InteractiveFly: GeneBrief

Utx: Biological Overview | References

Trimethylated lysine 27 of histone H3 (H3K27me3) is an epigenetic mark for gene silencing and can be demethylated by the JmjC domain of UTX. Excessive H3K27me3 levels can cause tumorigenesis, but little is known about the mechanisms leading to those cancers. Mutants of the Drosophila H3K27me3 demethylase dUTX display some characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. A disruption of the JmjC domain of dUTX results in a growth advantage for mutant cells over adjacent wild-type tissue due to increased proliferation. The growth advantage of dUTX mutant tissue is caused, at least in part, by increased Notch activity, demonstrating that dUTX is a Notch antagonist. Furthermore, the inactivation of Retinoblastoma (Rbf in Drosophila) contributes to the growth advantage of dUTX mutant tissue. The excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila melanogaster and may provide a model for UTX-dependent tumorigenesis in humans (Herz, 2010).

Mammalian UTX, UTY, and JmjD3 and Drosophila UTX (dUTX) are histone demethylases that specifically demethylate di- and trimethylated lysine 27 on histone H3 (H3K27me2 and H3K27me3, respectively). The catalytic domain of this activity is the Jumonji C (JmjC) domain, located at the C terminus of these proteins. The N-terminal domains of UTX, UTY, and dUTX contain several tetratricopeptide repeats (TPRs) thought to be required for protein-protein interactions (Herz, 2010).

H3K27me3 is a histone mark for Polycomb (Pc)-mediated genomic silencing and transcriptional repression and is associated with animal body patterning, X-chromosome inactivation, genomic imprinting, and stem cell maintenance. H3K27 methylation is catalyzed by Polycomb repressive complex 2 (PRC2), which in Drosophila is composed of the catalytic subunit enhancer of zeste [E(z)] (EZH2 in mammals), extra sex combs (Esc), suppressor of zeste 12 [Su(z)12], and nucleosome remodeling factor 55 (Nurf55). H3K27me3 is recognized by the chromodomain of Pc, which is a component of a different silencing complex, called PRC1, which, in addition to Pc, contains Polyhomeotic (Ph), posterior sex combs (Psc), and dRING. The wild-type function of UTX is to demethylate H3K27me3 and, thus, to antagonize Polycomb-mediated silencing (Herz, 2010).

UTX is also a component of mixed-lineage leukemia complex 3 (MLL3) and MLL4 (Cho, 2007; Issaeva, 2007; Patel, 2007). MLL complexes are histone methyltransferases for H3K4. The function of UTX in MLL3 and MLL4 is unknown. However, it appears that UTX is not required for the H3K4 methyltransferase activity of MLL3 and MLL4 (Lee, 2007). The best-characterized targets of H3K27me3/Pc-mediated silencing are homeotic genes, which are critical regulators of animal patterning. However, many other genes are also enriched for H3K27 methylation and Pc binding. Furthermore, elevated H3K27me3 levels due to an increased activity of the methyltransferase EZH2 could be a leading cause of certain human cancers. Recently, mutations that inactivate UTX, and which are thus expected to cause increased H3K27me3 levels, have been linked to the development and progression of human cancer (van Haaften, 2007). However, the precise mechanisms by which this occurs are largely unknown (Herz, 2010).

Notch is the receptor of a highly conserved signaling pathway involved in many biological processes, including lateral inhibition, stem cell maintenance, and proliferation control. The binding of Delta or Serrate, the two ligands in Drosophila melanogaster, triggers the proteolytic processing of Notch, resulting in the release and translocation of the Notch intracellular domain (NICD) into the nucleus, where it regulates gene expression. Aberrant, oncogenic Notch signaling has been linked to tumor development in humans, including T-cell acute lymphoblastic leukemias (T-ALLs), pancreatic cancer, medulloblastoma, and mucoepidermoid carcinoma. Thus, an improved understanding of Notch signaling will have significant implications for human health (Herz, 2010).

In Drosophila, the Notch signaling pathway also controls the growth of the eye primordium and wing margin formation during development. Although the mechanistic details are unclear, one way by which Notch signaling controls proliferation during Drosophila eye development is through the negative regulation of the Retinoblastoma (Rb) family member Rbf. Rbf inactivation has also been implicated in Notch-induced eye tumors in Drosophila. Rb is a tumor suppressor that negatively regulates cell cycle progression through the inhibition of the transcription factor E2F. Rb binds directly to E2F and represses its transcriptional activity. The release of Rb activates E2F to induce the transcription of cell cycle regulators such as cyclin E and PCNA. Therefore, the inactivation of Rbf by increased Notch signaling can trigger increased proliferation, which may lead to cancerous growth (Herz, 2010).

This study genetically characterizes loss-of-function mutations of dUTX. dUTX mutants display some of the characteristics of Trithorax group mutants and have increased H3K27me3 levels in vivo. Surprisingly, dUTX mutations also affect H3K4me1 levels in a JmjC-independent manner. dUTX mutant tissue has an H3K27me3-dependent growth advantage over wild-type tissue due to increased proliferation in the developing eye. The growth advantage of dUTX mutant tissue is caused by increased Notch activity, demonstrating that dUTX is a Notch antagonist. The inactivation of Rbf contributes to the growth advantage of dUTX mutant tissue. Moreover, an excessive activation of Notch in dUTX mutant cells leads to tumor-like growth in an Rbf-dependent manner. In summary, these data suggest that dUTX is a suppressor of Notch- and Rbf-dependent tumors in Drosophila and may provide a model for UTX-dependent tumorigenesis in humans (Herz, 2010).

Based on the enzymatic activity of the JmjC catalytic domain as H3K27me3 demethylases, UTX proteins are predicted to counteract Polycomb function. Consistently, it was found that dUTX mutants display genetic characteristics of Trithorax group genes. In vitro studies have shown that dUTX and UTX demethylate H3K27me2 and H3K27me3. However, dUTX mutants affect the global levels of only H3K27me3 but not of H3K27me2. Nevertheless, this observation does not mean that dUTX does not demethylate H3K27me2 in vivo. There may be fewer genes regulated by dUTX at the H3K27me2 level such that the global levels are not detectably altered in dUTX mutants (Herz, 2010).

Interestingly, dUTX mutants also affect global levels of H3K4me1, which are significantly reduced in mutant tissue. Mammalian UTX is a component of the MLL3 and MLL4 methyltransferase complexes, and based on the reduction of H3K4me1 levels, it is predicted that dUTX is also a component of the Drosophila equivalent of the MLL3/MLL4 methyltransferase complex, which contains Trithorax-related (Trr) as a histone methyltransferase. The function of UTX in MLL3 and MLL4 complexes is currently unknown. It was suggested previously that UTX is not required for H3K4 methylation, but in these studies, only H3K4me2 and H3K4me3 were investigated. Consistently, the global levels of H3K4me2 and H3K4me3 are not affected in dUTX mutant clones. The data demonstrate that dUTX is required for the monomethylation of H3K4. Interestingly, the JmjC demethylase domain of dUTX is not required for H3K4me1 methylation, suggesting that other domains of dUTX, such as the TPR domains, may be necessary for mediating this function. The finding that the global levels of H3K4me2 and H3K4me3 are not affected in dUTX mutants is also quite interesting, as it implies that the monomethylation of H3K4 is not required for the di- or trimethylation of H3K4 (Herz, 2010).

The epigenetic control of gene expression has been best studied for the control of homeotic gene expression, which is established during embryogenesis and maintained throughout animal life. However, not only homeotic genes are regulated through epigenetic modifications. Other genes in different developmental processes are also subject to epigenetic control. In this study, by analyzing the dUTX mutant phenotype, a role was establised of H3K27me3 levels in cell cycle control. The data suggest that increased H3K27me3 levels in dUTX clones cause the epigenetic silencing of several genes involved in Notch signaling. This includes both positive and negative regulators of Notch signaling activity as well as target genes that are either positively or negatively regulated by the Notch pathway. Such an incoherent control of gene expression by the Notch pathway has been reported previously, suggesting that the final outcome of Notch activity may be determined by the relative expression levels of positive or negative regulators. Because this study determined that the overrepresentation phenotype of dUTX clones is caused by elevated levels of Notch signaling, it appears that the silencing of Notch inhibitors is dominant over the silencing of Notch activators, resulting in a net increase of Notch activity. However, this increased Notch activity may be specific for the cell cycle phenotype of dUTX mutants, since increased Notch activity was not found for other Notch-dependent paradigms, such as E(spl)m8-lacZ. This is also consistent with the finding that E(spl) genes contain increased H3K27me3 levels in dUTX mutants. Thus, the wild-type function of dUTX is to restrict the cell cycle through the negative control of Notch. Therefore, the data link H3K27me3-dependent Notch activity with enhanced tissue growth, implying that dUTX is a Notch antagonist regarding the cell cycle and explaining the overrepresentation phenotype of dUTX mutant clones (Herz, 2010).

However, this phenotype is subtle compared to that of mutants in growth control pathways such as the Hippo pathway. Nevertheless, the overgrowth of dUTX clones is strongly potentiated by the additional activation of Notch. The expression of Delta in dUTX clones causes a strong tumor-like growth phenotype. Thus, dUTX functions as a suppressor of Notch-induced tumors under normal conditions. This synergistic interaction between the loss of dUTX and increased Notch activity is a clear example that tumor development requires several hits for progression (Herz, 2010).

The overrepresentation phenotype of dUTX clones can be dominantly enhanced by the genetic loss of Rbf, suggesting that the reduction of Rbf contributes to the overrepresentation phenotype. However, the reduction of Rbf activity in dUTX clones is not caused by direct epigenetic silencing at the Rbf locus. No increased H3K27me3 levels was found at the Rbf locus in dUTX mutants, and mRNA levels of Rbf were unchanged. Instead, Rbf is negatively regulated by the Notch pathway during eye growth. Thus, the increased activity of Notch in dUTX clones leads to a partial inactivation of Rbf and increased proliferation, causing the overrepresentation phenotype. Currently, it is unknown how Notch regulates Rbf (Herz, 2010).

The control of cell cycle progression by UTX proteins is likely conserved in mammals. A parallel study performed by Wang showed that the loss of mammalian UTX also results in elevated levels of proliferation (Wang, 2010). Consistent with the current work, that study also implicated the inactivation of Rb function in increased proliferation in response to UTX knockdown. Similar to the current study, Rb itself is not subject to increased H3K27m3 silencing, but the promoters of several genes in the Rb network were found to be occupied and likely controlled by UTX (Wang, 2010). Thus, although the mechanisms of Rb control by UTX proteins (Notch in this study and the Rb network in the study reported previously by Wang) are distinct, both studies established the control of the Rb pathway as a common element of cell cycle control by UTX proteins. Wang also demonstrated a link between UTX and Rb during vulval development in Caenorhabditis elegans. Thus, these studies combined suggest a well-conserved function of UTX proteins for Rb control (Herz, 2010).

Although these studies establish a link between UTX genes and Rb for cell cycle control, it should be noted that the loss of dUTX (and likely mammalian UTX) affects many genes. While the deregulation of individual genes may not cause a significant phenotype on its own, the combined deregulation may disrupt gene regulatory networks, which accounts for the growth phenotype of dUTX mutants. Thus, while aberrant Notch signaling was identified as an important element of the overrepresentation phenotype of dUTX mutants, other genes and signal transduction pathways may also contribute to this phenotype. For example, this study also identified genes involved in growth control by the Hippo pathway (four-jointed [fj] and warts) associated with increased H3K27me3 levels in dUTX mutants and showed reduced transcript levels for fj. Thus, it is possible that the Hippo pathway and other genes contribute to the overrepresentation phenotype of dUTX mutants (Herz, 2010).

These observations have important implications for the initiation and development of human tumors. Increased levels of H3K27me3 due to the elevated activity of the H3K27me3 methyltransferase EZH2 have been associated with human cancer. Furthermore, mutations that inactivate UTX have been linked to human cancer (van Haaften, 2009), and low UTX activity correlates with poor patient prognosis (Wang, 2010). This study establishes that increased levels of H3K27me3 affect Notch activity, which in turn affects Rbf activity. Rb is a well-known tumor suppressor, the loss of which causes human tumors. Therefore, tumors associated with the loss of UTX and, thus, increased H3K27me3 levels may be caused by decreased Rb activity. It should also be noted that aberrant Notch signaling is the cause of several human cancers, including T-cell acute lymphoblastic leukemias (T-ALLs), pancreatic cancer, medulloblastoma, and mucoepidermoid carcinoma. In summary, these data demonstrate that the appropriate control of H3K27 methylation is critical for normal tissue homeostasis, and increased H3K27me3 levels may contribute to cancer through the inactivation of Rb (Herz, 2010).

Drosophila UTX is a histone H3 Lys27 demethylase that colocalizes with the elongating form of RNA polymerase II

Histone H3 methylation at Lys27 (H3K27 methylation) is a hallmark of silent chromatin, while H3K4 methylation is associated with active chromatin regions. This study reports that a Drosophila JmjC family member, dUTX, specifically demethylates di- and trimethylated but not monomethylated H3K27. dUTX localization on chromatin correlates with the elongating form of RNA polymerase II (Pol II), and dUTX can associate with Pol II. Furthermore, heat shock induction results in the recruitment of dUTX to the hsp70 gene, like that of several other Pol II elongation factors. These data indicate that dUTX is intimately associated with actively transcribed genes and may provide a paradigm for how H3K27 demethylation is required for the activation of preinitiated Pol II on transcriptionally poised genes (Smith, 2008).

The human UTX protein has been shown to function as an H3K27 demethylase. In order to learn about the cellular and molecular properties of this enzyme, its Drosophila homolog, dUTX, was identified. UTX was originally discovered as an X-linked homolog of a ubiquitously transcribed gene on the mammalian Y chromosome, UTY. In addition to a C-terminal JmjC domain, both UTX and UTY contain an N-terminal domain with several tetratricopeptide repeats, versatile protein binding modules found in a variety of proteins with diverse cellular functions. Remarkably, UTX was recently identified as a component of trithorax-related MLL3 and MLL4 complexes that mediate the methylation of H3 on lysine 4. Together, these findings reveal a new level of complexity in the regulation of gene expression by Polycomb and trithorax proteins through histone methylation and demethylation (Smith, 2008).

To determine whether dUTX is a histone demethylase in vitro, recombinant dUTX was expressed in insect cells by using a baculovirus expression system. dUTX specifically demethylated di- and trimethylated but not monomethylated H3K27 when presented with a mixture of total histones. Furthermore, dUTX is not capable of removing other repressive chromatin marks, such as H3K9me3 or H4K20me3, or the sites of methylation marks correlated with active transcription, such as H3K4me3 and H3K36me3, in vitro. While H3K27 di- and trimethylated forms are found primarily at repressive chromatin sites, the monomethylated form of H3K27 is found in coding regions of transcribed genes. Polycomb has a strong preference for binding the trimethylated form of H3K27, suggesting that demethylation down to the di- or monomethylated form could disrupt Polycomb binding. To ensure that the observed demethylase activity was intrinsic to dUTX and not a copurifying protein, a dUTX mutant bearing double mutations in the JmjC domain was engineered and expressed in the baculovirus system. The purified mutant enzyme lacked catalytic activity, demonstrating that dUTX is an H3K27 demethylase (Smith, 2008).

H3K27 methylation has been classically associated with the stable maintenance of transcriptional silencing. Therefore, it was of interest to determine the genomic distribution of dUTX. Anti-dUTX antibodies were made against a C-terminal peptide and tested for reactivity against full-length dUTX. The antibody recognized dUTX in SF21 cells infected with the Drosophila UTX baculovirus. To help validate the specificity of the antibodies, a dUTX-targeted RNAi transgenic line was used. Significant reductions in levels of immunostaining with an anti-dUTX antiserum were observed for knockdown compared to control polytene chromosomes from salivary glands. This RNAi line was also used to assess H3K27 methylation levels in extracts from adult flies. Modest levels of enrichment of H3K27 methylation were observed in total histones from adult flies after normalization to total-H3 levels. However, no significant changes in H3K27 methylation levels was detected by immunofluorescence on polytene chromosomes of 3rd-instar larvae. This may indicate that other factors in addition to dUTX are involved in H3K27 demethylation in vivo or that dUTX may function at specific loci throughout development (Smith, 2008).

Since dUTX staining appeared to occur in interband regions, frequently sites of active transcription, chromosomes were costained with antibodies recognizing elongating and paused forms of Pol II. There was very extensive colocalization with the elongating form of RNA polymerase (Ser2-phosphorylated C-terminal domain [CTD]) and a lesser extent of colocalization with the engaged but paused form of RNA polymerase (Ser5-phosphorylated CTD). Although the Lys4 demethylase Lid also was present in interband regions, it did not colocalize with the elongating form of RNA polymerase, demonstrating distinct biological roles for these enzymes (Smith, 2008).

To test if dUTX could be recruited to a gene upon activation similarly to other elongation factors, the hsp70 gene was used as a model inducible gene. Chromatin immunoprecipitation was performed before heat shock and after 5 min of heat shock of Schneider 2 cells. Antibodies directed to dUTX and RNA polymerase both immunoprecipitated increased levels of the hsp70 gene after heat shock. Consistent with the chromatin immunoprecipitation results from Schneider 2 cells, dUTX became highly enriched at major heat shock loci (puffs) on polytene chromosomes. Furthermore, it was demonstrated that dUTX in cell extracts exists in complexes associated with Pol II (Smith, 2008).

The colocalization of an H3K27 demethylase with the elongating form of RNA polymerase was unexpected but is emblematic of how little is understand about the mechanisms of regulation of gene expression by histone modifications. The recent finding that human UTX associates with trithorax family members is consistent with a role for dUTX at transcribing genes. Although no direct evidence exists for demethylation of Lys27 in coding regions, it is intriguing that a recent study found a large number of genes with paused polymerases. It has been speculated that Polycomb group proteins could be responsible for the pausing of these polymerases. In this context, the finding of a Lys27 demethylase colocalizing with the elongating form of RNA polymerase suggests that demethylation could be a key step in the pathway by which these genes are activated (Smith, 2008).

Search PubMed for articles about Drosophila Utx

Cho, Y. W., et al. (2007). PTIP associates with MLL3- and MLL4-containing histone H3 lysine 4 methyltransferase complex. J. Biol. Chem. 282: 20395-20406. PubMed Citation: 17500065

Herz, H. M., et al. (2010). The H3K27me3 demethylase dUTX is a suppressor of Notch- and Rb-dependent tumors in Drosophila. Mol. Cell. Biol. 30(10): 2485-97. PubMed Citation: 20212086

Issaeva, I., et al. (2007). Knockdown of ALR (MLL2) reveals ALR target genes and leads to alterations in cell adhesion and growth. Mol. Cell. Biol. 27: 1889-1903. PubMed Citation: 17178841

Lee, M. G., et al. (2007). Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination. Science 318: 447-450. PubMed Citation: 17761849

Patel, S. R., et al. (2007). The BRCT-domain containing protein PTIP links PAX2 to a histone H3, lysine 4 methyltransferase complex. Dev. Cell 13: 580-592. PubMed Citation: 17925232

Smith, E. R., et al. (2008). Drosophila UTX is a histone H3 Lys27 demethylase that colocalizes with the elongating form of RNA polymerase II. Mol. Cell Biol. 28(3): 1041-6. PubMed Citation: 18039863

van Haaften, G., et al. (2009). Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer. Nat. Genet. 41: 521-523. PubMed Citation: 19330029

Wang, J. K., et al. (2010). The histone demethylase UTX enables RB-dependent cell fate control. Genes Dev. 24: 327-332. PubMed Citation: 20123895

date revised: 15 January 2011

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