRPGs). When one copy of an hRPG is removed, this gives rise to the 'Minute' phenotype characterized by a general developmental delay and improper bristle development. When intermingled with wild-type winner cells, cells heterozygous for an hRPG become losers and are eliminated via apoptosis. Various genetic manipulations of a tissue can result in different and widely documented cell competition responses. Several pathways, such as the BMP, Toll, Wnt, JAK/STAT, Ras/MAPK and Hippo pathways, have been implicated in cell competition, suggesting the existence of a complex framework of actions that serve to induce apoptosis and eliminate loser cells. However, what actually triggers elimination yet remains elusive (Baillon, 2018).

Multicellular organisms maintain genomic stability via the activation of DNA repair mechanisms to identify and correct DNA damages. During this process, cell cycle progression is arrested to prevent the expansion of damaged cells. However, when DNA repair fails, apoptosis is induced to eliminate irremediably damaged cells. The p53 transcription factor plays an evolutionarily conserved role in the induction of apoptosis following DNA damage, however evidence points towards the existence of alternative routes for the induction of apoptosis in response to DNA damage (Baillon, 2018).

This study shows that, in a cell competition context, a possible alternative route to P53 for the induction of apoptosis goes via Xrp1, a gene encoding a b-ZIP DNA binding protein. The expression of Xrp1 is induced in various stress conditions, for instance in response to irradiation. Notably, Xrp1 mutant animals have been reported to have higher levels of loss-of-heterozygosity after ionizing radiations. Additionally Xrp1 plays a role in repair of DNA breaks after transposase cleavage. Therefore Xrp1 may have a role in sensing and responding to DNA damage (Baillon, 2018).

This study reports the discovery, in an EMS-based screen, of Xrp1 mutations that suppress the elimination of loser cells. This is consistent with earlier reports that proposed Xrp1 might affect cell competition. For the first time this study has discerned how Xrp1 might regulate cell competition. Xrp1 is homologous to mammalian C/EBPs, a class of transcription factors that is known to autoregulate their own transcription, to prevent proliferation and induce apoptosis. It was further shown that Xrp1 expression is upregulated in loser cells in response to the removal of one copy of a haploinsufficient ribosomal protein gene, where, similarly to C/EBP homologs, it regulates its own expression via a positive autoregulatory loop, the expression of pro-apoptotic genes and that of other genes that were previously implicated in cell competition (Baillon, 2018).

In order to identify genes whose function is necessary for the elimination of RPG heterozygous mutant loser cells, a forward genetic screen was performed using ethyl methanesulfonate (EMS) in Drosophila melanogaster. A mosaic system was designed that allows direct screening through the larval cuticle for the persistence of otherwise eliminated RpL19+/- loser clones. This enabled screening of a high number of animals for mutations that either dominantly (anywhere in the genome) or recessively (on the right arm of the third chromosome) suppress cell competition. The induction of a single somatic recombination event between two FLP recognition targets (FRTs) generates a RPG heterozygous mutant cell that becomes homozygous for the mutagenized right arm of the third chromosome. Loser clones are induced at the beginning of larval development (L1). If no suppressive mutation is present, clones are efficiently eliminated over time and thus undetectable by the end of the third instar larval stage (L3) when the screening is performed. 20,000 mutagenized genomes were screened for the presence of mutations that prevent the elimination of loser clones. Eleven heritable suppressors were obtained, and attention was focused on three of the strongest suppressors that did not display any obvious growth-related phenotype. RpL19+/- clones were eliminated and little or no signal was observed. Their elimination, however, is prevented when cells are not heterozygous mutant for RpL19 or when different Xrp1 mutations (Xrp108 in the example) are additionally present. In the latter cases GFP signal is observed in wing discs (Baillon, 2018).

Xrp1 suppressors did not belong to a lethal complementation group and the causative mutations were identified using a combination of positional mapping and whole-genome re-sequencing. In particular, three independent mutations in the introns of CG17836/Xrp1 were identified, all caused by substitutions of single nucleotides. These nucleotides are conserved within the Drosophila genus and inspection of the alignment revealed an embedment of these nucleotides in conserved sequence motifs. Of particular interest are the polypyrimidine motifs containing the nucleotide mutations in Xrp120 and Xrp108. These motifs flank the alternative first exon and are potential splice regulators. The CTCTCT motif in proximity of the 5' splice site of Xrp1 has been identified as a putative intronic splicing enhancer (ISE) predicted to serve as binding site for the polypyrimidine-tract binding protein (PTB) splicing regulator. The presence of these motifs prompted an investigation of the consequences of the Xrp108 allele on exonic junctions. The most prominent effect of this allele is a strong and consistent reduction in the expression of two similar Xrp1 transcripts, RC and RE, which only differ in the composition of their 5' UTRs. They share the transcriptional start site and contain the same long open reading frame that codes for the short isoform of Xrp1 (Baillon, 2018).

The behavior of RpL19+/- clones was checked in the presence and absence of Xrp1 function. To this end the twin spot MARCM system was used that enables differently marking twin clones generated by the same recombination event. In the genetic set up, mCherry expression marks loser clones whereas two copies of GFP mark wild-type twin clones. As expected, RpL19+/- loser clones are eliminated from the tissue. Elimination is also observed when RpL19+/- cells within these clones are additionally mutant for Xrp108 but contain a transgene comprising the genomic region of Xrp1. Importantly, when Xrp1 mutations are not rescued cell competition-driven elimination of RpL19+/- losers no longer occurs. In particular, the Xrp108 intronic mutation retrieved from the EMS screen is able to prevent loser cell elimination, and a similar result is obtained with a newly generated complete loss-of-function allele, Xrp161, as well as with Xrp126. Xrp161 contains a frame shift mutation upstream of the Xrp1 basic region-leucine zipper domain (b-ZIP), and is considered a null allele. Like other Xrp1 alleles analyzed it is homozygous viable and does not impair the development of mutant animals. To confirm that Xrp1 function is of general importance for the elimination of hRPG+/- cells, and not limited to RpL19+/- loser cells, the effect of Xrp1 mutations on RpL14+/- loser clones was tested. Similarly to RpL19+/- cells, RpL14+/- cells are normally eliminated from wing discs during larval development. No elimination occurs if these cells express RpL14 from a transgene, or when Xrp1 is mutated (Xrp161) (Baillon, 2018).

To further explore this notion direct genomic targets of Xrp1 were determined by chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) on wing imaginal discs. In order to do this, Xrp1 expression was induced in wing discs. The top targets revealed by ChIP-seq comprise a number of genes that are already implicated in cell competition, cell cycle regulation and apoptosis. Among the interesting genes Xrp1 itself was identified, suggesting the existence of a potential autoregulatory loop. To test this notion Xrp1 was overexpressed in the posterior compartment of the wing disc, and the transcriptional behavior of Xrp1 was checked with the aforementioned Xrp1-lacZ reporter. The upregulation of lacZ expression was observed in response to Xrp1 overexpression, indicating that Xrp1 can boost its own expression in a positive autoregulatory loop. These observations were confirmed by measuring mRNA levels of Xrp1 upon forced Xrp1 expression. With a similar strategy the response was observed of other putative transcriptional targets from the ChIP-seq experiment. Xrp1 was shown to promote the transcription of Dif, a Drosophila NFκB homolog gene that has previously been implicated in the cell competition-dependent induction of apoptosis via the induction of rpr transcription7. puc, Upd3, Nedd4 and rad50 were also tested: all of these genes were upregulated upon induction of Xrp1 expression. puc, Upd3 and Nedd4 are involved in the JAK/STAT and Hippo signaling pathways, both of which have previously been implicated in cell competition. Rad50 is instead required for double strand break repair (Baillon, 2018).

The most prominent sequence motif of Xrp1 derived from ChIP-seq data shows a strong similarity with the b-ZIP binding motif of the human C/EBP protein family. It was therefore checked whether Xrp1 shows homology to C/EBP transcription factors, being itself a bona fide transcription factor. Xrp1 was shown to share a 40% identity with the human C/EBPs (PSI-BLAST). Phylogenetic reconstruction allowed recognition of three Drosophila C/EBP homologs, one of which is Xrp1. Interestingly, human C/EBP-alpha is retained in the nucleolus and binds to ribosomal DNA, a feature that may be evolutionarily conserved since Xrp1 binds rDNA loci with high affinity. The encoded rRNA is found in the nucleoli (Baillon, 2018).

Therefore a working model is proposed in which Xrp1, under normal conditions, sits on rDNA in the nucleolus. In the presence of genotoxic stress or of a ribosomal imbalance, as in the context of Minute cell competition, Xrp1 acts nuclearly as a C/EBP transcription factor that stimulates its own transcription and the expression of pro-apoptotic target genes. When intermingled with wild-type cells, cells with only one copy of an hRPG are eliminated in a Xrp1-dependent manner. In the experimental system, the deletion of one copy of the RpL19 gene is catalyzed by the Flp/FRT recombination system, which leaves no apparent lesion in the DNA. Therefore, the initial recruitment of Xrp1 into the nucleus may not depend on DNA damage per se, but rather on the unbalanced physiology of the cell resulting from the loss of one copy of the hRPG. The nucleolus is the site of ribosome biogenesis and a major stress sensor organelle. RpL19+/~ cells experience a related nucleolar stress, since their nucleoli are enlarged as revealed by anti-fibrillarin staining. The most likely explanation for this is partially stalled ribosome assembly. Since genotoxic stress triggers Xrp1 expression, it is speculated that Xrp1 acts as a caretaker of genomic integrity. In support of this hypothesis, the growth of salvador~/~ mutant tumor clones is suppressed by the concurrent loss of one copy of the RpL19 gene. However, this suppression fails in the absence of Xrp1 function, indicating that the presumptive protective function that RPGs haploinsufficiency provides can also operate within tumorous cells. In addition, according to a Monte-Carlo simulation, the likelihood that one hRPG locus becomes heterozygous mutant before any other gene gets mutated to homozygosity is very high. Together with the observation that hRPGs are broadly distributed within the genome, this further supports the potential role of Xrp1 as a caretaker of genomic integrity. Although further research is required to better elucidate this phenomenon, it is nevertheless proposed that RPG haploinsufficiency provides a simple, yet effective, mechanism to protect the organism from the emergence of potentially deleterious cells (Baillon, 2018).

A regulatory response to ribosomal protein mutations controls translation, growth, and cell competition

Ribosomes perform protein synthesis but are also involved in signaling processes, the full extent of which are still being uncovered. This study reports that phenotypes of mutating ribosomal proteins (Rps) are largely due to signaling. Using Drosophila, this study discovered that a bZip-domain protein, Xrp1, becomes elevated in Rp mutant cells. Xrp1 reduces translation and growth, delays development, is responsible for gene expression changes, and causes the cell competition of Rp heterozygous cells from genetic mosaics. Without Xrp1, even cells homozygously deleted for Rp genes persist and grow. Xrp1 induction in Rp mutant cells depends on a particular Rp with regulatory effects, RpS12, and precedes overall changes in translation. Thus, effects of Rp mutations, even the reductions in translation and growth, depend on signaling through the Xrp1 pathway and are not simply consequences of reduced ribosome production limiting protein synthesis. One benefit of this system may be to eliminate Rp-mutant cells by cell competition (Lee, 2018).

Ribosomes are the essential protein synthesis machines of the cell. Large and small subunits (LSU and SSU), 40S and 60S in eukaryotic cells, form an 80S complex together with mRNA and perform translation in the cytoplasm. Each ribosome subunit is a ribonucleoprotein complex containing one (SSU) or three (LSU) non-coding rRNA molecules and a battery of ribosomal proteins (Rps) and is assembled in the nucleolus for export to the cytoplasm. Rps can contribute to folding and assembly of the ribosomal subunits as well as their function in translation. Most Rps are essential, and cells homozygous for their mutations die, while heterozygous Rp mutants that lack one copy of the gene are abnormal in both humans and in Drosophila (Lee, 2018).

To what extent do the defects in Rp mutants reflect deficient translation, and to what extent do they reflect signaling pathways that monitor ribosome status? Aspects of Diamond-Blackfan Anemia, the ribosomopathy that occurs in humans heterozygous for mutations in a number of Rp genes, are thought to reflect chronic p53 signaling, activated by accumulation of a ribosome assembly intermediate and nucleolar stress. On the other hand, Diamond-Blackfan Anemia is also characterized by short stature and delayed maturation as well as skeletal defects, and it has sometimes been treated with L-leucine to stimulate protein synthesis. Reduced protein synthesis has been measured in both Drosophila embryos and in mouse fibroblasts and hematopoietic cells from heterozygous, Rp+/- genotypes (Lee, 2018).

This study made use of Drosophila to investigate the effects of Rp mutations further. Drosophila that are haploinsufficient for any of 66 of the 79 Rp genes exhibit a common phenotype, first recognized a century ago (the 'Minute' phenotype), which includes a reduction in the size and thickness of bristles on the adult body ('Minute' bristles) and a developmental delay associated with reduced translation and growth rate. Unlike the bristle structures, most mutant cells are of normal size, as are mutant flies themselves, suggesting that the extended growth period is sufficient to compensate for reduced cellular growth. In fact, mutant organs can be larger than normal, depending on the particular balance of growth between organs (Lee, 2018).

In Drosophila, and possibly in mammals, Rp+/- genotypes are subject to 'cell competition' in genetic mosaics. If growing imaginal discs (progenitor cells that grow in an undifferentiated state in the larva to give rise to the adult tissues) contain both wild-type and Rp+/- cells, the latter are progressively lost during growth. Conversely, wild-type cells growing in Rp+/- backgrounds come to dominate developmental compartments at the expense of the Rp+/- cells. Both competitive situations are associated with selective apoptosis of Rp+/- cells in proximity to wild-type, which is responsible for the loss of Rp+/- clones. There are other genotypes that can be competed from genetic mosaics, but neither is it clear that the mechanisms are the same nor whether deficits in translation or growth are required. There are also examples of 'super-competitor' genotypes that can eliminate nearby wild-type cells, even though wild-type cells should have normal ribosomes. In the mouse embryo, cells expressing more Myc or less p53 are super-competitors (Lee, 2018).

Cell competition is also seen in mammalian cell co-cultures, in many cases eliminating hyperplastic or preneoplastic cells. Such cells can also be eliminated from mosaics with otherwise normal tissues in vivo (Lee, 2018).

The current studies reported originated in a genetic screen designed to identify new components of cell competition. This led to isolation of a mutation affecting a basic leucine zipper (bZip)-domain protein gene, Xrp1 (Lee, 2016). Xrp1 was previously known as a putative transcription factor induced by p53 following X-irradiation of Drosophila and implicated in genome maintenance, although no point mutant alleles had been studied previously. Xrp1 was also characterized as a component of the protein complex that binds to the P element transposon in Drosophila and found to contribute to P element transposition (Franci, 2016). This study reports a major role for Xrp1 in multiple features of Rp mutants. Xrp1 expression is elevated in mutant cells by a signal from the ribosome and controls cellular translation rate and growth in addition to cellular competitiveness and almost the entire gene expression signature of Rp+/- cells. Xrp1 is even responsible for eliminating cells homozygously mutant for essential Rp genes that are deficient for new ribosome biogenesis. It is concluded that Xrp1 controls a cellular stress pathway that monitors Rps, regulates multiple cellular properties, and acts upstream of the major defects in global translation, which are in fact only indirectly related to the initial mutation of an Rp gene (Lee, 2018).

Xrp1, which behaved like a master-regulator of responses to Rp mutations. Even the acute lethality of Rp-/- cells depended on Xrp1. The only aspect of the Rp+/- phenotype that appeared largely independent of Xpr1 was the reduced size of the bristles, which was only slightly restored by Xrp1 mutations. Bristle size might depend on ribosome function directly or on a different regulatory gene that replaces Xrp1 in bristle precursors (Lee, 2018).

Rp/+ genotypes express a phenotype of reduced translation, slow cellular growth rate, and reduced competitiveness in comparison to wild-type cells. All of these effects of the Rp+/- genotypes depended on the bZip-domain protein Xrp1. The signal to increase Xrp1 transcription depended on the RpS12 protein, which appears to signal the existence of a ribosomal defect or ribosomal protein imbalance (Kale, 2018). Xrp1 was responsible for reducing the bulk translation rate in Rp+/- cells, and this must include a reduction in the translational activity of mature ribosomes. Reduced translation was likely responsible for the slow growth of Rp+/- cells, although Xrp1 might also affect growth independently of translation. Xrp1 also controlled the competitiveness of Rp+/- cells in mosaics with wild-type cells, which provides a possibility for eliminating Rp+/- cells in favor of non-mutant replacements. It is hypothesized that one or more target genes control competitiveness, either in response to Xrp1 itself, or indirectly in response to the changes in translation or growth rates. By utilizing cell competition, a decision could be made to eliminate defective cells only where better cells were available to replace them (Lee, 2018).

A null allele of Xrp1 was isolated in a screen for mutations preventing cell competition. Xrp1 transcription and Xrp1 protein were found to be selectively elevated in Rp+/- cells and to be required cell-autonomously to render these cells less competitive than wild-type cells. Later it was found that Xrp1 also acted to reduce the growth rate of Rp+/- cells. In the absence of Xrp1, or even when Xrp1 gene dose was reduced to one copy, Rp+/- cells grew more like wild-type cells. Xrp1 contributed substantially to the developmental delay of Rp+/- animals, which without Xrp1 could reach adulthood only slightly later than wild-type animals, despite lacking one copy of essential Rp genes (Lee, 2018).

Xrp1 probably reduces growth by reducing the overall translation rate. Rp+/- cells had lower translation rates than wild-type cells, but it was Xrp1, not haploinsufficiency for an important Rp gene, that reduced translation rate, because the difference from wild-type disappeared when Xrp1 was mutated simultaneously. Although ribosome numbers have not been counted directly, the proportion of rRNAs was not reduced in most Rp+/- genotypes, suggesting that an Xrp1-dependent reduction in translational activity per ribosome may occur. A mutation in RpL27A was the exception that did appear to reduce LSU number, but this was not rescued in RpL27A+/-Xrp1+/- discs and so was not responsible for the Xrp1-dependent growth inhibition. The persistence of Rp-/- mutant clones in the absence of Xrp1 also suggests changes in ribosome activity. Rp-/- cells should be deficient in synthesizing new ribosomes, so the prolonged survival of Rp-/-Xrp1-/- clones cannot easily be explained through restored ribosome biogenesis (Lee, 2018).

These findings suggest Xrp1 influences the rate of translation by cytoplasmic ribosomes, but they do not exclude additional roles in ribosome biogenesis. For example, late third instar wing discs from wild-type, RpS18+/-, and RpS18+/-Xrp1+/- larvae contained indistinguishable ribosome numbers, but these genotypes developed at different rates. Based on the time taken for adults to emerge, it is estimated that the ribosomes in late third instar wing discs had accumulated over ~80 hr, ~115 hr, and ~100 hr of larval life, respectively, so this is consistent with different rates of ribosome biogenesis generating similar absolute numbers of ribosomes over different durations of larval development. A reduced rate of ribosome biogenesis was reported previously in the mouse RpL24Bst/+mutant (Lee, 2018).

Regarding the overall rate of organismal development, it is well known that progress through the insect life cycle is controlled, in part, through systemic signals ultimately controlling ecdysone levels. This is not the primary means that Xrp1 affects imaginal disc growth because this occurs cell-autonomously. Additional non-autonomous effects of Xrp1 on organismal development are not ruled out. For example, Dilp8, a secreted factor that regulates organismal growth, undergoes Xrp1-dependent upregulation in Rp+/- wing discs (Lee, 2018).

Hundreds of genes show altered mRNA levels in Rp+/- wing discs, but it has not been clear how these changes arise. This study now reports that >80% of altered mRNA levels were Xrp1-dependent. Some of these genes might be indirect targets of Xrp1; for example, Xrp1-dependent changes in overall translation rate may change gene transcription through a variety of mechanisms. The Xrp1-dependent changes include oxidative stress responses, which are reported to make cells less competitive, and also DNA repair genes (Lee, 2018).

How is Xrp1 induced by Rp mutations? Xrp1 is a transcriptional target of p53 in the response to irradiation, but p53 is not required for the elimination of Rp+/- cells by cell competition (Kale, 2015). Accordingly, this study showed that p53 was not required to elevate Xrp1 in Rp+/- wing discs. Reduced overall translation was unlikely to induce Xrp1 because Xrp1 was actually responsible for this. Perhaps more subtle changes in the translation of specific mRNAs occur first and induce Xrp1 expression. Another possibility is that a signal is sent when ribosome assembly is altered, for example, through an accumulated assembly intermediate. This study reports that a particular Rp, RpS12, that was already recognized as a gene required for cell competition (Kale, 2018), was required to elevate Xrp1 transcription. Although the molecular mechanism by which RpS12 can affect transcription is not yet known, this demonstrates that a link between a particular Rp and the Xrp1 gene triggers most of the response that occurs to mutations in other Rp genes, upstream of overall changes in bulk translation rates, which are a later consequence of the Xrp1 pathway. All the DNA repair gene expression in Rp+/- wing discs was also downstream of Xrp1 and may reflect Xrp1's other role in the response to irradiation (Brodsky, 2004, Akdemir, 2007). How DNA repair genes contribute to aspects of the Rp+/- phenotype remains to be determined (Lee, 2018).

The importance of Xrp1 extends to homozygous Rp mutant cells. Remarkably, even Rp-/- cells survived and underwent limited growth if Xrp1 was completely removed. It is not believed that imaginal disc cells can grow and divide without ribosomes and protein synthesis, but since ribosome turnover occurs very slowly, Rp-/- recombinant cells probably retain most of the ribosome complement from the Rp+/- mother cell at first. They would be deficient in replenishing their ribosome complement, however, which would dilute with further growth and cell division. Previous studies indicate that when ribosome activity diminishes below a critical threshold, Rp-/- cells undergo apoptosis (Kale, 2015). The absence of Xrp1 allowed Rp-/- cells to survive longer, by allowing more translation by the remaining ribosomes and possibly by preventing competitive elimination of Rp-/- cells by Rp+/- cells (Lee, 2018).

Despite the unquestioned importance of Rps in ribosome structure and function, the current results indicate that the effects of Rp mutations in Drosophila are largely due to a regulatory response. Even the reduced translation in Rp mutant genotypes, which has also been observed in other organisms, is downstream of Xrp1 and does not play a primary role as a sensor of Rp mutations. Even when Rp genes are homozygously mutated, which seemingly should affect overall translation very quickly, Xrp1 normally kills the Rp-/-cells before such effects become evident. Rp+/- cells can also be killed by Xrp1, but indirectly, by cell competition, when wild-type cells are nearby (Lee, 2018).

Mutations that reduce translation or ribosome biogenesis by other routes are phenotypically distinct from Rp mutants. For example, mutations in the myc gene homolog or in components of the Drosophila TOR pathway lead to smaller flies, unlike Rp+/- flies. In mammals as well, mutations that affect ribosome biogenesis independently of Rp genes lead to human disease, but the symptoms of such ribosomopathies differ from Diamond-Blackfan Anemia. These differences may occur because translation is affected indirectly in mutations of Rp genes and is not the primary trigger for all the cellular responses leading to pathology (Lee, 2018).

Although it may at first seem surprising that mutations in Rps that are so directly involved in translation affect the cell through another mechanism, perhaps it is advantageous to mount such a coordinated response, for example to enable cell competition. Much as it is adaptive to eliminate cells with damaged DNA through apoptosis, perhaps cell competition is a mechanism to eliminate one or a few cells with defective ribosomes in favor of other, more normal cells (Lee, 2018).

A potential link between p53, cell competition and ribosomopathy in mammals and in Drosophila

The term cell competition has been used to describe the phenomenon whereby particular cells can be eliminated during tissue growth only when more competitive cells are available to replace them. Multiple examples implicate differential activity of p53 in cell competition in mammals, but p53 has not been found to have the same role in Drosophila, where the phenomenon of cell competition was first recognized. Recent studies now show that Drosophila cells harboring mutations in Ribosomal protein (Rp) genes, which are eliminated by cell competition with wild type cells, activate a p53 target gene, Xrp1. In Diamond Blackfan Anemia, human Rp mutants activate p53 itself, through a nucleolar stress pathway. These results suggest a link between mammalian and Drosophila Rp mutants, translation, and cell competition (Baker, 2019).

P53 is a transcription factor that is well known as the guardian of the genome. When activated by DNA-damage, p53 coordinates cell cycle arrest that facilitates DNA repair, or, if damage is more extreme, apoptosis that removes irretrievably damaged cells. In undamaged cells, baseline p53 activity is low, in part due to rapid turnover controlled by the E3 ubiquitin ligase MDM2. Activity increases rapidly after modification by the DNA Damage Response kinases ATM and Chk2. P53 regulates the expression of many genes, some direct targets but others indirect (Baker, 2019).

Recently, evidence has been accumulating for another function of p53, in the process of cell competition. Cell competition involves the selective elimination of particular cells based on intrinsic differences from their cellular context, e.g., elimination of certain genotypes of cells from genetic mosaics that would survive and proliferate in a genetically homogenous environment. Cell competition does not reflect a cell-autonomous cell death process, such as can be caused high levels of p53 activity, but requires interaction with distinct neighboring cells that are fitter and able to replace the out-competed cells. Cell competition is of interest because of its possible roles in tumor development and tumor surveillance, both situations where cells of distinct genotypes confront one another and influence one another's growth and survival. Cell competition may also help prevent developmental defects and monitor stem cell populations for inappropriate differentiation (Baker, 2019).

It is not yet directly demonstrated that mammalian Rp+/- cells are eliminated by cell competition. In Drosophila, DNA damage activates the putative transcription factor Xrp1 through p53. Rp+/- genotypes activate Xrp1 through RpS12 instead, which in mosaic tissues marks these cells for elimination and replacement by wild type (Rp+/+) cells. Both p53 in mammals and Xrp1 in Drosophila predispose cells to elimination by competition, if more normal cells are present (Baker, 2019).

The classic example of cell competition is the elimination in Drosophila of Rp+/- cells from mosaic imaginal discs (undifferentiated larval tissues that contain the rapidly proliferating progenitors for adult structures) that also contain wild type cells. Another example includes competition between imaginal disc cells that express different levels of the proto-oncogene Myc. In the latter case even wild type cells can be eliminated by cells expressing more Myc ('supercompetitors'). A further example concerns cells that are mutated for the scribbled gene (scrib). The conserved Scrib protein is required for epithelial cell polarity and in scrib mutants the imaginal discs become neoplastic. By contrast, clones of scrib mutant cells are eliminated from mosaic imaginal discs by competition with the wild type cells before they become neoplastic (Baker, 2019).

One of the first cell competition phenomena described from mammals involved hematopoietic stem and progenitor cells. Mild irradiation, that by itself would have negligible effect on hematopoietic stem cell viability and function, significantly disadvantages these cells for months afterwards in competitive situations where stem cells with less p53 activity also present. In a study of embryonic development, p53 knock-down ES cells injected into E3.5 day blastocysts strongly compete with co-injected control ES cells by E14.5, indicating that even the baseline p53 activity of normal blastocyst cells can be disadvantageous in the presence of cells lacking even that activity. One study also identified circumstances where p53 knockdown could be disadvantageous. Mild p53 activity is also shown to be disadvantageous in studies of mdm2 family members. These ubiquitin ligases are major negative regulators of p53. Whereas Mdm2+/- Mdm4+/- double heterozygous mice show only a mild increase in p53 activity and undergo normal embryogenesis, they are at a disadvantage in chimeras and outcompeted by normal cells in which p53 activity maintains a lower baseline (Baker, 2019).

In tissue cultures, mammalian Scribbled is also involved in cell competition. Scrib knock-down has little effect on MDCK cells in homogenous culture, but when these cells are co-cultured with wild-type MDCK cells then the knock-down cells are selectively eliminated by apoptosis. Gene profiling led to the discovery that p53 was activated during the competition, and in fact was required for the Scrib knockdown cells to be eliminated. Accordingly, even in otherwise normal MDCK cells, mild p53 activity by itself was sufficient for these cells to be eliminated in mixed culture. P53 activity is also found to be required for competitive elimination of mouse embryo cells mutated for Bmpr1a, and for tetraploid cells (Baker, 2019).

In summary, p53 activity is a common feature of cell competition in mammals. In addition to cell-autonomous roles in cell cycle arrest and apoptosis that follow DNA damage, low levels of p53 activity that normally are compatible with cell growth and survival have an effect in chimeras or mixed cultures where other cells are present that have lower p53 activity levels. In some cases, such as competition between MDCK cells with and without Scrib expression, or mouse embryo cells that are tetraploid or mutated for Bmpr1a, changes in p53 activity are induced by other genetic changes (Baker, 2019).

Until recently there had been little evidence for any molecular similarity between what little was known of the mechanisms of cell competition signaling in Drosophila and mammals. In particular, p53 is not required in Rp+/- cells for their elimination from mosaic imaginal discs (Kale, 2015), and cells lacking p53 do not eliminate wild type cells in Drosophila. The p53 gene is also not required in wild type cells for their elimination by cells expressing more Myc. P53 does seem to have another effect. In cells expressing more myc: under competitive conditions, it shifts their metabolism and promotes their survival and ability to eliminate nearby wild type cells (Baker, 2019).

Recently, however, a hint that cell competition in mammals and Drosophila might share something in common has emerged. Rp+/- cells that undergo cell competition in Drosophila elevate expression of a bZip domain protein, Xrp1, which contributes to many of the altered properties of Rp+/- genotypes. This includes their reduced overall translation rate, slower cellular growth rate, and slower rate of organismal developmental, as well as their susceptibility to elimination by cell competition (Lee, 2018). Importantly, the Xrp1 locus had already been identified as the most highly-induced transcriptional target of p53 following irradiation. Xrp1 may contribute to the DNA damage response downstream of p53, since it is required for aspects of genome stability (Akdemir, 2007). Multiple genes that were previously described as p53 targets are upregulated in Rp+/- cells in an Xrp1-dependent manner, suggesting that they may actually be Xrp1 targets (Kucinski, 2017; Lee, 2018). Thus, it is possible that in Drosophila p53 itself is not essential for elimination of Rp+/- cells by cell competition because relevant target genes can be activated by Xrp1, bypassing p53 (Baker, 2019).

Interestingly, p53 itself is activated in mammalian cells with Rp mutations, such as in patients with Diamond-Blackfan Anemia, one of several ribosomopathies associated with ribosome biogenesis defects, and in Rp knockout mouse models. The mammalian Rp+/- cells experience a nucleolar stress in which RpL5 and RpL11, rendered in excess by the reduced rate of ribosome biogenesis in cells with mutations in any of the other ribosomal protein genes, bind to and inhibit MDM2 (in mice; HDM2 in humans), thereby reducing p53 turnover. No DM2 protein is conserved in Drosophila, where p53 turnover is regulated by an unrelated ubiquitin ligase that has not been reported to interact with any ribosomal proteins, so Rp mutations could not activate p53 by this mechanism in Drosophila. Instead, the elevated expression of Xrp1 depends on a different ribosomal protein, RpS12, whose role in cell competition was recently discovered in parallel with that of Xrp1 (Kale, 2018). The molecular mechanism of Xrp1 activation by RpS12 is not known at present. RpL5 and RpL11 are special ribosomal proteins in that they complex with the 5 S rRNA that is transcribed separately from other rRNAs and by RNA polymerase III rather than RNA polymerase I. The resulting 5 S RNP is somewhat stable which facilitates accumulation in the presence of other ribosome biogenesis defects. By contrast, very little is yet known about the function and regulation of the RpS12 protein, which binds to the 18 S rRNA of the small subunit (Baker, 2019).

These new results suggest that cell competition mechanisms in Drosophila and mammals may not be as distinct as may have seemed. The cell interactions that lead to cell competition may depend on pathways that can be activated by p53 in both mammals and in Drosophila, but whereas they are activated by p53 in several mammalian examples, in Drosophila Rp+/- cells they are activated by a more downstream transcription factor, by-passing p53. Many questions remain. In the Drosophila pathway, how does RpS12 communicate ribosome biogenesis defects to the Xrp1 gene? How important is Xrp1 for the DNA Damage Response downstream of p53? To what extent does p53 act through downstream factors in mammalian cell competition, where there is an RpS12 protein but no obvious homolog of Xrp1? It would be interesting now to know whether Rp+/- cells are eliminated by cell competition in mammals, as might be expected since differential p53 activity can be a cause of cell competition. RpL24+/- cells are certainly disfavored in chimeras with wild-type cells, but it has not been distinguished whether this simply represents a passive consequence of their differential translation and growth rates, or an additional active process of targeted elimination as is seen in Drosophila (Baker, 2019).

While the translation defect of Rp+/- cells in mammals has often been considered independent of the p53 activity, in fact p53 does affect translation through a variety of mechanisms, including regulation of ribosome biogenesis, regulation of general translation initiation, and regulation of specific mRNAs. In Drosophila, Xrp1 reduces the translation rate of Rp+/- cells (Lee, 2018). Given the effect of Xrp1 on translation in Drosophila, the possible effect of p53, or other regulatory signals activated by ribosome biogenesis defects, on protein synthesis in Diamond-Blackfan Anemia patient cells would bear further investigation (Baker, 2019).

Cell competition represents an emerging aspect of p53 function that is not cell-autonomous but only apparent between cells with different p53 activity levels. Now it seems that at least one p53-independent example of cell competition may depend on common targets activated by a p53-independent route. It may be interesting to determine whether cell competition contributes to the tumor suppressor function of p53 in mammals, which appears not strictly dependent on well-known cell cycle and cell survival targets. It was recently reported that human RpS12 gene dose is frequently reduced in Diffuse Large B-Cell Lymphoma, in a manner mutually exclusive to loss of p53. In principle this could be consistent with a function of human RpS12 related to that of p53, although this remains to be investigated (Baker, 2019).

Roles of C/EBP class bZip proteins in the growth and cell competition of Rp ('Minute') mutants in Drosophila

Reduced copy number of ribosomal protein (Rp) genes adversely affects both flies and mammals. Xrp1 encodes a reportedly Drosophila-specific AT-hook, bZIP protein responsible for many of the effects including the elimination of Rp mutant cells by competition with wild type cells. Irbp18, an evolutionarily conserved bZIP gene, heterodimerizes with Xrp1 and with another bZip protein, dATF4. This study shows that Irbp18 is required for the effects of Xrp1, whereas dATF4 does not share the same phenotype, indicating that Xrp1/Irbp18 is the complex active in Rp mutant cells, independently of other complexes that share Irbp18. Xrp1 and Irbp18 transcripts and proteins are upregulated in Rp mutant cells by auto-regulatory expression that depends on the Xrp1 DNA binding domains and is necessary for cell competition. Xrp1 is conserved beyond Drosophila, although under positive selection for rapid evolution, and that at least one human bZip protein can similarly affect Drosophila development (Blanco, 2020).

Heterozygous mutation of ribosomal protein genes lead to cell-autonomous, deleterious phenotypes in both flies and mammals and provide the classic example of a genotype that is eliminated from mosaics by competition. There is increasing interest in the potential roles of cell competition in mammalian development, cancer development, and in regenerative medicine. A remarkable recent finding from Drosophila is that many of the phenotypic effects of mutating ribosomal protein genes are mediated by a putative transcription factor, Xrp1, rather than as a direct consequence of altered ribosome number. Accordingly, Xrp1 plays a key role in the elimination of Rp mutant cells by cell competition. Xrp1 transcription and protein expression are elevated in Rp mutant cells, restricting translation, cellular growth rate, and the rate of organismal development, and enabling cell competition with nearby wild type cells. Xrp1 had previously been implicated in the DNA damage response downstream of p53 and in the transposition of P elements, and contributes to the pathology of a Drosophila model of Amyotrophic Lateral Sclerosis, as well as to the coordination of organ growth in flies with Rp gene knockdowns (Blanco, 2020).

Xrp1 has been reported not to have homologs in other eukaryotes. This seems surprising given the highly conserved and fundamental roles of ribosomal proteins, and is a barrier to investigating the potential conservation of cell competition mechanisms and the roles of cell competition in mammals, for example in the development of cancer. Xrp1 binds to DNA as a heterodimer with Irbp18, the Drosophila homolog of the C/EBP protein family, which is a conserved protein and co-purifies with it in cultured cells. Irbp18 in turn heterodimerizes with the conserved protein dATF4, encoded by the crc gene in Drosophila). This led to an investigation od whether it is the Xrp1 heterodimer with the conserved Irbp18 protein that functions in Rp+/- cells, and if so whether Xrp1/Irbp18 acts positively; alternatively, Xrp1 could act as a competitive inhibitor of Irbp18 function with its other partner, dAtf4/Crc, in which case Xrp1 could represent a Drosophila-specific regulator of a more conserved pathway (Blanco, 2020).

The current data provide overwhelming genetic evidence that Xrp1 does function along with Irbp18. Like Xrp1 mutations, irbp18 mutation suppressed multiple effects of Rp mutations, including the elimination of Rp+/- mutant cells by competitive apoptosis in the proximity of wild type cells and the reduced growth of Rp+/- wing cells. Like Xrp1, irbp18 was also required for the prompt disappearance of Rp-/- cell clones, which survived in the irbp18 mutant background. All these data were consistent with the model that Xrp1/Irbp18 heterodimers are the active species in Rp mutant cells and were inconsistent with the idea that Xrp1 might act as a competitive inhibitor of other Irbp18-containing species, as this would have predicted that Irb18 mutations would have had phenotypes opposite to those of Xrp1 (Blanco, 2020).

The phenotype of Crc knockdown is different from those of Xrp1 and irbp18 mutations. Whereas Xrp1 and irbp18 mutations enhance the growth and competitiveness of Rp+/- cells, crc knockdown greatly diminished growth and survival of Rp+/- cells. If Xrp1 was a Drosophila-specific competitive inhibitor of a conserved Crc/Irbp18 heterodimer that was required for growth, both irbp18 and crc mutants would show reduced growth, similar to Rp+/- genotypes. In contrast to this, irbp18 mutants have little phenotype except in Rp+/- genotypes, where their effects closely resemble those of Xrp1 mutants. Also, whereas crc knockdown strongly and cell-autonomously affected the growth of Rp+/- cells, it had less effect on Rp+/+ cells (Blanco, 2020).

In addition to these findings in loss-of-function experiments, it was also found that Xrp1 over-expression phenotypes depended on IRBP18, as would be expected if these proteins function together. It was also found that Xrp1 over-expression at higher temperatures resulted in a still stronger eye phenotype where simultaneous irbp18 mutation restored normal eye size but did not completely restore eye morphology. This is consistent with some IRBP18-independent component to ectopic Xrp1 function that is either cold-sensitive or only apparent at the highest ectopic expression levels. There is not yet any evidence whether these over-expression effects are physiologically relevant (Blanco, 2020).

Taken together, these findings suggest that the Xrp1/IRBP18 and Crc/IRBP18 heterodimers have independent and perhaps unrelated functions. Consistent with this, ectopic expression of IRBP18 had no phenotypic effect, suggesting that IRBP18 is normally made in excess, so that it is Xrp1 that is limiting for the growth inhibiting activities of the Xrp1/IRBP18 heterodimers, which do not impact IRBP18 availability sufficiently to affect Crc/Irbp18 functions (Blanco, 2020).

As expected if Xrp1 functions in a heterodimer, the basic and Leucine Zipper domains were important for Xrp1 function, as was the AT hook. In over-expression assays only, there could be reduced activity of proteins deleted for any of these domains individually, but not of a truncation that precedes them all. When encoded from the endogenous locus, basic and AT-hook domains appeared absolutely required (Blanco, 2020).

Mutual auto-regulation may be a significant feature of Xrp1 and IRBP18 function. As noted previously, the elevated Xrp1 and irbp18 transcription observed in Rp+/- wing discs is dependent on Xrp1 function. This study shows that IRBP18 protein levels are also elevated in Rp+/- cells in an Xrp1-dependent fashion, and that irbp18 is also required for the autoregulation. In principle, autoregulation could have been the major or indeed the only transcriptional function of the Xrp1/IRBP18 heterodimer, ie perhaps these proteins could control cell competition through other mechanisms once levels were sufficient. This cannot be completely correct, however, because Xrp1 was still substantially dependent on the irbp18 gene and on the Leucine Zipper and DNA binding domains when expressed using GAL4-driven transgenes that are independent of auto-regulation, so by-passing the requirement of auto-regulation does not relieve the requirements for heterodimerization and DNA binding domains. It is also worth noting that Xrp1 and irbp18 are both required to promptly eliminate Rp-/- cells, where their expression does not require auto-regulation. Although implicating other transcriptional targets of Xrp1/IRBP18 in Rp+/- and Rp-/- cells, these studies do not rule out other functions besides transcription (Blanco, 2020).

Previously it was thought that Xrp1 was restricted to the genus Drosophila, a surprising finding for a protein that has an important cellular function. This study found, however, that Xrp1 genes have been under strong positive selection for rapid evolutionary change. Recurrent positive selection is often the sign of an evolutionary arms race, such as are often driven by host-pathogen interactions, sexual competition, or intra-genomic conflict. Possibly pathogens target Xrp1 to promote growth and survival of infected cells. It is interesting that Xrp1 is already documented to interact with one transposable element, the P element. However, none of these scenarios for positive selection, or indeed additional possibilities, can yet be ruled out (Blanco, 2020).

Rapid divergence makes homology difficult to detect, and accordingly this study has identify divergent Xrp1 homologs in other Dipteran insects that have not previously been annotated because their sequence similarities are restricted to the key DNA-binding portion of the protein, and to a more amino-terminal Xrp1-homology domain. The failure to identify still more distant homologs might be genuine, or might reflect further divergence beyond ones ability to recognize homology. Mammals do contain other members of the C/EBP protein family without identified Drosophila homologs, and this study shows that DDIT3 (aka CHOP and C/EBP-Z) can generate a similar phenotype to Xrp1 when expressed in Drosophila. Interestingly C/EBP-α, one of the mammalian proteins more related to Irbp18, has been implicated in a cell competition-like phenomenon, the elimination of cells from the multipotent hematopoietic stem cell niche following irradiation (Blanco, 2020).

Xrp1 and Irbp18 trigger a feed-forward loop of proteotoxic stress to induce the loser status

Cell competition induces the elimination of less-fit 'loser' cells by more fit 'winner' cells. In Drosophila, cells heterozygous mutant in ribosome genes, Rp/+, known as Minutes, are outcompeted by wild-type cells. Rp/+ cells display proteotoxic stress and the oxidative stress response, which drive the loser status. Minute cell competition also requires the transcription factors Irbp18 and Xrp1, but how these contribute to the loser status is partially understood. This study provided evidence that initial proteotoxic stress in RpS3/+ cells is Xrp1-independent. However, Xrp1 is sufficient to induce proteotoxic stress in otherwise wild-type cells and is necessary for the high levels of proteotoxic stress found in RpS3/+ cells. Surprisingly, Xrp1 is also induced downstream of proteotoxic stress, and is required for the competitive elimination of cells suffering from proteotoxic stress or overexpressing Nrf2. These data suggests that a feed-forward loop between Xrp1, proteotoxic stress, and Nrf2 drives Minute cells to become losers (Langton, 2021).

Cells within a tissue may become damaged due to spontaneous or environmentally induced mutations, and it is beneficial to organismal health if these cells are removed and replaced by healthy cells. During cell competition, fitter cells, termed winners, recognise and eliminate less-fit cells, termed losers, resulting in restoration of tissue homoeostasis. Cell competition therefore promotes tissue health and is thought to provide a level of protection against developmental aberrations and against cancer by removing cells carrying oncoplastic mutations. However, an increasing body of evidence indicates that cell competition can also promote growth of established tumours, enabling them to expand at the expense of surrounding healthy cells (Langton, 2021).

Minute cell competition was discovered through the study of a class of Drosophila ribosomal mutations called Minutes and initial work suggests that it is conserved in mammals. While homozygous Rp mutations are mostly cell lethal, heterozygosity for most Rp mutations gives rise to viable adult flies that exhibit a range of phenotypes including developmental delay and shortened macrochaete bristles. Rp/+ tissues display a higher cell-autonomous death frequency than wild-type tissues, and competitive interactions further elevate cell death in Rp/+ cells bordering wild-type cells, contributing to progressive loss of Rp/+ cells over time (Langton, 2021).

It was suggested that Rp/+ cells are eliminated by cell competition due to their reduced translation rate. However, it has beem shown that Rp/+ cells experience significant proteotoxic stress and this is the main driver of their loser status. Rp/+ cells have a stoichiometric imbalance of ribosome subunits, which may provide the source of proteotoxic stress. The autophagy and proteasomal machineries become overloaded and protein aggregates build up in Rp/+ cells, leading to activation of stress pathways. This includes activation of Nuclear factor erythroid 2-related factor 2 (Nrf2) and of the oxidative stress response, whichia sufficient to cause the loser status. Restoring proteostasis in Rp/+ cells suppresses the activation of the oxidative stress response and inhibits both autonomous and competitive cell death (Langton, 2021).

Genetic screening for suppressors of cell competition led to the identification of Xrp1, a basic leucine Zipper (bZip) transcription factor. Loss of Xrp1 rescues both the reduced growth and competitive cell death of Rp/+ cells in mosaic tissues. Consistently, loss of Xrp1 restores translation rates and abolishes the increased JNK pathway activity characteristic of Rp/+ cells. Xrp1 forms heterodimers with another bZip transcription factor called Inverted repeat binding protein 18kDa (Irbp18), and removal of Irbp18 also strongly suppresses the competitive elimination of Rp/+ cells in mosaic tissues. Irbp18 and Xrp1 are transcriptionally upregulated and mutually required for each other's expression in Rp/+ cells, suggesting they function together in Minute cell competition (Blanco, 2020). Irbp18 forms heterodimers with another bZip transcription factor, ATF4. However, knockdown of ATF4 in Rp/+ cells reduces their survival in mosaic tissues, which is the opposite effect to knockdown of Xrp1 or Irbp18. This has been interpreted to suggest that the ATF4-Irbp18 heterodimer acts independently to the Xrp1-Irbp18 heterodimer (Langton, 2021).

How the Xrp1/Irbp18 complex contributes to the loser status is not clear. Given the recently identified role of proteotoxic stress in cell competition this study sought to establish whether Xrp1/Irbp18 and proteotoxic stress act independently or in the same pathway to contribute to cell competition in Rp/+ cells. This study identified a feed-forward loop between Xrp1/Irbp18 and proteotoxic stress, which is required for downstream activation of the oxidative stress response and the loser status. The data suggests a model in which the initial insult in RpS3/+ cells is ribosomal imbalance-induced proteotoxic stress, which is Xrp1 independent. Xrp1 is then transcriptionally activated downstream of proteotoxic stress, by increased phosphorylated-eukaryotic Initiation Factor 2α (p-eIF2α), and possibly by Nrf2. The Xrp1-Irbp18 complex then induces further proteotoxic stress, completing the feed-forward loop. This work provides new insight into the interactions between the stress signalling pathways active in Rp/+ cells and provides a mechanism for how the Xrp1-Irbp18 heterodimer mediates the competitive elimination of Rp/+ cells by wild-type cells (Langton, 2021).

This study provided evidence that a feed-forward loop between proteotoxic stress, Nrf2 and the Xrp1/Irbp18 complex is operational in RpS3/+ cells (including in the absence of cell competition) and contributes to reducing their fitness during cell competition. The data suggests that an imbalance between SSU and LSU Ribosomal proteins generates an initial source of proteotoxic stress, independently of Xrp1. This leads to xrp1 transcriptional upregulation, likely via p-eIF2α. Xrp1, together with Irbp18, generates further proteotoxic stress, in a feed-forward loop. This causes LSU ribosome proteins to accumulate, exacerbating the stoichiometric imbalance between LSU and SSU subunit components in RpS3/+ cells. Knockdown of Xrp1 or Irbp18 rescues proteotoxic stress in RpS3/+ cells, suggesting that this feed-forward loop is essential for build-up of proteotoxic stress and to reduce the competitiveness of Rp/+ cells. It is noted that during the revision of this manuscript two other independent studies have reported relevant and complementary findings. Nrf2 is also activated by proteotoxic stress and contributes to this feedback loop, either independently of p-eIF2α, or downstream of p-eIF2α. The data cannot distinguish between these two possibilities (Langton, 2021).

The data indicate that xrp1 upregulation is likely mediated by increased p-eIF2α levels. p-eIF2α accumulates in Rp/+ cells, and increasing p-eIF2α in wild-type cells (by knocking down GADD34) leads to increased xrp1 transcription, suggesting that p-eIF2α does, at least partially, contribute to xrp1 transcription in Rp/+ cells. p-eIF2α induces many transcriptional targets via stabilization of the transcription factor ATF4. This suggested that ATF4 may activate xrp1. Consistent with this, it was found that ATF4 overexpression is sufficient to upregulate an xrp1 transcriptional reporter in wing disc cells. However, it was surprising to find that xrp1 upregulation does not seem to depend on ATF4 in RpS3/+ cells. Indeed, ATF4 knockdown did not reduce xrp1 transcription in RpS3/+ cells. Furthermore, it was not possible to detect stabilization of ATF4 in RpS3/+ cells using a translational reporter. These observations suggest that p-eIF2α upregulates xrp1 transcription in Rp/+ cells by an unknown, ATF4 independent, mechanism. Alternatively, the role of ATF4 may be masked by other inputs onto the xrp1 promoter. For example, ATF4 knockdown could increase proteotoxic stress in Rp/+ cells, by inhibiting the UPR, and this may upregulate other pathways that act on the xrp1 promoter, thus masking any effect of ATF4 knockdown. This mechanism could involve Nrf2, since Nrf2 is also induced by proteotoxic stress and since it has been shown that Nrf2 induces cellular toxicity via xrp1. However, it is also possible that other factors activate Xrp1 in Rp/+ cells (Langton, 2021).

Nrf2 plays a pro-survival role in many contexts, by activating a battery of genes that enable the metabolic adaptation to oxidative stress. It is therefore counterintuitive that Nrf2 overexpression should induce the loser status and, at high expression levels, cell death. The current work suggests that the toxicity of Nrf2 is at least in part due to Xrp1 function, as elimination of Nrf2 expressing cells is rescued by Xrp1 knockdown. Whether additional Nrf2 target genes contribute to the loser status remains to be established (Langton, 2021).

Besides Xrp1 or Irbp18 knockdown, the only other condition known thus far to rescue xrp1 transcriptional upregulation in Rp/+ cells is an RpS12 point mutation, RpS1297D. However, the mechanism by which RpS12 affects xrp1 transcription remains elusive. It will be important in future work to establish whether RpS12 mutations rescue xrp1 transcriptional activation upstream or downstream of proteotoxic stress (Langton, 2021).

The results provide compelling evidence that Xrp1 and Irbp18 are responsible for inducing proteotoxic stress in RpS3/+ cells. Firstly, knockdown of Xrp1 or Irbp18 rescues the accumulation of p62 labelled aggregates and rescues the increased p-eIF2α in RpS3/+ cells. Secondly, overexpression of Xrp1 is sufficient to upregulate markers of proteotoxic stress in wild-type cells. Third, the presence of Xrp1 in RpS3/+ cells worsens the imbalance of Ribosomal proteins, causing LSUs to accumulate. It will be crucial in future work to identify the relevant targets of Xrp1 that cause proteotoxic stress in Rp/+ cells. Xrp1 may alter expression of a single target, for example a gene encoding a component or regulator of the autophagy or proteasomal systems, which deregulates cellular proteostasis. Alternatively, several target genes may contribute to enhancing proteotoxic stress: if several subunits of multi-protein complexes are deregulated by increased Xrp1, this could lead to unassembled complexes, increasing the burden on the cellular degradation machinery in already stressed Rp/+ cells. There may also be Xrp1 targets that contribute to the loser status without affecting proteotoxic stress. It is remarkable that, in addition to rescuing competitive elimination of Rp/+ cells, loss of Xrp1 can rescue elimination of mahj deficient cells and Nrf2 overexpressing cells. In mahj deficient cells, loss of Xrp1 was able to rescue the upregulation of p-eIF2α, suggesting that Xrp1 also promotes proteotoxic stress in mahj cells. It will be interesting to establish whether this is the case for Nrf2 expressing cells (Langton, 2021).

Xrp1 has been shown to play a role in a Drosophila model of Amyotrophic lateral sclerosis (ALS), a debilitating and lethal neurodegenerative disorder that can be caused by aggregogenic mutations in genes encoding RNA binding proteins, including TDP-43 and FUS, a member of the FET family of proteins. TDP-43 and FUS also form cytoplasmic, ubiquitinated aggregates, in several other neurodegenerative disorders. Drosophila cabeza (caz) is the single ortholog of the human FET proteins. Xrp1 is upregulated in caz mutants, and the pupal lethality, motor defects and dysregulated gene expression of caz mutants is rescued by xrp1 heterozygosity. Therefore, it is possible that the feed-forward loop that this study has uncovered is also active in this context: formation of cytoplasmic proteotoxic aggregates could stimulate xrp1 expression, which could then induce further proteotoxic stress in a feed forward loop, resulting in neuronal toxicity. Understanding the relationship between Xrp1, proteotoxic stress and oxidative stress may thus be beneficial for the study of human proteinopathies (Langton, 2021).

The CRL4 E3 ligase Mahjong/DCAF1 controls cell competition through the transcription factor Xrp1, independently of polarity genes

Cell competition, the elimination of cells surrounded by more fit neighbors, is proposed to suppress tumorigenesis. Mahjong (Mahj), a ubiquitin E3 ligase substrate receptor, has been thought to mediate competition of cells mutated for lethal giant larvae (lgl), a neoplastic tumor suppressor that defines apical-basal polarity of epithelial cells. This study shows that Drosophila cells mutated for mahjong, but not for lgl [l(2)gl], are competed because they express the bZip-domain transcription factor Xrp1, already known to eliminate cells heterozygous for ribosomal protein gene mutations (Rp/+ cells). Xrp1 expression in mahj mutant cells results in activation of JNK signaling, autophagosome accumulation, eIF2α phosphorylation and lower translation, just as in Rp/+ cells. Cells mutated for damage DNA binding-protein 1 (ddb1; pic) or cullin 4 (cul4), which encode E3 ligase partners of Mahj, also display Xrp1-dependent phenotypes, as does knockdown of proteasome subunits. These data suggest a new model of mahj-mediated cell competition that is independent of apical-basal polarity and couples Xrp1 to protein turnover (Kumar, 2022).

Cell competition, which is the elimination of, in most cases, slower-growing cells by faster-growing cells in mosaics, is important for precise development, regeneration and physiological maintenance. Cell competition was first recognized in Drosophila in the case of cells lacking one copy of ribosomal protein genes (Rp/+). These mutants, which are known as 'Minutes' because of their thin body bristles, also display slow growth. Minute mutant cells are eliminated from mosaics with wild-type cells by caspase-dependent cell death. Super-competition, the name given to the process of eliminating wild-type cells, happens in mosaics with faster-growing Myc- or Yorkie (Yki)-expressing cells. Considered together, cell competition and super-competition suggest that comparison of cellular fitness leads to cell competition. Because the mammalian homologs of Myc and Yki are proto-oncogenes, it has been suggested that super-competition might contribute to tumor expansion, as several recent studies have confirmed (Kumar, 2022).

Cell competition may also be tumor suppressive. Global loss of apico-basal polarity genes such as lgl [l(2)gl)] or scribble (scrib) leads to polarity-deficient neoplasia of Drosophila imaginal discs, but clones of lgl or scrib cells are eliminated from mosaics. These lgl or scrib mutant clones do not form tumors unless cell competition is blocked and mutant cells remain in the epithelium (Kumar, 2022).

Competition of Rp/+ mutant cells might also serve a tumor-surveillance role. Rp genes are spread throughout the genome, and it has been shown they can serve as sensors for aneuploidy, leading to elimination of aneuploid cells containing monosomies that affect Rp gene dose. Mutants with disrupted cell competition accumulate aneuploid cells. These would be expected to be tumorigenic in mammals, where aneuploidy is associated with tumorigenesis. Competition of Rp/+ cells depends on the Drosophila bZip AT-hook domain transcription factor Xrp1. The Xrp1 expression induced in Rp/+ imaginal discs is also responsible for most of their altered gene expression, their slow growth and their reduced translation, in addition to their propensity to be eliminated by cell competition. Xrp1 is also expressed in the DNA damage response, where its transcription is p53 dependent. Xrp1 induction in Rp/+ cells is independent of p53 but dependent on a particular Rp protein, RpS12, which is thought to play a role in signaling the defect in ribosome biogenesis (Kumar, 2022).

The tumor-suppressive cell competition of polarity gene mutant cells has been proposed to go through Mahjong (Mahj) (Tamori, 2010), a CRL4 E3 ubiquitin ligase (Ly, 2019). mahj physically interacts with Lgl, and its overexpression in lgl mutant clones suppresses their elimination from mosaic tissues (Tamori, 2010). Interestingly, mahj knockdown in MDCK cell cultures also leads to their elimination by co-cultured normal MDCK cells, suggesting a cell competition mechanism that is conserved between Drosophila and mammalian cells (Tamori, 2010). The mammalian homolog of mahj, known as DDB1-Cul4-associated factor 1 (DCAF1) or human immunodeficiency virus type 1 accessory protein Vpr-binding protein (VprBP), is important for G2 cell cycle arrest and virus replication after HIV1 infection. Dcaf1 is required for mouse embryogenesis and its knockdown affects cell proliferation, cell cycle and cell survival in multiple cell types. Dcaf1 interacts with the Hippo pathway and its knockdown also stabilizes p53, but there has been no report of Dcaf1/VprBP affecting epithelial cell polarity in mammals (Kumar, 2022).

In Drosophila, the overall transcriptional signature of mahj mutant wing discs is unexpectedly similar to that of Rp/+ mutants, including upregulation of Xrp1 mRNA. Because mahj and Rp/+ cells were thought to represent distinct mechanisms of cell competition, this finding suggested a gene expression signature common to cells targeted by cell competition. Besides transcription, other similarities have been reported between mahj and Rp/+ mutant cells, including autophagosome accumulation and evidence of proteotoxic stress (Kumar, 2022).

This study shows that mahj mutant cells trigger cell competition through an Xrp1-dependent pathway like that in Rp/+ cells, and distinct from cell competition of lgl or scrib clones, which do not express or depend on Xrp1 function for elimination. Xrp1 expression also makes mahj mutant cells phenotypically like Rp/+ cells, that is, results in 'Minute-like' thin thoracic bristles, slow growth, reduced translation, altered autophagy and increased JNK signaling. Regulation of Xrp1 by mahj likely requires its E3 ligase activity, depending on DNA Damage Binding Protein 1 (Ddb1) and Cullin 4 (Cul4). These results show that mahj mutant cells suffer cell competition because of a transcriptional response to altered ubiquitinylation mediated by Xrp1 and therefore resembling Rp/+ mutant cells. This seems unrelated to elimination of scrib or lgl mutant cells - the polarity-defective cells. Thus, loss of mahj function is an additional genotype triggering elimination by the Xrp1-dependent pathway that also removes Minute cells, not the mechanism for eliminating tumorigenic polarity-deficient cells (Kumar, 2022).

This research explored the cell competition mechanisms of Mahj, a CRL4 ubiquitin ligase (Ly, 2019), the mutation of which triggers similar cellular effects to Rp/+ mutations, including similar changes in gene expression, global translation rates, JNK activity and autophagy, leading mahj cells to be eliminated by competition with wild-type cells, as Rp/+ cells are. The basis of the similarity is that mahj and Rp loss of function both activate expression of Xrp1, the transcription factor that coordinates these effects. Unlike Rp/+ genotypes, which activate Xrp1 through a rpS12-dependent mechanism, mahj regulates Xrp1 most likely through its ubiquitin ligase activity, which depends on DDB1, Cul4 and Roc1a, although the specific ubiquitylated target has not yet been identified. It is suggested that Xrp1 is likely to be activated by a protein, or proteins, that are normally degraded by Mahj-dependent ubiquitylation, because Xrp1 is also activated by inhibition of the proteasome, which is expected to affect the degradation of ubiquitylated proteins, but not other functional consequences of ubiquitylation. The relevant target does not seem to be Warts, despite the fact that levels of Warts and Hippo pathway activity also control cellular growth and global translation levels, and can stimulate cell competition. These studies support the notion that Xrp1 is a sensor of multiple cellular defects that cause cell competition, rather than that of a 'loser signature' common to distinct cell competition mechanisms (Kumar, 2022).

Mahj was previously thought to be responsible for the cell competition of cells mutated for lgl (Tamori, 2010; Baker, 2011; Levayer, 2013), a gene that controls apical basal cell polarity. mahj was originally linked to apical-basal polarity because of a physical interaction with Lgl, and because mahj overexpression can rescue lgl mutant clones for elimination, suggesting that mahj behaves as an intracellular signal transducer of lgl activity in cell competition (Tamori, 2010). As such, it was surprising when similar gene expression changes were observed in mahj mutant and Rp mutant wing discs, because these were assumed to reflect distinct cell competition pathways and suggested a common gene expression signature associated with competed cells. This study shows, however, that neither lgl nor scrib, another related cell polarity gene, affects cell competition by the same mechanism as mahj, because neither lgl nor scrib mutant cells express or require Xrp1. Interestingly, several distinct pathways have recently been described to mediate the elimination of scrib mutant cells in competition with wild-type cells, and none of these pathways are shown to be required for the elimination of Rp/+ mutant cells. In addition, mahj loss by itself does not result in apical-basal polarity defects (Tamori, 2010), and its mammalian homolog is implicated in cell cycle regulation, genome integrity and p53 activity (Hrecka, 2007; Cooper 2014; Lubow, 2020). Drosophila mahj, which is an essential gene, regulates neural stem cell reactivation (Ly, 2019) and may have other roles in non-neuronal tissues, as suggested by defects observed when mahj is depleted in posterior wing compartments. Accordingly, it is concluded that mahj mutants affect cellular growth and cell competition in a manner unrelated to lgl and scrib, and that the functional relationship of mahj to apical-basal polarity pathways, should any exist, is unclear. The functional importance of physical interaction between Mahj and Lgl remains to be explored. It is known that lgl clones are rescued by reduced Hippo signaling, although this study did not detect reduced Hippo signaling after mahj overexpression in the absence of lgl mutations (Kumar, 2022).

These studies provide further evidence for Xrp1 as an integrator of multiple seemingly independent cellular defects that each result in a common spectrum of cellular responses and predispose cells to competitive elimination by wild-type neighbors (Kiparaki, 2022). These functional roles for Xrp1 first became apparent through its role in the slow growth, reduced translation and competitive elimination of Rp/+ cells, in which Xrp1 expression is induced in an rpS12-dependent manner. In the case of mahj, Xrp1 protein expression is induced to confer a very similar spectrum of cellular effects, but independently of rpS12 and perhaps depending on stabilization of a protein normally targeted for proteasomal turnover by mahj-dependent ubiquitylation. Xrp1 expression was first found as a p53-regulated gene, perhaps part of the DNA damage response. Recently, Xrp1 induction has also been found as a response to ER stress, possibly through translational regulation downstream of eIF2α phosphorylation. It has been suggested that eIF2α phosphorylation, and Xrp1 expression, can also be triggered by a global, cytoplasmic proteotoxic stress, which is suggested to occur as a consequence of deficient ribosome assembly in Rp mutant cells. Xrp1 expression in response to proteasome inhibition is one piece of evidence for this model. This study shows, however, that Xrp1 is induced, and cell competition results after loss of mahj, a single E3-ligase adapter protein that probably targets only a moderate number of proteins for degradation. Thus, an alternative explanation of Xrp1 induction after proteasome inhibition is that this could reflect stabilization of one or a few specific proteins. Overall, a picture is emerging of Xrp1 as a stress-responsive transcription factor whose expression can be initiated by multiple distinct pathways, then leading to a common cellular response, including the elimination of the stressed cells by competition with nearby wild-type cells, when such cells are available (Kumar, 2022).

Importantly, cells depleted for DCAF1/VprBP, the mammalian homolog of Mahj, are eliminated by competition with wild-type cells in mammalian cell culture. Thus, cell competition of mahj mutant cells may be a conserved process. Conservation of cell competition has not yet been demonstrated for Rp/+ cells in mammals, although it may very well occur. In mammals, knockdown of either mahj or its binding partner ddb1 results in P53 activation, which is functionally required for the resulting phenotypes. Differences in p53 activity lead to cell competition in many mammalian systems. p53 is not required for mahj-mediated cell competition in Drosophila, but because Xrp1 is a target of Drosophila p53 in irradiated cells, it is possible Xrp1 is a p53 target that has replaced the cell competition role of p53 in Drosophila, as has already been suggested for the competition of Rp/+ cells, which is also p53 independent in Drosophila, although Rp mutations activate p53 in mammals. Thus, mahj-mediated cell competition may provide another example where Xrp1 mediates a process in Drosophila that is dependent on p53 in mammals (Kumar, 2022).

Inter-organ growth coordination is mediated by the Xrp1-Dilp8 axis in Drosophila

How organs scale with other body parts is not mechanistically understood. This question was addressed using the Drosophila imaginal disc model. When the growth of one disc domain is perturbed, other parts of the disc and other discs slow down their growth, maintaining proper inter-disc and intra-disc proportions. The relaxin-like Dilp8 is required for this inter-organ coordination. This work also reveals that the stress-response transcription factor Xrp1 plays a key role upstream of dilp8 in linking organ growth status with the systemic growth response. In addition, the small ribosomal subunit protein RpS12 is required to trigger Xrp1-dependent non-autonomous response. This work demonstrates that RpS12, Xrp1, and Dilp8 form an independent regulatory module that ensures intra- and inter-organ growth coordination during development (Boulan, 2019).

Xrp1 genetically interacts with the ALS-associated FUS orthologue caz and mediates its toxicity

Cabeza (caz) is the single Drosophila melanogaster orthologue of the human FET proteins FUS, TAF15, and EWSR1, which have been implicated in amyotrophic lateral sclerosis (ALS) and frontotemporal dementia. This study identified Xrp1, a nuclear chromatin-binding protein, as a key modifier of caz mutant phenotypes. Xrp1 expression was strongly up-regulated in caz mutants, and Xrp1 heterozygosity rescued their motor defects and life span. Interestingly, selective neuronal Xrp1 knockdown was sufficient to rescue, and neuronal Xrp1 overexpression phenocopied caz mutant phenotypes. The caz/Xrp1 genetic interaction depended on the functionality of the AT-hook DNA-binding domain in Xrp1, and the majority of Xrp1-interacting proteins are involved in gene expression regulation. Consistently, caz mutants displayed gene expression dysregulation, which was mitigated by Xrp1 heterozygosity. Finally, Xrp1 knockdown substantially rescued the motor deficits and life span of flies expressing ALS mutant FUS in motor neurons, implicating gene expression dysregulation in ALS-FUS pathogenesis (Mallik, 2018).

Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis

In Drosophila, P-element transposition causes mutagenesis and genome instability during hybrid dysgenesis. The P-element 31-bp terminal inverted repeats (TIRs) contain sequences essential for transposase cleavage and have been implicated in DNA repair via protein-DNA interactions with cellular proteins. The identity and function of these cellular proteins were unknown. Biochemical characterization of proteins that bind the TIRs identified a heterodimeric basic leucine zipper (bZIP) complex between an uncharacterized protein that is termed 'Inverted Repeat Binding Protein (IRBP) 18' and its partner Xrp1. The reconstituted IRBP18/Xrp1 heterodimer binds sequence-specifically to its dsDNA-binding site within the P-element TIRs. Genetic analyses implicate both proteins as critical for repair of DNA breaks following transposase cleavage in vivo. These results identify a cellular protein complex that binds an active mobile element and plays a more general role in maintaining genome stability (Francis, 2016).

A role for the IRBP complex in the P-element transposition reaction has been postulated since initial identification of its sequence-specific DNA-binding properties (Staveley, 1995; Beall, 1994; Beall, 1996; Rio, 1988). The organizational overlap between IRBP DNA binding and transposase cleavage sites makes the IRBP complex a prime cellular candidate to influence some aspect of the P-element transposition cycle. Several putative IRBP proteins copurified with observed IRBP DNA-binding activity or unambiguously promoted DNA repair posttransposase P-element cleavage (Ku 70 and mus309/DmBLM). None, however, could reconstitute site-specific DNA binding to the P-element 31-bp TIRs (Francis, 2016).

This report identifies a bZIP heterodimer between a C/EBP family member IRBP18 and a drosophilid-specific protein Xrp1 as the sequence-specific DNA-binding subunits of a larger multiprotein IRBP complex that binds to the P-element TIRs. These proteins work in concert to facilitate efficient DNA repair following P-element transposase-mediated DNA cleavage. Finally, these proteins are required more generally in the cellular DNA damage response and DSB repair in the absence of P elements (Francis, 2016).

Repair of P-element~induced DNA breaks occurs predominantly through two distinct DSB repair pathways: Non-homologous end joining (NHEJ) or a variant of classical homologous recombinational repair, synthesis-dependent strand annealing (SDSA), The choice between these two pathways is dictated by cell-cycle location, the availability of pathway substrates, and tissue type. Because the IRBP homozygous mutant males are sterile, it is not possible at present to use any of the established DNA repair reporter strains to determine in which DNA repair pathway IRBP18 and Xrp1 participates. Future would should determine if the IRBP18/Xrp1 heterodimer can bind directly to different DNA repair intermediates and thus provide a direct link between the bZIP heterodimer and DNA repair (Francis, 2016).

A role for bZIP proteins and specifically mammalian C/EBP proteins in DNA repair is well established. In human and mouse keratinocytes, UV-B UV DNA damage induces p53-dependent transcriptional activation of both the C/EBPα and β genes. C/EBPα expressed in prostate cancer cells where it interacts with the DNA repair proteins Ku p70/p80 heterodimer and the poly (ADP ribose) polymerase 1 (PARP-1). Notably, Drosophila p53 up-regulates three bZIP proteins (CG6272/IRBP18, CG17836/Xrp1, and CG15479/Mabiki) upon DNA damage. CG15479/Mabiki is a novel regulator of caspase-independent cell death of excess cells in the expanded head region of 6x-bcd embryos and is thought to work in concert with other caspase-independent cell death mechanisms to ensure proper development. Genetic deletion of the CG17836/Xrp1 gene resulted in a DNA repair phenotype when challenged with ionizing radiation (Akdemir, 2007). Additionally, the Dmp53 DNA damage-induced apoptotic response was unaffected in the Xrp1 mutants, suggesting that Xrp1 functions to preserve genome stability through a pathway independent of apoptosis (Akdemir, 2007). Although this study has demonstrated that IRBP18 and Xrp1 share a similar function in DNA repair, more experiments are needed to understand how these proteins work downstream of p53 transactivation (Francis, 2016).

D. melanogaster uses multiple endogenous mechanisms to limit P-element transposition. Expression of catalytically active transposase is restricted to the germline by tissue-specific pre-mRNA splicing regulation. The germline piwi-interacting RNA pathway has been demonstrated to repress transposition in trans and plays a critical role in host adaptation to newly invaded P elements. It is proposde that the IRBP18/Xrp1 heterodimer recognizes new P elements and that its native function is to facilitate repair of breaks to maintain genomic stability during a genotoxic event such as ionizing radiation or the massive P-element mobilization that occurs following a hybrid dysgenic cross (Francis, 2016).

bZIP proteins are well suited to recognize newly invaded foreign DNA due to their inherit ability to form multiple heterodimers. IRBP18 and Xrp1, for example, form heterodimers with other several other bZIP proteins; the net result is expansion of the repertoire of DNA sequences that can be bound. This library of bZIP dimers can be deployed to recognize foreign DNA as part of a survival mechanism against the genome instability created by foreign DNA invasion. In humans, mice and Drosophila p53 transactivate steady-state levels of several bZIP proteins in response to DNA damage. It is unclear how changes in steady-state levels of these DNA repair proteins determine dimer formation or function. What is clear is that bZIP proteins are important players in DNA repair and maintenance of genome stability. In this respect, the IRBP18/Xrp1 heterodimer is a newly identified component of the interconnected pathways to combat the genotoxic effects of mass invasion/mobilization of transposons (Francis, 2016).

Ectopic antenna induction by overexpression of CG17836/Xrp1 encoding an AT-hook DNA binding motif protein in Drosophila

Drosophila imaginal discs are an excellent model system for studies of developmental plasticity. In imaginal discs, most cells adhere strictly to their specific identity, but some cells undergo transdetermination, a process wherein the determined identity switches to another disc-specific identity. In this study, gain-of-function screening was performed and a gene, CG17836/Xrp1, was identified that induces ectopic antennae in the eye field upon overexpression at the early eye disc stage. An essential factor in the distalization process, Distalless, and its upstream regulators Wingless, Hedgehog, and Decapentaplegic, are ectopically induced by CG17836/Xrp1 overexpression in eye discs, and this provides molecular evidence of the formation of ectopic antennae. Further, forced expression of CG17836/Xrp1 induced severe cell-proliferation defects. These findings suggest that CG17836/Xrp1 is involved in the regulation of cell proliferation in eye discs and affects disc identity specification (Tsurui-Nishimura, 2013).

p53 directs focused genomic responses in Drosophila

p53 is a fundamental determinant of cancer susceptibility and other age-related pathologies. Similar to mammalian counterparts, Drosophila p53 integrates stress signals and elicits apoptotic responses that maintain genomic stability. To illuminate core-adaptive functions controlled by this gene family, the Drosophila p53 regulatory network was examined at a genomic scale. In development, the absence of p53 impacted constitutive expression for a surprisingly broad scope of genes. By contrast, stimulus-dependent responses governed by Drosophila p53 were limited in scope. The vast majority of stress responders were induced and p53 dependent (RIPD) genes. The signature set of 29 'high stringency' RIPD genes identified in this study were enriched for intronless loci, with a non-uniform distribution that includes a recently evolved cluster unique to Drosophila melanogaster. Two RIPD genes, with known and unknown biochemical activities, were functionally examined. One RIPD gene, designated XRP1, maintains genome stability after genotoxic challenge and prevents cell proliferation upon induced expression. A second gene, RnrL, is an apoptogenic effector required for caspase activation in a model of p53-dependent killing. Together, these studies identify ancient and convergent features of the p53 regulatory network (Akdemir, 2007).

Search PubMed for articles about Drosophila Xrp1

Akdemir, F., Christich, A., Sogame, N., Chapo, J. and Abrams, J. M. (2007). p53 directs focused genomic responses in Drosophila. Oncogene 26(36): 5184-5193. PubMed ID: 17310982

Baillon, L., Germani, F., Rockel, C., Hilchenbach, J. and Basler, K. (2018). Xrp1 is a transcription factor required for cell competition-driven elimination of loser cells. Sci Rep 8(1): 17712. PubMed ID: 30531963

Baker, N. E., Kiparaki, M. and Khan, C. (2019). A potential link between p53, cell competition and ribosomopathy in mammals and in Drosophila. Dev Biol 446(1): 17-19. PubMed ID: 30513308

Beall, E. L., Admon, A. and Rio, D. C. (1994). A Drosophila protein homologous to the human p70 Ku autoimmune antigen interacts with the P transposable element inverted repeats. Proc Natl Acad Sci U S A 91(26): 12681-12685. PubMed ID: 7809101

Beall, E. L. and Rio, D. C. (1996). Drosophila IRBP/Ku p70 corresponds to the mutagen-sensitive mus309 gene and is involved in P-element excision in vivo. Genes Dev 10(8): 921-933. PubMed ID: 8608940

Blanco, J., Cooper, J. C. and Baker, N. E. (2020). Roles of C/EBP class bZip proteins in the growth and cell competition of Rp ('Minute') mutants in Drosophila. Elife 9. PubMed ID: 31909714

Boulan, L., Andersen, D., Colombani, J., Boone, E. and Leopold, P. (2019). Inter-organ growth coordination is mediated by the Xrp1-Dilp8 axis in Drosophila. Dev Cell. PubMed ID: 31006647

Brodsky, M. H., Weinert, B. T., Tsang, G., Rong, Y. S., McGinnis, N. M., Golic, K. G., Rio, D. C. and Rubin, G. M. (2004). Drosophila melanogaster MNK/Chk2 and p53 regulate multiple DNA repair and apoptotic pathways following DNA damage. Mol Cell Biol 24(3): 1219-1231. PubMed ID: 14729967

Francis, M. J., Roche, S., Cho, M. J., Beall, E., Min, B., Panganiban, R. P. and Rio, D. C. (2016). Drosophila IRBP bZIP heterodimer binds P-element DNA and affects hybrid dysgenesis. Proc Natl Acad Sci U S A 113(46): 13003-13008. PubMed ID: 27799520

Kale, A., Ji, Z., Kiparaki, M., Blanco, J., Rimesso, G., Flibotte, S. and Baker, N. E. (2018). Ribosomal Protein S12e Has a Distinct Function in Cell Competition. Dev Cell 44(1): 42-55 e44. PubMed ID: 29316439

Kucinski, I., Dinan, M., Kolahgar, G. and Piddini, E. (2017). Chronic activation of JNK JAK/STAT and oxidative stress signalling causes the loser cell status. Nat Commun 8(1): 136. PubMed ID: 28743877

Kumar, A. and Baker, N. E. (2022). The CRL4 E3 ligase Mahjong/DCAF1 controls cell competition through the transcription factor Xrp1, independently of polarity genes. Development 149(22). PubMed ID: 36278853

Langton, P. F., Baumgartner, M. E., Logeay, R. and Piddini, E. (2021). Xrp1 and Irbp18 trigger a feed-forward loop of proteotoxic stress to induce the loser status. PLoS Genet 17(12): e1009946. PubMed ID: 34914692

Lee, C. H., Rimesso, G., Reynolds, D. M., Cai, J. and Baker, N. E. (2016). Whole-genome sequencing and iPLEX MassARRAY genotyping map an EMS-induced mutation affecting cell competition in Drosophila melanogaster. G3 (Bethesda) 6(10): 3207-3217. PubMed ID: 27574103

Lee, C. H., Kiparaki, M., Blanco, J., Folgado, V., Ji, Z., Kumar, A., Rimesso, G. and Baker, N. E. (2018). A regulatory response to ribosomal protein mutations controls translation, growth, and cell competition. Dev Cell 46(4): 456-469. PubMed ID: 30078730

Mallik, M., Catinozzi, M., Hug, C. B., Zhang, L., Wagner, M., Bussmann, J., Bittern, J., Mersmann, S., Klambt, C., Drexler, H. C. A., Huynen, M. A., Vaquerizas, J. M. and Storkebaum, E. (2018). Xrp1 genetically interacts with the ALS-associated FUS orthologue caz and mediates its toxicity. J Cell Biol. PubMed ID: 30209068

Rio, D. C. and Rubin, G. M. (1988). Identification and purification of a Drosophila protein that binds to the terminal 31-base-pair inverted repeats of the P transposable element. Proc Natl Acad Sci U S A 85(23): 8929-8933. PubMed ID: 2848246

Staveley, B. E., Heslip, T. R., Hodgetts, R. B. and Bell, J. B. (1995). Protected P-element termini suggest a role for inverted-repeat-binding protein in transposase-induced gap repair in Drosophila melanogaster. Genetics 139(3): 1321-1329. PubMed ID: 7768441

Tsurui-Nishimura, N., Nguyen, T. Q., Katsuyama, T., Minami, T., Furuhashi, H., Oshima, Y. and Kurata, S. (2013). Ectopic antenna induction by overexpression of CG17836/Xrp1 encoding an AT-hook DNA binding motif protein in Drosophila. Biosci Biotechnol Biochem 77(2): 339-344. PubMed ID: 23391928

date revised: 2 February 2023

Home page: The Interactive Fly © 2011 Thomas Brody, Ph.D.