InteractiveFly: GeneBrief

little imaginal discs: Biological Overview | References

The Myc oncoprotein is a potent inducer of cell growth, cell cycle progression, and apoptosis. While many direct Myc target genes have been identified, the molecular determinants of Myc's transcriptional specificity remain elusive. A genetic screen was carried out in Drosophila and the Trithorax group protein Little imaginal discs (Lid) was identified as a regulator of dMyc-induced cell growth. Lid was originally identified in intergenic noncomplementation with a mutation in ash1, a trithorax group gene (Gildea, 2000; full text of article). Lid binds to dMyc and is required for dMyc-induced expression of the growth regulatory gene Nop60B. The mammalian Lid orthologs, Rbp-2 (JARID1A) and Plu-1 (JARID1B), also bind to c-Myc, indicating that Lid-Myc function is conserved. Lid is a JmjC-dependent trimethyl H3K4 demethylase in vivo, and this enzymatic activity is negatively regulated by dMyc, which binds to Lid's JmjC domain. Because Myc binding is associated with high levels of trimethylated H3K4, it is proposed that the Lid-dMyc complex facilitates Myc binding to, or maintenance of, this chromatin context (Secombe, 2007). Identication of Lid as a histone H3 trimethyl-Lys4 demethylase has also been reported by Lee (2007) and Eissenberg (2007).

Lid is a 1838-amino-acid protein possessing numerous conserved motifs including an ARID (A/T-rich interaction domain), implicated in binding A/T-rich DNA; a single C5HC2 zinc finger; three PHD fingers (plant homeobox domain), implicated in forming protein-protein interactions; and Jumonji N and C (JmjN and JmjC) domains. JmjC-containing proteins have recently been shown to act as histone demethylase enzymes in a Fe2+ and -ketoglutarate-dependent manner (Klose, 2006). To test whether Lid can demethylate histones in vivo, Lid was overexpressed in fat body and in wing disc cells and the levels of mono-, di-, and tri-methylated histone H3K4 and H3K27 were examined. Di- and tri-methylated histone H4K20 and trimethylated histone H3K9 and H3K36 were also examined. Overexpression of Lid specifically decreased the levels of the tri-methylated form of H3K4 but had no effect on the other methylated histones examined in either GFP-marked fat body or wing disc cells. Significantly, expression of Lid in the wing disc reduced trimethyl H3K4 in a dose-dependent manner, with two copies of the UAS-Lid transgene reducing trimethyl H3K4 more efficiently than one copy. Moreover, levels of trimethylated H3K4 are increased in wing discs from lid homozygous mutant animals, consistent with the model that Lid regulates the levels of this histone modification during normal development. To determine whether the JmjC domain of Lid is required for the observed H3K4 demethylation, transgenic flies were generated carrying a Gal4-inducible form of full-length Lid containing Ala substitutions at His637 and Glu639 (Lid-JmjC*) that abolishes the protein's ability to bind the Fe2+ cofactor required for demethylase activity. Similar mutations have been shown to disrupt the demethylase function of the JmjC domains of JHDM2A, JHDM3A, JHDM1, and JMJD2A. Unlike wild-type Lid, expression of full-length Lid-JmjC* did not decrease levels of trimethylated H3K4 in fat body or in wing disc cells, demonstrating that an active JmjC domain is required for Lid-mediated H3K4 demethylation. Interestingly, expression of Lid-JmjC* resulted in increased levels of trimethyl H3K4 in the fat body, perhaps due to a dominant interfering effect on wild-type Lid function in these cells. Taken together, these results demonstrate that Lid is a trimethyl H3K4 demethylase that modifies nucleosomal histone H3 in vivo. The global regulation of H3K4 trimethylation status by Lid is not, however, likely to be mediated by recruitment by dMyc, since no effect was observed of reduced or increased dMyc expression on trimethyl H3K4 levels in either fat body or wing disc cells (Secombe, 2007).

Forty other genomic regions were identified that enhanced or suppressed the GMR-Gal4, UAS-dMyc (GMM) phenotype when heterozygous. Two of these regions delete genes encoding known regulators of dMyc stability, such as ago, or are involved in Myc transactivation, such as Pcaf. Specific mutations in both of these genes have been shown to enhance or suppress the GMM rough eye phenotype, respectively. Interestingly, none of the known direct transcriptional targets of dMyc were identified as genetic modifiers of the GMM phenotype, suggesting that the GMM phenotype arises from modulation of multiple genes and provides a powerful tool to identify proteins directly required for dMyc function in vivo (Secombe, 2007).

TrxG proteins are renowned for their essential role in maintaining homeotic (hox) gene expression during development, with mutations in many TrxG genes resulting in lethality due to homeotic transformations. Six TrxG protein complexes have been identified to date. While one function of these complexes is to antagonize Polycomb group (PcG) repression to maintain active hox gene expression, TrxG proteins are also recruited to other developmentally important genes to either activate or repress their transcription in a context-dependent manner. Based on the suppression of the GMM phenotype, the physical interaction between Lid and dMyc, and the requirement of Lid for dMyc-dependent activation of Nop60B, it is predicted that Lid acts as a dMyc coactivator involved in cell growth. The interaction between endogenous Lid and dMyc proteins is also likely to be essential for normal larval development since reducing the gene dose of lid is lethal in combination with the dmyc hypomorphic allele dm^P0. In addition, genetically reducing lid enhances a small bristle phenotype induced by expression of a dMyc RNAi transgene. The original small discs phenotype described for lid mutants also suggests a role for Lid in the regulation of cell growth or proliferation during larval development. Unfortunately, this phenotype occurs at a frequency far too low (<1% of lid mutant larvae) to allow characterization (Secombe, 2007).

It is expected that the function of the Lid-Myc complex is evolutionarily conserved, since the human orthologs of Lid, Rbp-2 (JARID1A) and Plu-1 (JARID1B), bind strongly to c-Myc and dMyc in vitro, and both have been implicated in transcriptional regulation. Originally described as a binding partner for the tumor suppressor protein Retinoblastoma (RB), Rpb-2 has been shown to behave as a coactivator for RB at some promoters while antagonizing RB function at others (Benevolenskaya, 2005). Rbp-2 has also been identified as a transcriptional coactivator for nuclear hormone receptors (NRs) (Chan, 2001) and for the LIM domain transcription factor Rhombotin-2 (Mao, 1997). In addition, Plu-1 acts as a transcriptional corepressor for BF1 and PAX9 (Lu, 1999; Tan, 2003). While the transcriptional repression activities of Rbp-2 and Plu-1 are likely to be linked to a conserved trimethyl H3K4 demethylase activity, the molecular mechanism by which they activate transcription remains unclear. The mechanism by which Lid functions is currently being addressed by carrying out genetic screens using phenotypes generated by gain or loss of lid function (Secombe, 2007).

Coimmunoprecipitation analyses revealed that dMyc is likely to form multiple distinct complexes comprising TrxG proteins: One includes the Brm (SWI/SNF) nucleosome remodeling complex, and another contains Lid and Ash2. Consistent with the physical interaction observed between dMyc and Brm, components of the Brm complex suppress the GMM phenotype when genetically reduced, indicating that they are required for dMyc-induced cell growth. An interaction between Myc and the Brm complex has been observed in mammalian cells, where c-Myc interacts with the Brm (Brg1) subunit Ini1, and expression of a dominant-negative Brg1 allele inhibits c-Myc-dependent activation of a synthetic E-box reporter. However, the interaction between dMyc and the Brm complex described in this study, using Drosophila, provides the first demonstration of a biological significance for this complex (Secombe, 2007).

The second dMyc-TrxG complex identified includes Lid and Ash2, with Ash2 being immunoprecipitated with both anti-dMyc and anti-Lid antisera. In addition, decreased levels of Ash2 suppress, and increased levels of Ash2 levels enhance, the GMM phenotype, suggesting that Lid and Ash2 are limiting for dMyc-induced cell growth. In Schizosaccharomyces pombe, the orthologs of Ash2 and Lid (Ash2p and Lid2p) interact in vivo. While Ash2 has no known enzymatic activity, it is an integral component of several conserved complexes, including the SET1 histone methyltransferase complex (TAC1 in Drosophila; MLL in mammals) that is essential for methylation of histone H3K4. Biochemical purification of SET1, Lid2p, and Ash2p complexes from S. Pombe has demonstrated that the Lid2p-Ash2p complex is distinct from the SET1-Ash2 complex. Reducing the gene dose of the SET1 ortholog trx does not affect the GMM phenotype, consistent with the Drosophila Lid-Ash2-dMyc complex also being independent of TAC1 methyltransferase complex. The observation that Ash2 is a component of both H3K4 methylating (MLL) and demethylating (Lid) complexes is intriguing and suggests that it may be a crucial modulator of H3K4 methylation status. Whether Ash2 is required for Lid-mediated H3K4 demethylation is currently being tested (Secombe, 2007).

Lid is the first enzyme characterized that specifically demethylates trimethylated histone H3K4 in vivo. Based on the similarity between Lid and its mammalian orthologs Rbp-2 and Plu-1, it is expected that this demethylase activity to be conserved. The enzymatic activity of Lid requires a functional JmjC domain; however, Lid's specificity for a trimethylated lysine target is likely to be determined by the presence of a conserved N-terminal JmjN domain. Evidence to date suggests that proteins that possess both a JmjN and a JmjC domain prefer di- or trimethylated lysine substrates, whereas JmjC proteins that lack a JmjN domain demethylate mono- or dimethylated lysines. Indeed, analysis of the crystal structure of JMJD2A, which targets trimethylated H3K9 and K36, has revealed that the JmjN domain makes extensive contacts within the catalytic core of the JmjC domain, presumably accounting for the differences in target specificity between JmjC and JmjN/JmjC proteins (Secombe, 2007).

Trimethylated H3K4 is often found surrounding the transcriptional start site of active genes and is strongly correlated with binding by c-Myc. The trimethyl H3K4 demethylase activity of Lid would predict that Lid/Rbp-2 proteins may act as transcriptional repressors in a similar manner to LSD1, which demethylates mono- and di-methylated H3K4. Consistent with this hypothesis, it is observed that a large number of genes are derepressed in microarrays of homozygous lid mutant wing discs. However, expression of dMyc abrogates Lid's enzymatic activity, indicating that Lid is not acting as a demethylase when bound to dMyc. This is consistent with the observation that expression of Lid-JmjC* (a Gal4-inducible form of full-length Lid containing Ala substitutions at His637 and Glu639 that abolishes the protein's ability to bind the Fe2+ cofactor required for demethylase activity) enhances the GMM eye phenotype. Indeed, Lid behaves as a dMyc coactivator based on the requirement for Lid in dMyc-induced expression of the growth regulator Nop60B. Both activation and repression functions have been previously suggested for Rbp2. Interestingly, LSD1's demethylase activity is also negatively regulated by an associated protein, BHC80, in a similar manner to the inhibition of Lid's enzymatic activity by dMyc. Dynamic regulation of histone demethylase activity is therefore likely to be a common feature of regulated gene expression in vivo (Secombe, 2007).

Recently, analysis of c-Myc target gene promoters revealed a strong dependence on trimethylated H3K4 for E-box-dependent c-Myc binding. Based on this observation, it is tempting to speculate that although Lid is likely to be enzymatically inactive when complexed with dMyc, Lid may retain its ability to recognize trimethylated H3K4 (perhaps through its JmjN domain) and thereby facilitate appropriate E-box selection. The inhibition of Lid demethylase activity by dMyc may also result in maintenance of local H3K4 trimethylation to permit binding of additional dMyc molecules or other transcription factors. The maintenance of trimethylated H3K4 by dMyc may allow binding of the NURF chromatin remodeling complex that specifically recognizes trimethylated H3K4. NURF binding, through its large BPTF subunit, has been correlated with spatial control of Hox gene expression and is thought to link H3K4 methylation to ATP-dependent chromatin remodeling. Finally, considering the fact that Lid contains multiple domains potentially involved in DNA binding and protein interaction, it is likely that interaction of Lid/Rbp-2 with Myc in Drosophila and mammalian cells will promote association of other proteins with the Myc-Lid complex, allowing further diversification of Myc function (Secombe, 2007).

The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing

Epigenetic chromatin marks restrict the ability of differentiated cells to change gene expression programs in response to environmental cues and to transdifferentiate. Polycomb group (PcG) proteins mediate gene silencing and repress transdifferentiation in a manner dependent on histone H3 lysine 27 trimethylation (H3K27me3). However, macrophages migrated into inflamed tissues can transdifferentiate, but it is unknown whether inflammation alters PcG-dependent silencing. This study shows that the JmjC-domain protein Jmjd3 is a H3K27me demethylase expressed in macrophages in response to bacterial products and inflammatory cytokines. Jmjd3 binds PcG target genes and regulates their H3K27me3 levels and transcriptional activity. The discovery of an inducible enzyme that erases a histone mark controlling differentiation and cell identity provides a link between inflammation and reprogramming of the epigenome, which could be the basis for macrophage plasticity and might explain the differentiation abnormalities in chronic inflammation (De Santa, 2007).

Since maintenance of cell identity and differentiation depends on H3K27me3 and Polycomb-enforced gene silencing, the existence of enzymes that can erase the methyl mark on which cellular memory relies is counterintuitive at first sight. It is even more counterintuitive the idea that the expression of such an enzyme can be induced by stimuli as common as bacterial components and inflammatory cytokines. In this regard it is important to notice the different properties of Utx, which is a constitutively expressed H3K27me3 histone demethylase (HDM), and Jmjd3, which is endowed with a potent HDM activity but is expressed mainly in an inducible and cell-type restricted fashion. Moreover, Utx escapes X inactivation, and the requirement for a biallelic expression is suggested by the presence in males of a highly related gene (Uty) encoded by the Y chromosome. The properties of the two H3K4me3 HDMs Smcx and Smcy are remarkably similar to those of Utx and Uty: they are both encoded by sex chromosomes, and Smcx escapes X inactivation. Overall, it is likely that Utx/Uty (and Smcx/Smcy) act as 'housekeeping' HDMs whose constant availability allows for continuous surveillance over global and gene-specific H3 Lys 27 (or H3K4) methylation. Conversely, tissue-specific and inducible expression of Jmjd3 restricts its field of competence to specific and well-defined developmental stages or functional states, thus limiting the intrinsic dangerousness of the increased availability of a potent H3K27me3 demethylase (De Santa, 2007).

Considering the risks, what is the advantage of the potential increase in epigenomic plasticity afforded by the elevation in Jmjd3 levels? A limited level of plasticity may allow cells to reprogram their properties according to environmental cues, which is a well-known event in flies (Lee, 2005). Tissue regeneration that follows injury requires an efficient reconstitution of the damaged parts, and in some cases the ability of resident stem cells to reconstitute the tissue may be exceeded. When such a deficiency occurs, restoration of tissue integrity may require the recruitment of circulating cells whose plasticity enables them to transdifferentiate and generate a variety of tissue-specific cell types. However, plasticity of recruited cells may itself represent a risk, as it may result in the generation of abnormally differentiated cells and even tumorigenesis. A striking example of the progression through chronic inflammation > recruitment of circulating cells > transdifferentiation and tumor development, is provided by a mouse model of chronic gastritis caused by Helicobacter Felis, a relative of the major cause of gastric cancer in humans, H. Pilori. In this model, chronic inflammation associated with bacterial infection causes extensive apoptosis of the gastric epithelium, followed by the invasion of the stomach by circulating bone marrow-derived cells (BMDCs). BMDCs proliferate in the inflamed stomach and undergo a transdifferentiation-like event known as metaplasia: although metaplasia is supposed to be an entirely epigenetic event not associated with mutations, it leads to displasia and eventually to cancer (Houghton, 2004 ) (De Santa, 2007).

Macrophages are among the most abundant cells in an inflamed tissue, and they may help tissue repair also by transdifferentiation. Although several claims of macrophage transdifferentiation in the past could be explained by cell fusion, the case of lymphoendothelial transdifferentiation in inflamed tissues is supported by strong experimental evidence. In a mouse model of corneal transplantation macrophages infiltrating the transplanted cornea start expressing lymphoendothelial markers and the fate-determining transcription factor Prox1, which is essential for lymphatic vessel development. Later on, macrophage markers are lost and infiltrating cells retaining only lymphoendothelial markers generate a new network of lymphatic vessels. Similarly, neo-lymphoangiogenesis in transplanted and rejected kidneys in humans depends on recipient-derived myeloid cells. Interestingly, the Prox1 promoter in macrophages bears both high H3K4me3 and H3K27me3, and transcription is undetectable. However, it was not possible to observe H3K27me3 demethylation and Prox1 gene reactivation in the conditions used, which suggests the requirement for additional stimuli present in inflamed tissues. Lymphoendothelial trans-differentiation of macrophages during inflammation may help removal of tissue debris and enhance transport of microbial components to lymph nodes for presentation to lymphocytes. Therefore, it may not represent an accidental event but a controlled and desirable outcome (De Santa, 2007).

The direct targets of Jmjd3 include late HoxA cluster genes in differentiating bone marrow cells and Bmp-2 in activated macrophages. While regulation of Hox genes tentatively identifies Jmjd3 as a component of the MLL system (an assumption supported by the inclusion of Jmjd3 in MLL complexes), regulation of Bmp-2 provides interesting mechanistic and biological clues. In macrophages the Bmp-2 gene promoter contains a bivalent chromatin domain with high H3K4me3 and H3K27me3 levels. It has been proposed that in ES cells genes in this configuration are silenced but poised for rapid activation when differentiation is triggered, which implies a functional dominance of the inhibitory mark (H3K27me3) over the active one (H3K4me3). In differentiated cells bivalent domains may have a more complex function in fine-tuning gene expression, as suggested by a genome-wide analysis in T lymphocytes: genes bearing H3K4me3 could be clustered according to their H3K27me3 levels in genes with low transcriptional activity (high H3K27me3), genes with high transcriptional activity (low H3K27me3), and finally genes with intermediate levels of H3K27me3 and intermediate degrees of activity. Therefore, not only the presence or absence of the H3K27me3 mark is relevant, but also its abundance relative to H3K4me3. The data on Bmp-2 are consistent with this model. Bmp-2 gene transcription is rapidly activated in both WT and Jmjd3-depleted cells, although initial activation is rather low. Between 8 and 24 hr poststimulation a sharp increase in Bmp-2 mRNA is observed, but only in WT cells, in which H3K27me3 levels at the Bmp-2 promoter are reduced in a Jmjd3-dependent manner (De Santa, 2007).

The observation that newly synthesized Jmjd3 is incorporated in RbBP5-containing complexes suggests a possible model for the control of histone marks at genes bearing a bivalent domain. An attractive possibility is that a constitutively bound MLL complex devoid of a H3K27me3 HDM component is exchanged for a Jmjd3-containing MLL complex generated after LPS stimulation, thus allowing H3K27me3 demethylation while keeping constant the levels of H3K4me3 (De Santa, 2007).

Bmp-2 is a morphogen essential for embryonic development. It controls cell growth and promotes tumorigenesis, and it induces the differentiation of mesenchymal cells into osteoblasts. Bmp-2 production by activated macrophages participates in healing of bone fractures but also underlies heterotopic bone formation in inflamed joints and other tissues. Therefore, control of macrophage production of Bmp-2 by Jmjd3 is an indirect mechanism by which H3K27me3 demethylation in activated macrophages can impinge on the physiology of inflamed tissues (De Santa, 2007).

In conclusion, these data support the conceptually challenging idea that a histone mark involved in the control of differentiation and in the maintenance of cellular memory can be erased by a rapidly inducible enzyme whose expression is regulated by common environmental stimuli (De Santa, 2007).

Search PubMed for articles about Drosophila Little imaginal discs

Benevolenskaya, E. V., Murray, H. L., Branton, P., Young, R. A., and Kaelin, W. G. (2005). Binding of pRB to the PHD protein RBP2 promotes cellular differentiation. Mol. Cell 18: 623-635. Medline abstract: 15949438

Chan, S. W. and Hong, W. J. (2001). Retinoblastoma-binding protein 2 (Rbp2) potentiates nuclear hormone receptor-mediated transcription. J. Biol. Chem. 276: 28402-28412. Medline abstract: 11358960

De Santa, F., et al. (2007). The histone H3 lysine-27 demethylase Jmjd3 links inflammation to inhibition of polycomb-mediated gene silencing. Cell 130(6): 1083-94. Medline abstract: 17825402

Eissenberg, J. C., Lee, M. G., Schneider, J., Ilvarsonn, A., Shiekhattar, R., Shilatifard, A. (2007). The trithorax-group gene in Drosophila little imaginal discs encodes a trimethylated histone H3 Lys4 demethylase. Nat. Struct. Mol. Biol. 14(4): 344-6. Medline abstract: 17351630

Gildea, J. J., Lopez, R. and Shearn, A. (2000). A screen for new trithorax group genes identified little imaginal discs, the Drosophila melanogaster homologue of human retinoblastoma binding protein 2. Genetics 156(2): 645-63. Medline abstract: 11014813

Klose, R. J., Yamane, K., Bae, Y. J., Zhang, D. Z., Erdjument-Bromage, H., Tempst, P., Wong, J. M., and Zhang, Y. (2006). The transcriptional repressor JHDM3A demethylates trimethyl histone H3 lysine 9 and lysine 36. Nature 442: 312-316. Medline abstract: 16732292

Lee, N, Maurange, C., Ringrose, L. and Paro, R. (2005). Suppression of Polycomb group proteins by JNK signalling induces transdetermination in Drosophila imaginal discs. Nature 438: 234-237. Medline abstract: 16281037

Lee, N., Zhang, J., Klose, R. J., Erdjument-Bromage, H., Tempst, P., Jones, R. S., Zhang, Y. (2007). The trithorax-group protein Lid is a histone H3 trimethyl-Lys4 demethylase. Nat. Struct. Mol. Biol. 14(4): 341-3. Medline abstract: 17351631

Lu, P. J., Sundquist, K., Baeckstrom, D., Poulsom, R., Hanby, A., Meier-Ewert, S., Jones, T., Mitchell, M., Pitha-Rowe, P., Freemont, P., et al. (1999). A novel gene (PLU-1) containing highly conserved putative DNA chromatin binding motifs is specifically up-regulated in breast cancer. J. Biol. Chem. 274: 15633-15645. Medline abstract: 10336460

Mao, S. F., Neale, G. A. M. and Goorha, R. M. (1997). T-cell oncogene rhombotin-2 interacts with retinoblastoma-binding protein 2. Oncogene 14: 1531-1539. Medline abstract: 9129143

Secombe, J., Li, L., Carlos, L., and Eisenman, R. N. (2007). The Trithorax group protein Lid is a trimethyl histone H3K4 demethylase required for dMyc-induced cell growth. Genes Dev. 21: 537-551. Medline abstract: 17311883

Tan, K., Shaw, A. L., Madsen, B., Jensen, K., Taylor-Papadimitriou, J., and Freemont, P. S. (2003). Human PLU-1 has transcriptional repression properties and interacts with the developmental transcription factors BF-1 and PAX9. J. Biol. Chem. 278: 20507-20513. Medline abstract: 12657635

date revised: 17 January 2008

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