InteractiveFly: GeneBrief

lethal (3) malignant brain tumor: Biological Overview | References

BIOLOGICAL OVERVIEW

In Drosophila, defects in asymmetric cell division often result in the formation of stem cell derived tumors. This study shows that very similar terminal brain tumor phenotypes arise through a fundamentally different mechanism. Brain tumors in l(3)mbt mutants originate from overproliferation of neuroepithelial cells in the optic lobes caused by de-repression of target genes in the Salvador-Warts-Hippo (SWH) pathway. ChIP-seq was used to identify L(3)mbt binding sites, and it was shown that L(3)mbt binds to chromatin insulator elements. Mutating l(3)mbt or inhibiting expression of the insulator protein gene mod(mdg4) results in upregulation of SWH-pathway reporters. As l(3)mbt tumors are rescued by mutations in bantam or yorkie or by overexpression of expanded the deregulation of SWH pathway target genes is an essential step in brain tumor formation. Therefore, very different primary defects result in the formation of brain tumors, which behave quite similarly in their advanced stages (Richter, 2011).

Drosophila nervous system recapitulates many steps in mammalian neurogenesis. Neurons in the adult fly brain arise from stem cells called neuroblasts which undergo repeated rounds of asymmetric cell division during larval stages. After division, one daughter cell remains a neuroblast while the other is called the ganglion mother cell (GMC) and divides just once more into two differentiating neurons. Most larval neuroblasts are inherited from the embryo but the so-called optic lobe neuroblasts (NB) located laterally on each brain lobe pass through a neuroepithelial (NE) stage and are therefore a particularly suitable model for mammalian neurogenesis. During early larval stages, the NE cells of the optic lobes (OL) proliferate and separate into the inner (IOA) and outer (OOA) optic anlagen. During late larval stages, NE cells switch to a neurogenic mode. On the medial side, they generate optic lobe neuroblasts (OL NBs), which generate the neurons of the medulla, the second optic ganglion. OL neurogenesis is controlled by a wave of lethal of scute (l(1)sc) expression passing through the neuroepithelium from medial to lateral. The activity of the Jak/STAT pathway inhibits neural wave progression and thereby controls neuroblast number. Differentiation of neuroepithelial cells also involves the Notch, Epidermal Growth Factor (EGF) and Salvador-Warts-Hippo (SWH) pathways (Richter, 2011).

Characterization of Drosophila genes identified in brain tumor suppressor screens has demonstrated that defects in neuroblast asymmetric cell division result in the formation of stem cell derived tumors that metastasize and become aneuploid upon transplantation. These screens also identified lethal (3) malignant brain tumor (l(3)mbt) (Gateff, 1993; Wismar, 1995), a conserved transcriptional regulator (Bonasio, 2010) that is also required for germ-cell formation in Drosophila (Yohn, 2003). L(3)mbt binds to the cell cycle regulators E2F (Lewis, 2004) and Rb (Trojer, 2007) but the relevance of these interactions is unclear. This study shows that in Drosophila, L(3)mbt regulates target genes of the Salvador-Warts-Hippo (SWH) pathway that are important in proliferation and organ size control. The SWH-pathway is regulated by the membrane proteins Expanded (Ex) and Fat, which activate a protein complex containing the kinases Hippo and Warts to phosphorylate the transcriptional co-activator Yorkie. Yorkie acts together with the transcription factors Scalloped and Homothorax to activate proliferative genes like Cyclin E and the microRNA bantam (ban) and Drosophila inhibitor of apoptosis 1 (diap1: thread). Upon phosphorylation, Yorkie is retained in the cytoplasm and its target genes are not activated. In Drosophila the main role of the SWH-pathway is to limit proliferation in imaginal discs and its absence leads to tumorous overgrowth. In vertebrates, many homologs of key pathway members are tumor suppressors indicating that this function is conserved (Richter, 2011).

L(3)mbt contains three MBT domains which bind mono- or dimethylated histone tails (Trojer, 2007; Grimm, 2009). Biochemical experiments in vertebrates have suggested a role in chromatin compaction (Trojer, 2007) but whether this role is conserved is not known. Results published while this paper was under review have shown that germline genes are upregulated in l(3)mbt mutant brains and are necessary for tumor formation (Janic, 2010). The current data indicate that L(3)mbt is bound to insulator sequences, which affect promoter-enhancer interactions and influence transcription. In Drosophila, the proteins CTCF, CP190, BEAF-32, Su(Hw), Mod(mdg4) and GAF are found at insulator sequences but how these factors act is largely unknown (Richter, 2011).

The data presented in this study show that tumor formation in l(3)mbt mutants is initiated by the uncontrolled overproliferation of neuroepithelial cells in the optic lobes due to the upregulation of proliferation control genes normally repressed by the SWH-pathway. L(3)mbt is located at DNA sequences bound by chromatin insulators and we propose that the function of L(3)mbt as a chromatin insulator is essential for repressing SWH target genes and preventing brain tumor formation (Richter, 2011).

brat, lgl and dlg were previously identified as Drosophila brain tumor suppressors. In all cases, defects in asymmetric cell division cause a huge expansion of the neuroblast pool. In l(3)mbt mutants, however, the neuroblast pool is expanded because an upregulation of SWH target genes results in a massive expansion of neuroepithelial tissue. Why those neuroblasts proliferate indefinitely upon transplantation is currently not understood for any of those mutants (Richter, 2011).

While the SWH-pathway is essential for tumorigenesis in l(3)mbt mutants, its overactivation can not recapitulate the neuroblast tumor phenotype seen in l(3)mbt mutants (this study and Reddy, 2010). Similar to the multifactorial origin of mammalian tumors, therefore, the combined deregulation of several signaling pathways could be required. The Notch pathway could be involved as it regulates the formation of OL neuroblasts from neuroepithelia and Notch pathway gene insulator sequences are bound by L(3)mbt. Increased activity of the Jak/STAT pathway, a major regulator of OL development, was also observed. Finally, the deregulation of germline genes in l(3)mbt mutants that has been described while this manuscript was under review (Janic, 2010) could provide another exciting explanation (Richter, 2011).

The results indicate that L(3)mbt acts on insulator elements, which isolate promoters from the activity of nearby enhancers acting on other genes. the analysis showed that L(3)mbt binding sites overlap with CP190, CTCF and BEAF-32, placing the protein into what has been called the class I of chromatin insulators (Negre, 2010; Richter, 2011 and references therein).

The identification of a DNA consensus motif for a histone binding protein like L(3)mbt is highly unexpected as insulators are typically nucleosome free. Currently, the activity of these important transcriptional regulators could be explained in several ways. Either, they form physical barriers blocking the interaction between enhancers and promoters. Alternatively, they mimic promoters and compete with endogenous promoters for enhancer interaction. Finally, they could interact with each other or nuclear structures to form loop domains that regulate transcriptional activity. The data suggests another model in which insulators interact with histones on nearby nucleosomes and influence the structure of higher order chromatin. Importantly, in the regions flanking CTCF binding sites nucleosomes are enriched for histones that are mono- and di-methylated on H3K4 or mono-methylated on H3K9 or H4K20, the variants to which MBT domains can bind in vitro (Bonasio, 2010; Grimm, 2009; Klymenko, 2006). As the human L(3)mbt homolog L3MBTL1 was shown to compact nucleosome arrays in vitro (Trojer, 2007), a model becomes feasible in which simultaneous binding to insulators and the surrounding nucleosomes reduces flexibility and thereby restricts the ability of nearby enhancers to interact with promoters on the other side of the insulator. However, the data could equally well be worked into the other prevalent models for insulator activity. Since L(3)mbt is currently the only chromatin insulator besides CTCF that is conserved in vertebrates, analysis of its homologs will certainly allow to distinguish between those possibilities (Richter, 2011).

OL development resembles vertebrate neurogenesis. Both processes consist of an initial epithelial expansion phase followed by neurogenesis through a series of asymmetric divisions. Together with previous findings, these data demonstrate that l(3)mbt and the SWH-pathway are crucial regulators of the initial neuroepithelial proliferation phase. Interestingly, the SWH-pathway has been implicated in regulating neural progenitors in the chicken embryo and it will be exciting to test the role of mammalian L(3)mbt in this process. It is remarkable that YAP is upregulated and L3MBTL3 is deleted in a subset of human medulloblastomas. Medulloblastoma is the leading cause of childhood cancer death and investigating the role of the SWH-pathway might contribute to the progress in fighting this disastrous disease (Richter, 2011).

LINT, a novel dL(3)mbt-containing complex, represses malignant brain tumour signature genes

Mutations in the l(3)mbt tumour suppressor result in overproliferation of Drosophila larval brains. Recently, the derepression of different gene classes in l(3)mbt mutants was shown to be causal for transformation. However, the molecular mechanisms of dL(3)mbt-mediated gene repression are not understood. This study identified LINT, the major dL(3)mbt complex of Drosophila. LINT has three core subunits -- dL(3)mbt, dCoREST, and l(3)mbt interacting protein 1 (dLint-1) -- and is expressed in cell lines, embryos, and larval brain. Using genome-wide ChIP-Seq analysis, it was shown that dLint-1 binds close to the TSS of tumour-relevant target genes. Depletion of the LINT core subunits results in derepression of these genes. By contrast, histone deacetylase, histone methylase, and histone demethylase activities are not required to maintain repression. These results support a direct role of LINT in the repression of brain tumour-relevant target genes by restricting promoter access (Meier, 2012).

LINT subunit composition differs from the human L3MBTL1 complex which contains pRb, HP1γ, H1b and core histones. dLint-1 has no apparent homolog in mammals. The mammalian homologs of dCoREST exist in complexes containing LSD1 and HDAC1/2. dLsd1 and dRpd3 are not stably associated with LINT. Nevertheless, the LINT subunit dLint-1 associates with dCoREST, dLsd1 and dRpd3 arguing for the existence of complexes in Drosophila that are related to mammalian CoREST/LSD1 complexes. Two observations are consistent with the view that these complexes might associate with chromatin and occupy sites that are not bound by LINT. First, dLint-1 is associated with approximately 50 bands on polytene chromosomes that show no dL(3)mbt binding. Second, ChIP-Seq analysis has revealed 2,902 dL(3)mbt binding sites but more than 8,000 dLint-1 binding sites. The functional relationship between these different dLint-1-containing complexes is unclear (Meier, 2012).

Comparison of genomewide binding profiles of dL(3)mbt in larval brain and dLint-1 in S2 and Kc cells strongly argues that LINT subunits bind to a large set of common binding sites. In particular, MBTS germline-related genes are bound and often repressed by the three LINT subunits. The finding that LINT exists in larval brain strongly implies that it is the LINT complex that is inactivated in l(3)mbt^ts mutants. In addition to malignant brain tumour signature (MBTS) genes, genes targeted by the Salvador-Warts-Hippo (SWH) pathway have recently been shown to be deregulated in l(3)mbt^ts brains (Richter 2011). Although binding of dLint-1 to about half of the SWH targets was detected, changes in SWH target gene expression following depletion of dL(3)mbt or dLint-1 has not been detected in Kc cells. It is possible that protein depletion was not sufficient to derepress these genes under the conditions used. Also, SWH target genes might be regulated differently in larval brain compared to cell lines (Meier, 2012).

The results suggest that maintenance of MBTS germline gene repression by LINT is largely independent of repressive histone modifying activities. Depletion of the dLint-1-associated histone demethylase dLsd1 and dRpd3 enzymes does not lead to derepression of LINT targets. An increase of the active H3K4me2 mark was detected at derepressed LINT target genes but this is most likely a result of active transcription rather than a direct consequence of the loss of LINT associated chromatin modifying activities. In agreement with this view, depletion of dLsd1 does not result in changes of H3K4me2 levels at LINT target genes. Microarray analysis also did not detect significant changes in the expression of genes recently shown to be repressed by dLsd1 in S2 cells and developing flies. This suggests that LINT and dLsd1 target different sets of genes (Meier, 2012).

Chromatin association and the repressive potential of human L3MBTL1 is enhanced by PR-SET7 and H4K20 monomethylation (Trojer, 2007; Kalakonda, 2008). Depletion of dPR-Set7, the sole Drosophila enzyme responsible for H4K20 monomethylation, did not result in derepression of LINT targets. Also no significant levels of H4K20me1 was detected at promoters of LINT target genes. This strongly suggests that even though dL(3)mbt can bind H4K20me1 in vitro this interaction does not play an important role in LINT complex targeting and repression (Meier, 2012).

dL(3)mbt does also bind to H4K20me2 in vitro. Indeed, H4K20me2 is present at LINT-regulated genes. However, H4K20me2 levels are are not elevated at LINT target gene promoters compared to control regions. This finding was not surprising given that 85%-90% of all histone H4 molecules are dimethylated at K20 and, therefore, H4K20me2 levels might be expected to be uniformely high along the chromosome. This makes it unlikely that an interaction between the MBT domains and H4K20me2 specifically directs the LINT complex to its target genes. However, it remains possible that after recruitment of LINT by other means, an interaction between dL(3)mbt and H4K20me2 contributes to transcriptional repression (Meier, 2012).

Depletion of other enzymes setting repressive histone marks such as H3K9me3 and H3K27me3 has likewise no effect on LINT-mediated repression. Although it was not possible to test all histone modifying enzymes for their roles in LINT target gene repression, the results argue for a largely histone modification independent mode of repression. LINT subunits bind predominantly near TSSs suggesting that LINT might inhibit transcription by restricting the access of RNA polymerase II or transcription factors to promoters. In support of this model, recruitment of LINT subunits to the promoter of a reporter gene is sufficient for repression even under conditions where the levels of repressive histone modification enzymes are reduced. Two modes of promoter access restriction by LINT can be envisioned that are not mutually exclusive. First, LINT might bind to the promoter segments required for RNA polymerase II recruitment. Second, as has been suggested for human L3MBTL1, LINT might locally compact nucleosomes. Two of the findings are inconsistent with the latter hypothesis. Nucleosome compaction by L3MBTL1 is dependent on the presence of the H4K20me1 modification. However, as discussed above, ablation of this modification does not result in derepression of LINT target genes. In addition, as a consequence of nucleosome compaction at LINT bound promoters one might expect a local increase in nucleosome density. However, histone H3 ChIP experiments have shown that the promoters of LINT target genes are generally depleted of nucleosomes. While these findings do not rule out a local nucleosome compaction that is, once established, independent of H4K20 monomethylation and undetectable by H3 ChIP, we favour the simpler hypothesis that LINT association with promoter sequences prevents transcription factors and RNA polymerase II from promoter binding (Meier, 2012).

The dL(3)mbt and dCoREST subunits of LINT are well conserved. Similar to the derepression of germline-related genes in l(3)mbt^ts tumours, misexpression of testis-specific genes (so-called cancer testis antigens) have been described in many human tumours. Based on this study, it is conceivable that L3MBTL1 or CoREST play a role in the repression of cancer testis antigens (Meier, 2012).

The histone H4 lysine 20 monomethyl mark, set by PR-Set7 and stabilized by L(3)mbt, is necessary for proper interphase chromatin organization

Drosophila PR-Set7 or SET8 is a histone methyltransferase that specifically monomethylates histone H4 lysine 20 (H4K20). L(3)MBT has been identified as a reader of methylated H4K20. It contains several conserved domains including three MBT repeats binding mono- and dimethylated H4K20 peptides. Depletion of PR-Set7 was found to block de novo H4K20me1 resulting in the immediate activation of the DNA damage checkpoint, an increase in the size of interphase nuclei, and drastic reduction of cell viability. L(3)mbt, in contrast, stabilizes the monomethyl mark, as L(3)mbt-depleted S2 cells show a reduction of more than 60% of bulk monomethylated H4K20 (H4K20me1) while viability is barely affected. Ploidy and basic chromatin structure show only small changes in PR-Set7-depleted cells, but higher order interphase chromatin organization is significantly affected presumably resulting in the activation of the DNA damage checkpoint. In the absence of any other known functions of PR-Set7, the setting of the de novo monomethyl mark appears essential for cell viability in the presence or absence of the DNA damage checkpoint, but once newly assembled chromatin is established the monomethyl mark, protected by L(3)mbt, is dispensable (Sakaguchi, 2012).

In these studies it was established that PR-Set7 sets the H4K20 monomethyl mark in vivo and that at least in S2 cells K20 is the only amino acid that is methylated. It was further found that depleting PR-Set7 in Drosophila S2 cells leads to the activation of the DNA damage checkpoint and within about 10 days to cell death. When the DNA damage checkpoint is abrogated by double knock-down of PR-Set7 and the checkpoint genes mei-41 or grp, the half/life of the cells is increased by 1 to two days, but ultimately the cells still die, suggesting that whatever is perturbed in the absence of PR-Set7 cannot be repaired. No double strand breaks were observed when staining for anti-phosphorylated histone H2A. This does not agree with results observed in vertebrate cells and may be because, in Drosophila, H2Av is not phosphorylated in the absence of H4K20me1 or the specific epitope is obscured. Alternatively, double strand breaks may not exist and the checkpoint is activated because of abnormal chromatin organization or because protein complexes are not removed in a timely manner as observed in Saccharomyces cerevisiae (Sakaguchi, 2012).

In this context it is interesting to note that in vertebrates H4K20 me2 is implicated in double strand break repair. Because H4K20me1 is the likely substrate for Suv4-20H1 and H2, the di- and trimethyltransferases, an additional link between H4K20 methylation and double strand breaks seems to exist. However, besides potentially setting the monomethyl mark at double strand breaks, PR-Set7 would have to have additional functions, because in both flies and vertebrates PR-Set7 mutants have a substantially stronger phenotype than the loss of the Suv4-20 enzymes (Sakaguchi, 2012).

The increase in nuclear volume, together with the changes in the number of FISH signals per nucleus observed in interphase cells following PR-Set7 RNAi would be consistent with a role for PR-Set7 in chromosome compaction and higher-order chromatin organization. Interestingly, mass spectrometry experiments show that the H4K20 monomethyl mark is set at the G2/M transition well after newly synthesized histone H4 is incorporated into chromatin in S phase. These findings suggest that the abnormalities in chromosome compaction and organization evident in interphase nuclei might be due to defects arising during the G2/M transition. Consistent with this possibility, cells depleted for only Pr-Set7 appear to arrest mostly in early mitosis. But unlike what is observed in larval brains, there is also a subset of cells that arrest in S phase; these may represent cells that despite the abnormalities in higher order chromatin organization are able to continue through the cell cycle until a checkpoint is activated during S. The discrepancy between the brain and tissue culture cells may be a reflection of differences in their cell cycle and developmental potential (Sakaguchi, 2012).

Results from several laboratories suggest that PR-Set7 function is coupled to DNA replication based on its targeting to the dividing fork via its interaction with PCNA. The current findings indicate that while abnormalities in chromatin organization and compaction appear to accumulate after growth without Pr-Set7 activity, these defects are inconsistent with massive disruptions in de novo nucleosome assembly during replication. Instead, the DNA damage checkpoint activation must arise from more subtle abnormalities in chromatin or DNA structure (Sakaguchi, 2012).

As for the l(3)mbt, its functional requirement does not appear to overlap with that of PR-Set7, neither in tissue culture as shown in this study, nor in flies. In larvae the loss of l(2)mbt results in an expansion of the neuroblast pool and subsequent tumorous overgrowth of the optic lobe while in PR-Set7 mutants the cell cycle of neuroblasts arrests in early mitosis resulting in fewer cells. PR-Set7 is essential for de novo methylation of H4K20. While the loss of H4K20me1 could occur either because in the absence of L(3)mbt protection the H4K20me1 is lost, or it could be a secondary effect. Consistent with the latter explanation, recent results show that L(3)mbt binds to DNA boundary elements and affects the level of transcription of Salvador-Wart-Hippo pathway genes both positively and negatively. That L(3)mbt possibly controls expression of many genes is also supported by the observation that the transcription level of all genes tested was reduced compared to wild type (Sakaguchi, 2012).

Ectopic expression of germline genes drives malignant brain tumor growth in Drosophila

Model organisms such as the fruit fly Drosophila melanogaster can help to elucidate the molecular basis of complex diseases such as cancer. Mutations in the Drosophila gene lethal (3) malignant brain tumor cause malignant growth in the larval brain. This study shows that l(3)mbt tumors exhibited a soma-to-germline transformation through the ectopic expression of genes normally required for germline stemness, fitness, or longevity. Orthologs of some of these genes were also expressed in human somatic tumors. In addition, inactivation of any of the germline genes nanos, vasa, piwi, or aubergine suppressed l(3)mbt malignant growth. These results demonstrate that germline traits are necessary for tumor growth in this Drosophila model and suggest that inactivation of germline genes might have tumor-suppressing effects in other species (Janic, 2010).

The Drosophila tumor-suppressor gene l(3)mbt was identified as a temperature-sensitive mutation that caused malignant growth in the larval brain (Gateff, 1993). Other l(3)mbt mutant alleles obtained later show the same temperature-sensitive phenotype (Yohn, 2003). L(3)mbt's closest homologs, Drosophila Scm (Sex comb on midleg) and Sfmbt (Scm-related gene containing four mbt domains), encode Polycomb Group proteins (Bonasio, 2010). L3MBTL1, the human homolog of Drosophila L(3)MBT (Bonasio, 2010), is a transcriptional repressor (Boccuni, 2003) that is found in a complex with core histones, heterochromatin protein 1γ (HP1γ), and RB (Retinoblastoma protein) and can compact nucleosomes (Trojer, 2007). Drosophila L(3)MBT is a substoichiometric component of the dREAM-MMB complex, which includes the two Drosophila Retinoblastoma-family proteins and the Myb-MuvB (MMB) complex (Lewis, 2004). Depletion of components of the dREAM/MMB complex in Drosophila Kc cells by RNA interference results in genome-wide changes in gene expression. These data strongly suggest that l(3)mbt function might contribute to establishing and maintaining certain differentiated states through the stable silencing of specific genes (Janic, 2010).

To identify the genes whose misexpression might account for the growth of l(3)mbt tumors (henceforth referred to as mbt tumors), genome-wide gene expression profiling was carried out of l(3)mbt^E2 and l(3)mbt^ts1 homozygous and transheterozygous larval brains raised at restrictive temperature (29°C). l(3)mbt^ts1 tumors were also analyzed at the 1st, 5th, and 10th rounds of allograft culture in adult flies (T1, T5, and T10, respectively). Brains from homozygous white¹¹¹⁸ (w¹¹¹⁸), l(3)mbt^E2, or l(3)mbt^ts1 larvae raised at permissive temperature (17°C) were used as controls. For comparison, larval brain malignant neoplasms caused by mutation in brain tumor (brat) as well as allograft cultures at T1,T5, and T10 of tumors caused by mutants in brat, lethal giant larvae (lgl), miranda (mira), prospero (pros), and partner of inscuteable (pins), were also profiled (Janic, 2010).

Hierarchical clustering plots of these data reveal three distinct clusters that include control larval brains, mbt larval brain tumors, and cultured l(3)mbt^ts1 tumors, respectively. From these data, 151 genes were identified that were either overexpressed or underexpressed in all three larval mbt tumor types compared to all three controls. From this list, those genes were removed that were also up- or down-regulated) in larval brat neoplasms and, hence, likely to encode functions generally required for larval brain tumor growth. The expression levels of the remaining 102 up-regulated genes are referred to as as the mbt signature (MBTS). MBTS is notably enhanced in cultured mbt tumors and can be used unequivocally to distinguish mbt tumors from other cultured malignant brain neoplasms like lgl, mira, pros, pins, or brat. Individual MBTS genes, however, are also up-regulated in some of these tumors (Janic, 2010).

The function of most MBTS genes remains unknown. However, a quarter of them (26 of 102) are genes required in the germ line. For instance, nanos (nos), female sterile(1)Yb (fs(1)Yb), and zero population growth (zpg) function in the establishment of the pole plasm in the egg and cystoblasts differentiation. The gonad-specific thioredoxins ThioredoxinT (TrxT) and deadhead (dhd), giant nuclei (gnu), corona (cona), hold'em (hdm), matotopetli (topi), and the female germline-specific γTUB37C isoform function during oocyte differentiation, meiosis, and syncytial embryo development. Also piwi, aubergine (aub), krimper (krimp), and tejas (tej) are involved in the biogenesis of Piwi-interacting RNAs (piRNAs) that protect germline cells against transposable elements and viruses. Some of these genes also have functions that are not germline related. For instance, some piwi alleles display synthetic lethality), and nos is required during nervous system development (Janic, 2010).

Driven by the high percentage of MBTS genes that have germline functions, other germline-related genes were sought that do not meet the stringent criteria applied to select the 102 MBTS genes, but are overexpressed in mbt tumors. Among these, the genes were found that encode the synaptonemal complex protein Crossover suppressor on 3 of Gowen [C(3)G] and the cell cycle kinase Pan gu (PNG), which interact with the proteins encoded by the MBTS genes cona and gnu, respectively. The same applies to Squash (SQU), Spindle-E (SPN-E), Maelstrom (MAEL), and AGO3, components of the piRNA machinery, which colocalize with other MBTS proteins in nuage (Janic, 2010).

To determine whether the mRNAs found ectopically expressed in mbt tumors are translated, protein expression was examined with a selected number of currently available antibodies. Given the key role of VASA in the assembly of the pole plasm and germline development, it was included in this study, even though vasa mRNA levels are not significantly increased in mbt tumors. By Western blot, it was confirmed that PIWI, AUB, and VASA are ectopically expressed in mbt tumors. Immunofluorescence studies also revealed the ectopic expression in l(3)mbt^ts1 brains raised at 29°C of C(3)G, SQU, and VASA. These results show that some of the germline genes ectopically expressed in mbt tumors are translated. However, it has not been possible to confirm the expression of other proteins, including MAEL, ORB, BAM, GNU, and TOPI, which suggests that, possible technical problems aside, either the corresponding mRNAs are not translated or these proteins might be unstable in such an ectopic environment. The expression of VASA, by contrast, suggests that other mRNAs whose levels are not appreciably increased in mbt tumors might actually be ectopically translated (Janic, 2010).

Prompted by the expression in l(3)mbt^ts1 brains of several genes involved in the biogenesis and regulation of piRNAs, 23- to 30-nucleotide RNAs were sequenced from l(3)mbt^ts1 larval brain tumors and from wild-type brains and ovaries. 117 known piRNAs and microRNAs (miRNAs) were detected in l(3)mbt^ts1 larval brain tumor samples. Of these, 31 are either not expressed in wild-type brains or are expressed there at less than 10% their level in larval brain tumors. Most of them are highly expressed in wild-type ovaries, thus substantiating further the ectopic acquisition of germline traits that characterizes mbt tumors (Janic, 2010).

It is not known which, if any, of the germline genes that are up-regulated in mbt tumors are direct targets of l(3)mbt or if their ectopic expression is a downstream consequence of intermediate events. The putative direct targets of l(3)mbt are many. The dREAM-MMB complex, of which L(3)MBT is a substoichiometric component, has been found to be promoter-proximal to 32% of Drosophila genes, and MMB factors are known to regulate transcription of a wide range of genes in Drosophila Kc cells (Georlette, 2007). In addition, there is no estimate for the number of proteins like VASA that, despite their low mRNA expression levels, might be up-regulated in mbt tumors. Indeed, many of these genes, as well as the piRNAs and miRNAs expressed in mbt tumors, might themselves regulate the basal transcription and translation machineries, adding a further layer of gene expression modulation (Janic, 2010).

The extent to which ectopic expression of germline genes contributes to mbt tumor growth was determined. To this end, larval brain growth was quantified in individuals that were mutant for l(3)mbt^ts1 alone, or double mutant for l(3)mbt^ts1 and one of several of the germline genes that are ectopically expressed in mbt tumors. Measured as the total amount of protein, the average brain size in l(3)mbt^ts1 is about seven times as large as that in control w¹¹¹⁸ larvae, a difference that is not significantly reduced by the additional loss of zpg, Pxt, or AGO3. However, brain overgrowth is reduced to a size similar to that of the control in l(3)mbt^ts1 larvae that are also mutant for either piwi, vasa, aub, or nos. The loss of piwi does not prevent brain overgrowth in brat ^k06028 mutant larvae. Then tumor growth was quantified after allograft in adult flies. The frequency with which l(3)mbt^ts1 homozygous larval brain tissue develops tumors in this assay is not significantly reduced by the additional loss of zpg or AGO3 and is only moderately reduced by the loss of Pxt, but it is markedly reduced by the additional loss of piwi, vasa, aub), or nos. The frequency of brat ^k06028 tumor formation is not affected by the loss of piwi or nos. These results demonstrate that the ectopic expression of germline genes, particularly piwi, vasa, nos, and aub, significantly contributes to mbt tumor growth (Janic, 2010).

A closely reminiscent soma-to-germline transformation observed in mutants in the Caenorhabditis elegans Rb homolog LIN-35, as well as in long-lived C. elegans strains, has led some to propose that the acquisition of germline characteristics by somatic cells might contribute to increased fitness and survival, a mechanism that could contribute to the transformation of mammalian cells. Also in humans, some genes that are predominantly expressed in germline cells and have little or no expression in somatic adult tissues become aberrantly activated in various malignancies, including melanoma and several types of carcinomas. These are known as cancer-testis (CT) genes or cancer-germline (CG) genes. A subset of these CG genes encode antigens that are immunogenic in cancer patients and are being pursued as biomarkers and as targets for therapeutic cancer vaccines (Janic, 2010 and references therein).

Human CG genes are suspected to contribute to oncogenesis germline traits like immortality, invasiveness, and hypomethylation, but their actual role in cancer remains unknown. The current results demonstrate that ectopic germline traits are necessary for tumor growth in Drosophila mbt tumors, suggesting that their inactivation might have tumor-suppressing effects in other species. Some germline genes up-regulated in mbt tumors are orthologs of human CG genes like PIWIL1/piwi, NANOS1/nanos, and SYCP1 /c(3)G. The list of genes up-regulated in mbt tumors includes many other germline genes that might also be relevant in human cancer (Janic, 2010).

3MBTL1, a histone-methylation-dependent chromatin lock

Distinct histone lysine methylation marks are involved in transcriptional repression linked to the formation and maintenance of facultative heterochromatin, although the underlying mechanisms remain unclear. This study demonstrated that the mammalian malignant-brain-tumor (MBT) protein L3MBTL1 is in a complex with core histones, histone H1b, HP1gamma, and Rb. The MBT domain is structurally related to protein domains that directly bind methylated histone residues. Consistent with this, it was found that the L3MBTL1 MBT domains compact nucleosomal arrays dependent on mono- and dimethylation of histone H4 lysine 20 and of histone H1b lysine 26. The MBT domains bind at least two nucleosomes simultaneously, linking repression of transcription to recognition of different histone marks by L3MBTL1. Consistently, L3MBTL1 was found to negatively regulate the expression of a subset of genes regulated by E2F, a factor that interacts with Rb (Trojer, 2007).

The transcriptional repressor L3MBTL1 compacts chromatin in a manner that is strictly dependent on histone methylation marks -- specifically H4K20me1/2 and H1K26me1/2. The apparent exclusivity of L3MBTL1 for mono- and dimethylated states supports a model in which different degrees of methylation at a particular site can give rise to different readouts. The chromodomains found in several proteins provide a paradigm for this model, given their preference for di- and trimethylated lysines as compared to the monomethyl state (Trojer, 2007).

The binding specificities of L3MBTL1 raise an important question. How do the MBT repeats bind specifically to two different methylated lysine residues (H4K20me1/2 and H1K26me1/2)? Even more intriguing is that the MBT domains bind H1K26me1/2 but not H3K9me2/3 or H3K27me1/2/3, yet these lysine residues are located within a conserved consensus sequence (ARKS). The simplest explanation would be that each one of the three MBT domains binds a different ligand. However, the data suggest otherwise. The second MBT domain is important for H1K26me1/2 as well as for H4K20me1/2 binding, as pre binding with H1K26me2 peptides abolished 3MBT binding to H4K20me1. Moreover, since a mutant was identified in the second MBT domain that abolishes binding to both ligands and a second mutant that selectively abolishes H1K26me, but not H4K20me, binding, we suggest that both methylated residues are bound via the second MBT domain but that different aromatic residues are involved in caging the methylated lysine residue. Caging through aromatic residues is a property of all proteins that specifically recognize methylated lysine residues (Trojer, 2007).

With respect to chromatin compaction, two different scenarios are invisioned, taking into account that the P2 domain of L3MBTL1 can bind H4K20me1 or H1K26me1/2 but apparently not both modifications simultaneously. Importantly, monomeric 3MBT can still compact chromatin in the assay conditions, suggesting that the P2 domain can accommodate two modified histone marks on two nucleosomes. However, given that pre binding with H1K26me2 peptides abolished 3MBT binding to H4K20me1, the two marks accommodated by the monomer must be identical. Thus, in the case of the dimeric L3MBTL1, each of the monomers would bind two identical marks such that four H4K20me1, four H1K26me1/2, or two of each mark are bound (Trojer, 2007).

In the 'bridging model' L3MBTL1 functions either as a monomer or a dimer, and adjacent nucleosomes or chromatosomes are bound simultaneously, thereby bridging the linker DNA and moving the nucleosomes closer together. L3MBTL1 does exist as a homodimer in vivo (Boccuni, 2003), and dimerization is one mechanism by which two L3MBTL1 molecules bind to H4K20me1 and H1K26me1/2 on adjacent nucleosomes/chromatosomes. This is also supported by looping experiments. Yet, repression does not depend on the SPM domain responsible for L3MBTL1 homodimerization, and a monomeric 3MBT molecule lacking the SPM domain still shows compaction in the assays and can repress transcription when directed to a reporter (Boccuni, 2003). Nonetheless, chromatin compaction by a single 3MBT molecule is still consistent with the bridging model. A similar mechanism for binding and compacting of multiple nucleosomes by a single molecule was reported recently in the case of the PRC1 component PSC, but the histone tails or histone lysine methylation marks were not required. In this case, the compacted particles resembled the chromatin structures that were observed in the presence of L3MBTL1 (Trojer, 2007).

In the 'association model' a single L3MBTL1 molecule (monomer or dimer) could compact chromatin by positioning itself on the surface of the nucleosome/chromatosome in a fashion that promotes bending of the linker DNA or facilitates histone-histone interactions (see 'Alternate Models for L3MBTL1 Compaction of Nucleosomal Arrays,' Figure 6 in Trojer, 2007). In this model, correct positioning of L3MBTL1 is accomplished by the specific recognition of H4K20me1 or H1K26me1/2 on the nucleosome or chromatosome surface, respectively. The linker histone itself functions in a similar manner. Moreover, proteins containing HMG boxes, the transcription factor HNF3, the myeloid and erythroid nuclear termination stage-specific protein (MENT), or PARP-1 can also bind to nucleosomes and alter chromatin conformation. In the case of L3MBTL1, compaction is dependent on specific methylated lysine residues adding a distinct regulatory parameter (Trojer, 2007).

Linker histone H1 functions as a transcriptional repressor in vitro and is important in chromatin folding in vitro. The C-terminal region of H1 is required for its binding to DNA between nucleosomes, and H1 phosphorylation changes its ability to bind to chromatin. This study shows that L3MBTL1 interacts with H1 in a methylation-dependent manner and that H1bK26me1/2 is important for chromatin compaction by the MBT domains. H1 has been detected on both transcriptionally active and inactive genes, and its binding to chromatin is dynamic in viv, yet the number of factors affecting H1 mobility is unknown. It is possible that methylation of histone H1 at lysine-26 in the presence of L3MBTL1 increases H1 residence time on chromatin, thereby facilitating a compacted chromatin state (Trojer, 2007).

The specificity of the proteins involved in establishing a type of facultative heterochromatin is likely dictated by the interaction of regulators (E2F) with other regulators (Rb) and factors that function in compacting chromatin. L3MBTL1 is a member of a large family of mammalian MBT proteins that contain variable numbers of MBT domains. These domains are not identical in sequence, as is also the case with the chromo- and bromo-domains. Given this, the different members of the MBT family might recognize different patterns of histone methyl marks to establish repression of specific genes through the formation of facultative heterochromatin (Trojer, 2007).

One of the L3MBTL1 targets shown in this study is c-myc, the expression of which is tightly regulated, with increased myc expression often correlated with cancer. Reduction of L3MBTL1 levels significantly increases MYC protein levels. Ectopic expression of L3MBTL1, however, does not affect MYC protein levels. This is perhaps not surprising given that the in vitro data show a specific requirement for histone methylation marks in binding; thus, increased expression of L3MBTL1 would not necessarily lead to its increased chromatin binding. It remains to be investigated if overexpression of myc in cancer correlates with aberrant L3mbtl1 gene expression and/or localization. The human L3MBTL1 gene is located on chromosome 20q within the region commonly deleted in patients with myeloproliferative disorders. It is also possible that the chromatin signature (e.g., histone methylation marks) of the c-myc promoter region is abnormal in malignant cells, thereby altering L3MBTL1 binding and the regulated expression of c-myc (Trojer, 2007).

Identification of a Drosophila Myb-E2F2/RBF transcriptional repressor complex

The Drosophila Myb complex has roles in both activating and repressing developmentally regulated DNA replication. To further understand biochemically the functions of the Myb complex, Drosophila embryo extracts were fractionated relying upon affinity chromatography. it was found that E2F2, DP, RBF1, RBF2, and the Drosophila homolog of LIN-52, a class B synthetic multivulva (synMuv) protein, copurify with the Myb complex components to form the Myb-MuvB complex. In addition, it was found that the transcriptional repressor protein, Lethal (3) malignant brain tumor protein, L(3)MBT, and the histone deacetylase, Rpd3, associate with the Myb-MuvB complex. Members of the Myb-MuvB complex localize to promoters and corepress transcription of developmentally regulated genes. These and other data now link together the Myb and E2F2 complexes in higher-order assembly to specific chromosomal sites for the regulation of transcription (Lewis, 2004).

The Drosophila melanogaster tumor suppressor gene lethal(3)malignant brain tumor encodes a proline-rich protein with a novel zinc finger

The lethal(3)malignant brain tumor [l(3)mbt] gene causes, when mutated, malignant growth of the adult optic neuroblasts and ganglion mother cells in the larval brain and imaginal disc overgrowth. Via overlapping deficiencies a genomic region of approximately 6.0 kb was identified, containing l(3)mbt⁺ gene sequences. The l(3)mbt⁺ gene encodes seven transcripts of 5.8 kb, 5.65 kb, 5.35 kb, 5.25 kb, 5.0 kb, 4.4 kb and 1.8 kb. The putative MBT163 protein, encompassing 1477 amino acids, is proline-rich and contains a novel zinc finger. In situ hybridizations of whole mount embryos and larval tissues revealed l(3)mbt⁺ RNA ubiquitously present in stage 1 embryos and throughout embryonic development in most tissues. In third instar larvae l(3)mbt⁺ RNA is detected in the adult optic anlagen and the imaginal discs, the tissues directly affected by l(3)mbt mutations, but also in tissues, showing normal development in the mutant, such as the gut, the goblet cells and the hematopoietic organs (Wismar, 1995).

A temperature-sensitive brain tumor suppressor mutation of Drosophila melanogaster: developmental studies and molecular localization of the gene

The recessive-lethal, temperature-sensitive (ts) mutation of the tumor suppressor gene lethal(3)malignant brain tumor (l(3)mbt) causes in a single step the malignant transformation of the adult optic neuroblasts and ganglion mother cells in the larval brain at the restrictive temperature of 29 degrees C. The transformed cells are differentiation-incompetent and grow autonomously in a lethal and invasive fashion in situ in the brain as well as after transplantation in vivo into wild-type adult hosts. The imaginal discs show epithelial overgrowth. At the permissive temperature of 22 degrees C development is completely normal. The ts-period of gene activity responsible for 100% brain tumor suppression and normal imaginal disc development encompasses the first six hours of embryonic development. The l(3)mbt gene function is, however, also required thereafter for the proper differentiation of the brain and the imaginal discs. The l(3)mbt gene is located cytologically in the salivary gland chromosome bands 97E8-F11, and in molecular terms in 29 kb of DNA detected via a P-element insertional deletion (Gateff, 1993).

Combinatorial control of recruitment of a variant PRC1.6 complex in embryonic stem cells

Though genetic data suggest that Polycomb group proteins (PcGs) are central chromatin modifiers and repressors that have been implicated in control of embryonic stem cell (ESC) pluripotency, the precise mechanism of PcG complex recruitment remains elusive, especially in mammals. This study reports that the first and second MBT repeats of L3mbtl2 (see Drosophila L(3)mbt) are important structural and functional features that are necessary and sufficient for L3mbtl2-mediated recruitment of PRC1.6 complex to target promoters. Interestingly, this region of L3mbtl2 harbors the evolutionarily conserved Pho-binding pocket also present in Drosophila Sfmbt, and mutation of the critical residues within this pocket completely abolishes its interaction with target promoters. Additionally, decreased PRC1.6 chromatin occupancy was observed following loss of individual components (L3mbtl2, Pcgf6, and Max) of the complex. These findings suggest that the recruitment of noncanonical PRC1.6 complex in ESCs might be the result of L3mbtl2's interaction with multiple components of the complex (Huang, 2018).