Utx histone demethylase: Biological Overview | References
Gene name - Utx histone demethylase
Cytological map position- 31C7-31C7
Function - enzyme, chromatic component
Symbol - Utx
FlyBase ID: FBgn0260749
Genetic map position - 2L:10,272,627..10,278,811 [-]
Classification - JmjC domain protein, Tetratricopeptide repeat domain protein, H3K27me3 demethylase
Cellular location - nuclear
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-relatedTrr) 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).
Trithorax group (TrxG) proteins antagonize Polycomb silencing and are required for maintenance of transcriptionally active states. Previous studies have shown that the Drosophila acetyltransferase CREB-binding protein (CBP; Nejire) acetylates histone H3 lysine 27 (H3K27ac), thereby directly blocking its trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) in Polycomb target genes. This study shows that H3K27ac levels also depend on other TrxG proteins, including the histone H3K27-specific demethylase Utx and the chromatin-remodeling ATPase Brahma (Brm). Utx and Brm are physically associated with CBP in vivo, and Utx, Brm, and CBP colocalize genome-wide on Polycomb response elements (PREs) and on many active Polycomb target genes marked by H3K27ac. Utx and Brm bind directly to conserved zinc fingers of CBP, suggesting that their individual activities are functionally coupled in vivo. The bromodomain-containing C terminus of Brm binds to the CBP PHD finger, enhances PHD binding to histone H3, and enhances in vitro acetylation of H3K27 by recombinant CBP. brm mutations and knockdown of Utx by RNA interference (RNAi) reduce H3K27ac levels and increase H3K27me3 levels. It is proposed that direct binding of Utx and Brm to CBP and their modulation of H3K27ac play an important role in antagonizing Polycomb silencing (Tie, 2012).
Acetylation of histone H3K27, a strong predictor of active genes, has emerged as one of the central mechanisms for antagonizing/reversing Polycomb silencing since it directly prevents trimethylation of H3K27 by PRC2. Interestingly, H3K27ac is present in animals, plants, and fungi, but H3K27me3 and PRC2 homologs are present only in animals and plants, clearly indicating that H3K27ac has functions other than preventing H3K27 methylation. This study provides evidence that the Drosophila TrxG proteins Utx and Brm modulate H3K27 acetylation by CBP. Utx and Brm are physically associated with CBP in vivo and bind directly to ZF1 and the PHD finger of CBP. Genome-wide ChIP-chip analysis revealed that the chromatin binding sites of Utx, Brm, and CBP coincide on many genes and that strong peaks of all three are highly correlated with the presence of high levels of H3K27ac. Importantly, brm mutants and RNAi knockdown of Brm in vivo result in a decrease of H3K27ac and a concomitant increase of H3K27me3. Similarly, knockdown and overexpression of Utx with no change in the CBP level are sufficient to promote, respectively, a decrease and increase in the bulk H3K27ac level. This suggests that coupled H3K27 deacetylation/trimethylation and demethylation/acetylation are dynamically antagonistic. It further suggests that regulating the balance of these opposing activities is likely to play an important role in determining whether active and silent chromatin states will be maintained or switched (Tie, 2012).
This is the first report that Utx is physically associated with CBP. While functional collaboration between Utx and CBP is required to execute the sequential reactions required to switch Polycomb target genes from silent to active states, it was not obvious that this should require that they be physically associated. The fact that they are suggests that their two reactions are more efficiently coupled. It also suggests that despite the many histone and nonhistone substrates of CBP, H3K27 acetylation on Polycomb target genes to prevent their silencing is sufficiently critical to have evolved a Utx-CBP methyl-to-acetyl switching module, perhaps to counter the complementary coupling effect of the physical association of the H3K27 deacetylase RPD3 with PRC2 to create the antagonistic acetyl-to-methyl switch. Coupling of the Utx and CBP activities may increase the fidelity of maintenance of active chromatin states of Polycomb target genes by ensuring rapid reversal of H3K27ac deacetylation and methylation by RPD3 and PRC2 that may occur either adventitiously or as part of an ongoing dynamic balance between these antagonistic activities. Such coupling could also increase the efficiency of switching Polycomb target genes from a transcriptionally silent to an active state in response to developmentally programmed signals or other cellular signals, ensuring definitive establishment of the new active transcriptional state (Tie, 2012).
While Brm is well known for the chromatin remodeling activity associated with its highly conserved ATPase domain, the current findings identify another activity of Brm associated with its highly conserved BrD-containing C terminus [Brm(1417-1634)], which binds histone H3, enhances H3 binding to the CBP PHD finger, and enhances acetylation of H3K27 by CBP in vitro. The latter effect is most likely due to the simultaneous binding of Brm to H3 and the CBP PHD finger, thereby stabilizing the H3 interaction with the CBP HAT domain. However, it cannot be ruled out that it may also reflect a direct stimulatory effect of Brm on the intrinsic activity of the CBP HAT domain. In any case, this suggests that the physical association and genome-wide colocalization of CBP and Brm do not simply reflect a spatial and temporal coordination of their separate acetylation and chromatin remodeling activities but also reflect regulatory interactions. The reduced H3K27ac level in brm2 mutants and after RNAi knockdown is consistent with the observed enhancement of CBP HAT activity in vitro. However, at this time the possibility cannot be ruled out that the loss of Brm chromatin-remodeling activity in brm2 mutants may also contribute to their reduced H3K27ac level (Tie, 2012).
The physical association of Brm with CBP and with Utx reported in this study is consistent with recent reports that BRG1 can be coimmunoprecipitated with human CBP and p300 from tumor cells and with Utx/Jmjd3 in murine EL4 T cells. However, the region of human CBP reported to bind BRG1 differs from the current findings. It was reported that BRG1 interacts directly with the human CBP fragment containing ZF3, and the proline-rich region of BRG1 is required for this interaction. The current results indicate that only ZF1 and the PHD finger of Drosophila CBP bind directly to the BrD-containing C terminus of Brm. Since the proline-rich region of Drosophila Brm was not assayed due to its insolubility, the possibility cannot be ruled out that it mediates additional contact(s) with CBP (Tie, 2012).
CBP was not found in the previously purified Drosophila Brm-containing complexes, suggesting that only a portion of Brm is physically associated with CBP or that this association may be stabilized only on chromatin and/or may be regulated by other cellular signals. Consistent with this, there are some sites detected by ChIP-chip that are enriched for Brm and Utx without CBP and there are some column fractions containing Brm and Utx without CBP. It is possible that the CBP-Brm association may also serve to recruit Brm to some sites, e.g., recruitment of human Brm or BRG1 to the beta interferon (IFN-β) promoter depends on the prior presence of CBP and leads to subsequent nucleosome remodelin (Tie, 2012).
This study found that Utx, Brm, and CBP colocalize not only with H3K27ac at many regions, including promoters, transcribed regions, PREs, and other presumed cis-regulatory elements. They are also present, albeit at lower levels, on repressed genes marked by strong H3K27me3 domains (e.g., the ANT-C and BX-C). At such sites, the H3K27me3 may be protected from Utx-mediated demethylation by the binding of PC/PRC1. Alternatively, their lower levels at repressed genes may simply result in a dynamic balance of deacetylation/methylation and demethylation/acetylation that overwhelmingly favors the former. It is also possible that Utx and CBP may also be involved in Brm-dependent transcriptional repression at some sites. Recent evidence suggests that maintenance of steady-state levels of histone acetylation is highly dynamic, and the reciprocal changes in H3K27me3 and H3K27ac levels that occur upon altering Utx or E(Z) levels suggest that maintenance of histone methylation levels may also be highly dynamic. Much remains to be discovered about the factors that regulate the demethylase and acetyltransferase activities of Utx and CBP in different chromatin environments (Tie, 2012).
The bromodomains of yeast SWI2/SNF2 and human BRG1 bind specifically to H3K14ac, indicating that this binding specificity has been highly conserved during evolution. Brm(1417-1634) also binds specifically to H3K14ac, and the BrD of Brm is required for CBP binding and histone H3 binding. The BrD-containing C termini of human and plant Brm have been reported to be functionally important in vivo. Surprisingly, the BrD of Drosophila Brm has been reported to be dispensable for viability. A brm transgene containing a deletion of the central 72 residues of the BrD can rescue the late embryonic lethality of brm2 mutants, allowing them to develop into adults. Whether the reduced H3K27ac level of these brm2 mutants is also rescued has not been determined. This rescue could indicate that the Brm BrD is functionally redundant or at least not critical for achieving adequate expression of the genes responsible for the inviability of brm2 mutants. The brm2 mutation behaves genetically as a strong hypomorphic or null allele, but the sequence alteration responsible for its phenotype has not been determined and so the precise nature of its functional deficit is unknown (Tie, 2012).
In summary, this study has shown that Utx and Brm interact directly with CBP and modulate H3K27 acetylation. Utx presumably does so indirectly, at Polycomb target genes, by providing demethylated H3K27 substrate for acetylation by CBP. Their direct physical coupling could provide obvious gains in the efficiency of their two sequential reactions and their consequent H3K27ac yield. The interaction between Brm and CBP may similarly couple their activities, but this study also presents initial evidence to suggest that the binding of the Brm BrD-containing C terminus to H3 and the CBP PHD finger may also directly enhance H3K27 acetylation through its effect on H3 binding by the CBP HAT domain. It is expected that additional TrxG proteins will modulate H3K27 acetylation, including KIS and ASH1, which have recently been shown to affect H3K27me3 levels. The broad distributions of H3K27me3 over many Polycomb target genes is mirrored by similar broad distributions of H3K27ac when those genes are active, suggesting that in addition to its general genome-wide association with active genes, it may play a more specialized dual role at Polycomb target genes, where it also dynamically antagonizes the encroachment of Polycomb silencing (Tie, 2012).
Adult stem cells reside in microenvironments called niches, where they are regulated by both extrinsic cues, such as signaling from neighboring cells, and intrinsic factors, such as chromatin structure. This study reports that in the Drosophila testis niche an H3K27me3-specific histone demethylase encoded by Ubiquitously transcribed tetratricopeptide repeat gene on the X chromosome (dUTX) maintains active transcription of the Suppressor of cytokine signaling at 36E (Socs36E) gene by removing the repressive H3K27me3 modification near its transcription start site. Socs36E encodes an inhibitor of the Janus kinase signal transducer and activator of transcription (JAK-STAT) signaling pathway. Whereas much is known about niche-to-stem cell signaling, such as the JAK-STAT signaling that is crucial for stem cell identity and activity, comparatively little is known about signaling from stem cells to the niche. The results reveal that stem cells send feedback to niche cells to maintain the proper gene expression and architecture of the niche. dUTX acts in cyst stem cells (CySCs) to maintain gene expression in hub cells through activating Socs36E transcription and preventing hyperactivation of JAK-STAT signaling. dUTX also acts in germline stem cells to maintain hub structure through regulating DE-Cadherin levels. Therefore, these findings provide new insights into how an epigenetic factor regulates crosstalk among different cell types within an endogenous stem cell niche, and shed light on the biological functions of a histone demethylase in vivo (Tarayrah, 2013).
This study identified a new epigenetic mechanism that negatively regulates the JAK-STAT signaling pathway in the Drosophila testis niche: the H3K27me3-specific demethylase dUTX acts in CySCs to remove the repressive H3K27me3 histone modification near the TSS of Socs36E to allow its active transcription. Socs36E acts upstream to suppress Stat92E activity and to restrict CySCs from overpopulating the testis niche. In addition, dUTX acts in CySCs to prevent hyperactivation of Stat92E in hub cells, which would otherwise ectopically turn on Zfh1 expression. When zfh1 cDNA was ectopically droven in hub cells using the upd-Gal4 driver, no obvious defect could be identified. Therefore, the biological consequence of ectopic Zfh1 expression in hub cells remains unclear. However, ectopic Zfh1 expression in hub cells and the overpopulation of Zfh1-expressing cells around the hub are two connected phenomena because both phenotypes are caused by loss of dUTX in CySCs (Tarayrah, 2013).
UTX also acts in GSCs to regulate DE-Cadherin levels to maintain proper GSC-hub interaction and normal morphology of the hub. It has been reported that differential expression of different cadherins causes cells with similar cadherin types and levels to aggregate. In wt testes, hub cells express higher levels of DE-Cadherin and therefore tightly associate with each other. Loss of dUTX in germ cells leads to higher levels of DE-Cadherin in GSCs, which probably allows them to intermingle with hub cells and causes disrupted hub architecture. It has also been demonstrated that the major role of JAK-STAT in GSCs is for GSC-hub adhesion, suggesting that the expression and/or activity of cell-cell adhesion molecules, such as DE-Cadherin, depends on JAK-STAT signaling. Therefore, the abnormal DE-Cadherin activity in GSCs in dUTX testis could also result from misregulated JAK-STAT signaling in the testis niche. dUTX is a new negative epigenetic regulator of the JAK-STAT signaling pathway (Tarayrah, 2013).
The JAK-STAT signaling pathway plays crucial roles in stem cell maintenance in many different stem cell types across a wide range of species. These studies identify the histone demethylase dUTX as a new upstream regulator of the JAK-STAT pathway, which directly controls the transcription of Socs36E. In addition to acting as an antagonist of JAK-STAT signaling, Socs36E has been reported to be a direct target gene of the Stat92E transcription factor (Terry, 2006). Therefore, increased Stat92E would be expected to upregulate Socs36E expression, but this was not observed in dUTX mutant testes. Instead, the data revealed that Socs36E expression decreased, whereas Stat92E expression increased, in dUTX testes, consistent with the hypothesis that Socs36E is a direct target gene of dUTX and acts upstream of Stat92E (Tarayrah, 2013).
Sustained activity of the JAK-STAT pathway in cyst cells has been reported to activate BMP signaling, which leads to GSC self-renewal outside the niche and gives rise to a tumor-like phenotype in testis. To examine BMP pathway activity, immunostaining experiments were performed using antibodies against phospho-SMAD (pSMAD), a downstream target of BMP signaling. No obvious difference was detected in the pSMAD signal between the dUTX testes and wt control, nor were any germline tumors detected in dUTX testes. It is speculated that germline tumor formation upon activation of the JAK-STAT pathway is secondary to the overproliferation of Zfh1-expressing cells, which was not observed in dUTX testes (Tarayrah, 2013).
This study also provides an example of the multidimensional cell-cell communication that takes place within a stem cell niche. Many studies of the stem cell niche have focused on understanding niche-to- stem cell signaling in controlling stem cell identity and activity. For example, in the Drosophila female GSC niche, Upd secreted from terminal filaments activates the JAK-STAT pathway in cap cells and escort cells, which subsequently produce the BMP pathway ligand Decapentaplegic (Dpp) to activate BMP signaling and prevent transcription of the differentiation factor bag-of-marbles (bam) in GSCs. In the Drosophila intestinal stem cell (ISC) niche, the visceral muscle cells underlying the intestine secrete Wingless to activate Wnt signaling and Upd to activate JAK-STAT signaling in ISCs, which are required to maintain ISC identity and activity (Tarayrah, 2013).
More studies have now revealed the multidirectionality of signaling within the stem cell niche. For example, in the Drosophila female GSC niche, GSCs activate Epidermal growth factor receptor (Egfr) signaling in the neighboring somatic cells, which subsequently represses expression of the glypican Dally, a protein required for the stabilization and mobilization of the BMP pathway ligand Dpp. Through this communication between GSCs and the surrounding somatic cells, only GSCs maintain high BMP signaling. The current studies establish another example of the multidimensional cell-cell communications that occur within the testis stem cell niche, where CySCs and GSCs have distinct roles in regulating hub cell identity and morphology (Tarayrah, 2013).
The data identified new roles of a histone demethylase in regulating endogenous stem cell niche architecture and proper gene expression. Previous studies have reported in vivo functions of histone demethylases in several model organisms. For example, mammalian UTX has been shown to associate with the H3K4me3 histone methyltransferase MLL2, suggesting its potential antagonistic role to the PcG proteins. The PcG proteins play a crucial role in Hox gene silencing in both Drosophila and mammals. Consistently, mammalian UTX has been reported to directly bind and activate the HOXB1 gene locus. In addition to antagonizing PcG function, H3K27me3 demethylases play crucial roles during development. For example, in zebrafish, inactivating the UTX homolog (kdm6al) using morpholino oligonucleotides leads to defects in posterior development, and in C. elegans the dUTX homolog (UTX-1) is required for embryonic and postembryonic development, including gonad development. Furthermore, loss of UTX function in embryonic stem cells leads to defects in mesoderm differentiation, and somatic cells derived from UTX loss-of-function human or mouse tissue fail to return to the ground state of pluripotency. These reports demonstrate that UTX is not only required for proper cellular differentiation but also for successful reprogramming. However, despite multiple reports on the in vivo roles of H3K27me3-specific demethylases, little is known about their functions in any endogenous adult stem cell system (Tarayrah, 2013).
Whereas mammals have multiple H3K27me3 demethylases, dUTX is the sole H3K27me3-specific demethylase in Drosophila. This unique feature, plus the well-characterized nature of Drosophila adult stem cell systems, make interpretation of the endogenous functions of histone demethylases in Drosophila unambiguous. Because mammalian UTX has been reported as a tumor suppressor, understanding the endogenous functions of dUTX in an adult stem cell system might facilitate the use of histone demethylases for cancer treatment. In summary, these results demonstrate that stem cells send feedback to the niche cells to maintain their proper gene expression and morphology. Furthermore, this feedback is regulated through the JAK-STAT signaling pathway, the activity of which is controlled by a chromatin factor, providing an example of crosstalk between these two regulatory pathways (Tarayrah, 2013).
UTX is known as a general factor that activates gene transcription during development. This study demonstrates an additional essential role of UTX in the DNA damage response, in which it upregulates the expression of ku80 in Drosophila, both in cultured cells and in third instar larvae. UTX mediates the expression of ku80 by the demethylation of H3K27me3 at the ku80 promoter upon exposure to ionizing radiation (IR) in a p53-dependent manner. UTX interacts physically with p53, and both UTX and p53 are recruited to the ku80 promoter following IR exposure in an interdependent manner. In contrast, the loss of utx has little impact on the expression of ku70, mre11, hid and reaper, suggesting the specific regulation of ku80 expression by UTX. Thus, these findings further elucidate the molecular function of UTX (Zhang, 2013).
To understand the mechanism underlying UTX function in tumorgenesis, this study explored whether UTX is involved in DNA damage response in Drosophila. This study found that UTX plays an essential role in DNA damage response by upregulation of ku80, which is uniquely required for p53 activated ku80 expression. In addition, the gene activity of utx is correlated with loss of histone demethylation at H3K27, suggesting that UTX could function as a histone demethylase and serve a gene-specific co-activator of p53 gene activation. This study therefore provides an example that p53 target genes expression may be regulated at the level of histone modifications (Zhang, 2013).
It is clear that p53 plays a pivotal role in the DNA damage response (DDR). One of the functions of p53 is to activate its target gene after DNA damage as transcription factor. For instance, p53 has been best characterized in regualting expression of cell cycle genes and apoptosis gene. However, the precise regulation mechanism of p53 is still not clear. It is interesting that in Drosophila ku80 upregulation mediated by p53 requires UTX, but not other genes in related to DNA repair and apoptosis. However, reduced H3K27me3 levels were found in apoptotic genes, which raises the possibility that there could be additional histone demethylases participating in DDR pathways that coordinate with p53 regulating expression of hid and reaper after DNA damage, and remaining to be determined in further studies. In contrast, reduced H3K27me3 levels were not detected in the ku70 promoter region following IR treatment. Further analysis revealed that the H3K27me3 level in the ku70 promoter region was lower than at the ku80 promoter. The expression of ku70 is independent of UTX, possibly due to the extremely low levels of H3K27me3 in the ku70 promoter region, which might not require demethylation for the expression of ku70 to occur. Thus, the data demonstrate the complexity of the function of p53 in the activation of target genes in response to DNA damage, particularly in terms of histone modification and the action of different demethylases (Zhang, 2013).
UTX has been reported to participate in many biological processes, including cell fate determination and animal development, largely depending on the transcriptional regulation of the target genes of UTX. UTX appears to play an important role in orchestrating several histone markers, including acetylation at H3K27 and ubiquitination at H2A, and mediates derepression of polycomb (Pc) target genes, such as HOX genes, by affecting Pc recruitment. These roles are consistent with UTX being a histone demethylase specific for H3K27. However, sporadic mutations of UTX have been linked to many types of human cancers and it remains to be elucidated whether this is also sufficiently explained by its enzymatic activity. Indeed, several studies have proposed a role of UTX independent of its demethylase activity in chromatin remodeling and embryonic development. This study found UTX is also involved in DDR by upregulation of ku80 in Drosophila after IR. Although there are no available data demonstrating that ku80 mRNA levels are increased following DSBs in human cells, the current data provide evidence that UTX functions to maintain genome stability and sheds light on the mechanism underlying the function of UTX in human cancer. Recent studies suggest that loss of polycomb-mediated silencing might promote the upregulation of DNA repair genes and facilitate the recovery of cells from genotoxic insults. UTX might therefore be required for various cell defense mechanisms under environmental stress, thereby contributing to tumor suppression (Zhang, 2013).
Trimethylation of histone H3 at lysine 27 (H3-K27me3) by Polycomb repressive complex 2 (PRC2) is a key step for transcriptional repression by the Polycomb system. Demethylation of H3-K27me3 by Utx and/or its paralogs has consequently been proposed to be important for counteracting Polycomb repression. To study the phenotype of Drosophila mutants that lack H3-K27me3 demethylase activity, UtxΔ), a deletion allele of the single Drosophila Utx gene, was created. UtxΔ homozygotes that contain maternally deposited wild-type Utx protein develop into adults with normal epidermal morphology but die shortly after hatching. By contrast, UtxΔ homozygotes that are derived from Utx mutant germ cells and therefore lack both maternal and zygotic Utx protein, die as larvae and show partial loss of expression of HOX genes (Ubx and Abd-B) in tissues in which these genes are normally active. This phenotype classifies Utx as a trithorax group regulator. It is proposed that Utx is needed in the early embryo to prevent inappropriate installment of long-term Polycomb repression at HOX genes in cells in which these genes must be kept active. In contrast to PRC2, which is essential for, and continuously required during, germ cell, embryonic and larval development, Utx therefore appears to have a more limited and specific function during development. This argues against a continuous interplay between H3-K27me3 methylation and demethylation in the control of gene transcription in Drosophila. Furthermore, this analyses do not support the recent proposal that Utx would regulate cell proliferation in Drosophila as Utx mutant cells generated in wild-type animals proliferate like wild-type cells (Copur, 2013).
Correct spatial and temporal induction of numerous cell type-specific genes during development requires regulated removal of the repressive histone H3 lysine 27 trimethylation (H3K27me3) modification. This study shows that the H3K27me3 demethylase dUTX is required for hormone-mediated transcriptional regulation of apoptosis and autophagy genes during ecdysone-regulated programmed cell death of Drosophila salivary glands. dUTX binds to the nuclear hormone receptor complex Ecdysone Receptor/Ultraspiracle, and is recruited to the promoters of key apoptosis and autophagy genes. Salivary gland cell death is delayed in dUTX mutants, with reduced caspase activity and autophagy that coincides with decreased apoptosis and autophagy gene transcripts. It was further shown that salivary gland degradation requires dUTX catalytic activity. These findings provide evidence for an unanticipated role for UTX demethylase activity in regulating hormone-dependent cell death and demonstrate how a single transcriptional regulator can modulate a specific complex functional outcome during animal development (Denton, 2013).
UTX function is known to be critical in mammalian embryonic development and somatic and germ cell reprogramming. This study found a novel role for dUTX in steroid hormone-mediated cell death during development. dUTX, together with nuclear hormone receptor EcR/Usp, is capable of regulating gene expression both spatially and temporally in a hormone-dependent manner. UTX gene mutations are frequently observed in malignancies including lethal castration-resistant prostate cancer, although a role for UTX in androgen receptor-mediated transcription has not yet been identified. This study indicates that UTX is a good candidate to extend the investigation to examine the role of UTX in coordinating nuclear hormone receptor-regulated gene expression, particularly in androgen receptor-mediated transcription during mammalian development and hormone-dependent cancers (Denton, 2013).
The complete degradation of larval salivary glands during metamorphosis utilizes both apoptosis and autophagy and by coordinately controlling the expression of critical genes in these two distinct biological pathways, dUTX ensures timely removal of salivary glands in response to temporal ecdysone pulse. The majority of studies addressing induction of autophagy have focused upon autophagosome formation and protein degradation. The transcriptional regulation of autophagy induction remains poorly understood. Indeed, several Atg genes are transcriptionally upregulated following autophagy induction; however, the molecular pathways are only beginning to be revealed. For example, the master gene controlling lysosomal biogenesis, transcription factor EB, coordinates the expression of both autophagy and lysosomal genes to induce autophagy in response to starvation. Induction of autophagy has been linked to reduced histone H4 lysine 16 acetylation (H4K16ac) through downregulation of the histone acetyltransferase hMOF. Downregulation of H4K16 deacetylation was associated with the downregulation of several Atg genes, whereas antagonizing H4K16ac downregulation upon autophagy induction resulted in cell death. The study indicates that a specific histone modification during autophagy modulates the expression of Atg genes, and is important for survival versus death responses upon autophagy induction. This work now describes dUTX as another regulator of autophagy and cell death in the context of developmental PCD and in concert with the steroid hormone response. Future studies to understand the complex nuclear events regulating both repression and induction of autophagy gene expression in response to particular signals will be important (Denton, 2013).
Despite the opposing roles of H3K27 and H3K4 methylation in transcriptional regulation, UTX has been identified in association with H3K4 methyltransferase and to play demethylase-independent functions. This study suggests that the demethylase activity of dUTX is necessary for hormone-mediated cell death. The nuclear hormone receptor response to ecdysone initiates a hierarchical transcription cascade by induction of transcription factors, including BR-C, E74 and E93. These transcription factors drive expression of downstream genes including cell death genes. The data show that dUTX regulates E93 and suggests that this HDM can regulate cell death both directly, through the transcription of apoptosis and autophagy genes through direct recruitment via EcR/Usp, as well as indirectly through key transcription factor E93. This additional level of regulation through the stage-specific transcription factor E93 may provide temporal control of ecdysone response during metamorphosis (Denton, 2013).
The role of autophagy in cell death is a matter of considerable debate as autophagy is generally a cell survival mechanism in response to cellular stress and nutrient limitations. Studies in Drosophila have provided perhaps some of the strongest evidence for a role of autophagy in developmental cell death in vivo. The data presented in this paper demonstrating coordinate regulation of both key apoptosis and autophagy genes by a single histone-modifying enzyme further provide genetic and molecular evidence linking autophagy and apoptosis in PCD during metamorphosis (Denton, 2013).
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).
Cell-fate reprogramming is at the heart of development, yet very little is known about the molecular mechanisms promoting or inhibiting reprogramming in intact organisms. In the C. elegans germline, reprogramming germ cells into somatic cells requires chromatin perturbation. This study shows that such reprogramming is facilitated by GLP-1/Notch (see Drosophila Notch) signaling pathway. This is surprising, since this pathway is best known for maintaining undifferentiated germline stem cells/progenitors (see Drosophila germline). Through a combination of genetics, tissue-specific transcriptome analysis, and functional studies of candidate genes, a possible explanation for this unexpected role of GLP-1/Notch was uncovered. The study proposes that GLP-1/Notch promotes reprogramming by activating specific genes, silenced by the Polycomb repressive complex 2 (PRC2) (see Drosophila E(z)), and identifies the conserved histone demethylase UTX-1 (see Drosophila Utx) as a crucial GLP-1/Notch target facilitating reprogramming. These findings have wide implications, ranging from development to diseases associated with abnormal Notch signaling (Seelk, 2016).
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 ID: 17500065
Copur, O. and Muller, J. (2013). The histone H3-K27 demethylase Utx regulates HOX gene expression in Drosophila in a temporally restricted manner. Development 140: 3478-3485. PubMed ID: 23900545Denton, D., Aung-Htut, M. T., Lorensuhewa, N., Nicolson, S., Zhu, W., Mills, K., Cakouros, D., Bergmann, A. and Kumar, S. (2013). UTX coordinates steroid hormone-mediated autophagy and cell death. Nat Commun 4: 2916. PubMed ID: 24336022
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 ID: 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 ID: 17178841
Lee, M. G., et al. (2007). Demethylation of H3K27 regulates polycomb recruitment and H2A ubiquitination. Science 318: 447-450. PubMed ID: 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 ID: 17925232
Seelk, S., Adrian-Kalchhauser, I., Hargitai, B., Hajduskova, M., Gutnik, S., Tursun, B. and Ciosk, R. (2016). Increasing Notch signaling antagonizes PRC2-mediated silencing to promote reprogramming of germ cells into neurons. Elife [Epub ahead of print]. PubMed ID: 27602485
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 ID: 18039863
Tarayrah, L., Herz, H. M., Shilatifard, A. and Chen, X. (2013). Histone demethylase dUTX antagonizes JAK-STAT signaling to maintain proper gene expression and architecture of the Drosophila testis niche. Development 140: 1014-1023. PubMed ID: 23364332
Terry, N. A., Tulina, N., Matunis, E. and DiNardo, S. (2006). Novel regulators revealed by profiling Drosophila testis stem cells within their niche. Dev Biol 294: 246-257. PubMed ID: 16616121
Tie, F., Banerjee, R., Conrad, P. A., Scacheri, P. C. and Harte, P. J. (2012). Histone demethylase Utx and chromatin remodeler Brm bind directly to CBP and modulate acetylation of histone H3 lysine 27. Mol Cell Biol 32: 2323-2334. PubMed ID: 22493065
van Haaften, G., et al. (2009). Somatic mutations of the histone H3K27 demethylase gene UTX in human cancer. Nat. Genet. 41: 521-523. PubMed ID: 19330029
Wang, J. K., et al. (2010). The histone demethylase UTX enables RB-dependent cell fate control. Genes Dev. 24: 327-332. PubMed ID: 20123895
Zhang, C., Hong, Z., Ma, W., Ma, D., Qian, Y., Xie, W., Tie, F. and Fang, M. (2013). Drosophila UTX coordinates with p53 to regulate ku80 expression in response to DNA damage. PLoS One 8: e78652. PubMed ID: 24265704
date revised: 30 September 2016
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