Diverse tumor pathology due to distinctive patterns of JAK/STAT pathway activation caused by different Drosophila polyhomeotic alleles

Drosophila polyhomeotic (ph) is one of the important polycomb group genes that is linked to human cancer. In the mosaic eye imaginal discs, while ph(del), a null allele, causes only non-autonomous overgrowth, ph(505), a hypomorphic allele, causes both autonomous and non-autonomous overgrowth. These allele-specific phenotypes stem from the different sensitivities of ph mutant cells to the Upd homologs that they secrete.     

The Polyhomeotic protein induces hyperplastic tissue overgrowth through the activation of the JAK/STAT pathway

Epigenetic mechanisms controlling cellular proliferation are essential to animal development. Moreover, altered levels of expression of the epigenetic regulator proteins are associated with the development and progression of human diseases like cancer. We have studied the effects of high levels of Polyhomeotic (PH) protein, a member of the Polycomb Group (PcG), during the proliferation of the imaginal discs in Drosophila. Over expression of PH protein causes induction of proliferation, accompanied with induction of JNK-dependent apoptosis. As a result, massive hyperplastic overgrowth is produced and the corresponding differentiated tissues show phenotypes related with mis-regulation of homeotic gene expression. We have found that high levels of PH up-regulate the JAK/STAT pathway through the de-repression of Unpaired (UPD), the extracellular ligand of the Drosophila JAK/STAT signalling cascade. Moreover, inactivation of the JAK/STAT pathway in the presence of a large amount of PH protein greatly reduces the tissue overgrowth, demonstrating a functional role of JAK/STAT in PH-induced hyperplasia. Finally, we have observed that decapentaplegic and d-myc, two growth genes and putative targets of the JAK/STAT pathway, are also over expressed in the PH-induced tumors. We propose that during normal development, the PcG proteins act to maintain inactive the JAK/STAT pathway. Upon cellular stress, changes in the levels of PcG proteins expression are induced and JAK/STAT is activated leading to tumor development. Our results show a functional relationship between the PcG gene expression and the JAK/STAT pathway, both of which are found to be perturbed in tumorigenesis.     

Drosophila chromosome condensation proteins Topoisomerase II and Barren colocalize with Polycomb and maintain Fab-7 PRE silencing

Mechanisms of cellular memory control the maintenance of cellular identity at the level of chromatin structure. We have investigated whether the converse is true; namely, if functions responsible for maintenance of chromosome structure play a role in epigenetic control of gene expression. We show that Topoisomerase II (TOPOII) and Barren (BARR) interact in vivo with Polycomb group (PcG) target sequences in the bithorax complex of Drosophila, including Polycomb response elements. In addition, we find that the PcG protein Polyhomeotic (PH) interacts physically with TOPOII and BARR and that BARR is required for Fab-7-regulated homeotic gene expression. Conversely, we find defects in chromosome segregation associated with ph mutations. We propose that chromatin condensation proteins are involved in mechanisms acting in interphase that regulate chromosome domain topology and are essential for the maintenance of gene expression.     

Trl-GAGA directly interacts with lola like and both are part of the repressive complex of Polycomb group of genes

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

dBRWD3 Regulates Tissue Overgrowth and Ectopic Gene Expression Caused by Polycomb Group Mutations

To maintain a particular cell fate, a unique set of genes should be expressed while another set is repressed. One way to repress gene expression is through Polycomb group (PcG) proteins that compact chromatin into a silent configuration. In addition to cell fate maintenance, PcG proteins also maintain normal cell physiology, for example cell cycle. In the absence of PcG, ectopic activation of the PcG-repressed genes leads to developmental defects and malignant tumors. Little is known about the molecular nature of ectopic gene expression; especially what differentiates expression of a given gene in the orthotopic tissue (orthotopic expression) and the ectopic expression of the same gene due to PcG mutations. Here we present that ectopic gene expression in PcG mutant cells specifically requires dBRWD3, a negative regulator of HIRA/Yemanuclein (YEM)-mediated histone variant H3.3 deposition. dBRWD3 mutations suppress both the ectopic gene expression and aberrant tissue overgrowth in PcG mutants through a YEM-dependent mechanism. Our findings identified dBRWD3 as a critical regulator that is uniquely required for ectopic gene expression and aberrant tissue overgrowth caused by PcG mutations.     

Regulation of larval hematopoiesis in Drosophila melanogaster: a role for the multi sex combs gene

Drosophila larval hematopoietic organs produce circulating hemocytes that ensure the cellular host defense by recognizing and neutralizing non-self or noxious objects through phagocytosis or encapsulation and melanization. Hematopoietic lineage specification as well as blood cell proliferation and differentiation are tightly controlled. Mutations in genes that regulate lymph gland cell proliferation and hemocyte numbers in the body cavity cause hematopoietic organ overgrowth and hemocyte overproliferation. Occasionally, mutant hemocytes invade self-tissues, behaving like neoplastic malignant cells. Two alleles of the Polycomb group (PcG) gene multi sex combs (mxc) were previously isolated as such lethal malignant blood neoplasm mutations. PcG genes regulate Hox gene expression in vertebrates and invertebrates and participate in mammalian hematopoiesis control. Hence we investigated the need for mxc in Drosophila hematopoietic organs and circulating hemocytes. We show that mxc-induced hematopoietic hyperplasia is cell autonomous and that mxc mainly controls plasmatocyte lineage proliferation and differentiation in lymph glands and circulating hemocytes. Loss of the Toll pathway, which plays a similar role in hematopoiesis, counteracted mxc hemocyte proliferation but not mxc hemocyte differentiation. Several PcG genes tested in trans had no effects on mxc hematopoietic phenotypes, whereas the trithorax group gene brahma is important for normal and mutant hematopoiesis control. We propose that mxc provides one of the regulatory inputs in larval hematopoiesis that control normal rates of plasmatocyte and crystal lineage proliferation as well as normal rates and timing of hemocyte differentiation.     

The E1 ubiquitin-activating enzyme Uba1 in Drosophila controls apoptosis autonomously and tissue growth non-autonomously

Ubiquitination is an essential process regulating turnover of proteins for basic cellular processes such as the cell cycle and cell death (apoptosis). Ubiquitination is initiated by ubiquitin-activating enzymes (E1), which activate and transfer ubiquitin to ubiquitin-conjugating enzymes (E2). Conjugation of target proteins with ubiquitin is then mediated by ubiquitin ligases (E3). Ubiquitination has been well characterized using mammalian cell lines and yeast genetics. However, the consequences of partial or complete loss of ubiquitin conjugation in a multi-cellular organism are not well understood. Here, we report the characterization of Uba1, the only E1 in Drosophila. We found that weak and strong Uba1 alleles behave genetically differently with sometimes opposing phenotypes. Whereas weak Uba1 alleles protect cells from cell death, clones of strong Uba1 alleles are highly apoptotic. Strong Uba1 alleles cause cell cycle arrest which correlates with failure to reduce cyclin levels. Surprisingly, clones of strong Uba1 mutants stimulate neighboring wild-type tissue to undergo cell division in a non-autonomous manner giving rise to overgrowth phenotypes of the mosaic fly. We demonstrate that the non-autonomous overgrowth is caused by failure to downregulate Notch signaling in Uba1 mutant clones. In summary, the phenotypic analysis of Uba1 demonstrates that impaired ubiquitin conjugation has significant consequences for the organism, and may implicate Uba1 as a tumor suppressor gene.     

Role of polycomb group proteins in stem cell self-renewal and cancer

Polycomb group proteins (PcG) form part of a gene regulatory mechanism that determines cell fate during normal and pathogenic development. The mechanism relies on epigenetic modifications on specific histone tails that are inherited through cell divisions, thus behaving de facto as a cellular memory. This cellular memory governs key events in organismal development as well as contributing to the control of normal cell growth and differentiation. Consequently, the dysregulation of PcG genes, such as Bmi1, Pc2, Cbx7, and EZH2 has been linked with the aberrant proliferation of cancer cells. Furthermore, at least three PcG genes, Bmi1, Rae28, and Mel18, appear to regulate self-renewal of specific stem cell types suggesting a link between the maintenance of cellular homeostasis and tumorigenesis. In this review, we will briefly summarize current views on PcG function and the evidence linking specific PcG proteins with the behavior of stem cells and cancer cells.     

The role of Polycomb Group Proteins in Cell Cycle Regulation During Development

Polycomb group (PcG) and trithorax group (trxG) proteins are evolutionarilyconserved chromatin modifiers that have well known roles in the maintenance ofsilent and active expression states of homeotic genes. PcG proteins may also beinvolved in the control of cellular proliferation, as several PcG complexes have beenshown to act either as proto-oncogenes or as tumor suppressors in vertebrates. InDrosophila, PcG factors associate with specific DNA regions termed PcG responseelements (PREs), and a PRE was recently identified in the gene encoding Cyclin A.Still, it is not yet clear how PcG complexes could control cell cycle progression.Beyond acting as stable silencers of cell cycle genes during the differentiationprocess, PcG complexes might also be integrators and/or modulators of cell cyclecheckpoints in dividing cells. Here, we discuss this dual aspect of PcG involvement inepigenetic cell cycle control.     

Selective interactions between vertebrate polycomb homologs and the SUV39H1 histone lysine methyltransferase suggest that histone H3-K9 methylation contributes to chromosomal targeting of Polycomb group proteins

Polycomb group (PcG) proteins form multimeric chromatin-associated protein complexes that are involved in heritable repression of gene activity. Two distinct human PcG complexes have been characterized. The EED/EZH2 PcG complex utilizes histone deacetylation to repress gene activity. The HPC/HPH PcG complex contains the HPH, RING1, BMI1, and HPC proteins. Here we show that vertebrate Polycomb homologs HPC2 and XPc2, but not M33/MPc1, interact with the histone lysine methyltransferase (HMTase) SUV39H1 both in vitro and in vivo. We further find that overexpression of SUV39H1 induces selective nuclear relocalization of HPC/HPH PcG proteins but not of the EED/EZH2 PcG proteins. This SUV39H1-dependent relocalization concentrates the HPC/HPH PcG proteins to the large pericentromeric heterochromatin domains (1q12) on human chromosome 1. Within these PcG domains we observe increased H3-K9 methylation. Finally, we show that H3-K9 HMTase activity is associated with endogenous HPC2. Our findings suggest a role for the SUV39H1 HMTase and histone H3-K9 methylation in the targeting of human HPC/HPH PcG proteins to modified chromatin structures.     

Common and Distinct Genetic Properties of ESCRT-II Components in Drosophila

Background

Genetic studies in yeast have identified class E vps genes that form the ESCRT complexes required for protein sorting at the early endosome. In Drosophila, mutations of the ESCRT-II component vps25 cause endosomal defects leading to accumulation of Notch protein and increased Notch pathway activity. These endosomal and signaling defects are thought to account for several phenotypes. Depending on the developmental context, two different types of overgrowth can be detected. Tissue predominantly mutant for vps25 displays neoplastic tumor characteristics. In contrast, vps25 mutant clones in a wild-type background trigger hyperplastic overgrowth in a non-autonomous manner. In addition, vps25 mutant clones also promote apoptotic resistance in a non-autonomous manner.

Principal Findings

Here, we genetically characterize the remaining ESCRT-II components vps22 and vps36. Like vps25, mutants of vps22 and vps36 display endosomal defects, accumulate Notch protein and – when the tissue is predominantly mutant – show neoplastic tumor characteristics. However, despite these common phenotypes, they have distinct non-autonomous phenotypes. While vps22 mutations cause strong non-autonomous overgrowth, they do not affect apoptotic resistance. In contrast, vps36 mutations increase apoptotic resistance, but have little effect on non-autonomous proliferation. Further characterization reveals that although all ESCRT-II mutants accumulate Notch protein, only vps22 and vps25 mutations trigger Notch activity.

Conclusions/Significance

The ESCRT-II components vps22, vps25 and vps36 display common and distinct genetic properties. Our data redefine the role of Notch for hyperplastic and neoplastic overgrowth in these mutants. While Notch is required for hyperplastic growth, it appears to be dispensable for neoplastic transformation.