UTX Gene Research Articles

Identification of Different miRNAs and Their Relevant miRNA Targeted Genes Involved in Sister Chromatid Cohesion and Segregation (SCCS)/chromatin Remodeling Pathway on T1G3 Urothelial Carcinoma (UC) Response to BCG Immunotherapy.

Till now, no definite clinical or laboratory marker can predict the recurrence or progression of T1 G3 urothelial carcinoma (UC). Genetic aberrations of the chromatin remodeling genes and sister chromatid cohesion and segregation (SCCS) were identified in UC. Here we investigated the impact of novel miRNAs and their targeted expressed SCCS and chromatin remodeling genes on T1G3 UC response to Bacillus Calmette-Guérin (BCG) therapy. One hundred tissue samples were obtained from NMIBC patients. Gene expression and immunohistochemical assay of STAG2, ARID1A, NCOR1and UTX were assessed. MiRNA analysis for their targeting miRNAs (miR-21, miR-31, Let7a and miR-199a) was carried out. Assessed genes were compared between responders and no responders to BCG. Univariate and multivariate analysis of predictors of disease recurrence and progression were performed using cox regression analysis. Thirty-two and 22 patients developed recurrence and progression to MIBC (BCG non-responders). BCG non-responders showed statistically significant higher expression of miR-21 and their targeted STAG2, miR-199a and NCOR1 gene (P < .001), and lower expression of miR-31, Let7a, ARID1A and UTX genes (P < .001). Higher miR-199a (P=.006) and lower miR-31 (P=.01), ARID1A (P=.008) and UTX (P=.03) were independent predictor of higher tumor recurrence. Recurrent disease (P=.003), higher expression of STAG2 (P=.01), NCOR1 (P=.01) and miR-21 (P=.03) genes and lower expression of miR-31 (P=.02), Let7a (P=.04) and ARID1A (P=.04) genes were the independent predictor of disease progression. Upregulation of STAG2 and NCOR1 and down regulation of ARID1A and UTX genes and their targeting miRNAs were associated with UC non-response to BCG.

UTX Affects Neural Stem Cell Proliferation and Differentiation through PTEN Signaling.

SummaryNeural stem cell (NSC) proliferation and differentiation in the developing brain is a complex process precisely regulated by intrinsic and extrinsic signals. Although epigenetic modification has been reportedly involved in the regulation of the cerebral cortex, whether UTX, an H3K27me3 demethylase, regulates the development of cerebral cortex during the embryonic period is unclear. In this study, we demonstrate that Utx deficiency by knockdown and conditional knockout increases NSC proliferation and decreases terminal mitosis and neuronal differentiation. Furthermore, we find that impairment of cortical development caused by lack of Utx is less significant in males than in females. In addition, UTX demethylates H3K27me3 at the Pten promoter and promotes Pten expression. P-AKT and P-mTOR levels are increased in the Utx conditional knockout cortices. Finally, Utx or Pten overexpression can rescue the impairment of brain development caused by Utx loss. These findings may provide important clues toward deciphering brain diseases.

Epigenetic Dysfunction in Turner Syndrome Immune Cells.

Turner syndrome (TS) is a chromosomal condition associated with partial or complete absence of the X chromosome that involves characteristic findings in multiple organ systems. In addition to well-known clinical characteristics such as short stature and gonadal failure, TS is also associated with T cell immune alterations and chronic otitis media, suggestive of a possible immune deficiency. Recently, ubiquitously transcribed tetratricopeptide repeat on the X chromosome (UTX), a histone H3 lysine 27 (H3K27) demethylase, has been identified as a downregulated gene in TS immune cells. Importantly, UTX is an X-linked gene that escapes X-chromosome inactivation and thus is haploinsufficient in TS. Mice with T cell-specific UTX deficiency have impaired clearance of chronic viral infection due to decreased frequencies of T follicular helper (Tfh) cells, which are critical for B cell antibody generation. In parallel, TS patients have decreased Tfh frequencies in peripheral blood. Together, these findings suggest that haploinsufficiency of the X-linked UTX gene in TS T cells underlies an immune deficit, which may manifest as increased predisposition to chronic otitis media.

Expression of Myogenin and MCK genes regulated by PI3K/AKT pathway

Many intracellular signaling pathways regulate skeletal muscle differentiation. Among them, PI3K/AKT pathway plays an important role. But the mechanisms of chromatin regulation remain unclear. In this study, the murine C2C12 myoblast cell line was used to investigate the expression of Myogenin and MCK genes during the skeletal muscle differentiation. Western blotting analysis showed that the expression of Myogenin and MCK protein was increased significantly after PI3K/AKT activator treatment for 24 h during the C2C12 cell differentiation and the expression of H3K27me3 demethylase UTX was also increased. Chromatin immunoprecipitation (ChIP) and quantitative PCR (Q-PCR) analysis showed that the enrichment of H3K27me3 on the promoter regions of Myogenin and MCK genes and the enhancer region of MCK gene were decreased. It was opposite to the PI3K/AKT inhibitor treatment. We concluded that the PI3K/AKT pathway maybe regulate skeletal muscle differentiation by regulating the expression of UTX gene to change the enrichment of H3K27me3 on the target genes.

The histone H3-K27 demethylase Utx regulates HOX gene expression in Drosophila in a temporally restricted manner

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, we created UtxΔ, a deletion allele of the single Drosophila Utx gene. 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 in tissues in which these genes are normally active. This phenotype classifies Utx as a trithorax group regulator. We propose that Utx is needed in the early embryo to prevent inappropriate instalment 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, our 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.

Abstract 1042: Down-regulation of UTX contributes to lung cancer progression

Abstract Smoking, the main risk factor leading to lung cancer, causes both genetic and epigenetic changes resulting in aberrant gene expression. UTX is a histone demethylase that has been implicated in many cancers and has been shown to regulate both cell cycle and differentiation genes. However, the role of UTX has not been elucidated in lung cancer. We hypothesized that smoking injury leads to a down regulation in UTX expression. This results in an inability of repairing airway epithelial cells to differentiate, resulting in premalignant lesions and ultimately invasive lung cancer. Examination of published lung cancer data sets revealed a down-regulation of Utx gene expression in patients with poorer prognosis, tumor grade, tumor recurrence and in smokers. To assess the involvement of UTX protein expression in lung cancer we stained a lung cancer tissue microarray and found a significant correlation between down-regulation of UTX protein expression and poorer prognosis in metastasis (p=0.0005) and smokers (p= 0.02). UTX expression was found to be significantly lower in squamous cell carcinoma compared to adenocarcinoma, linking down-regulation of UTX in a diseased caused solely by smoking. To test if smoking directly affects Utx expression we treated mouse airway epithelial cells at air-liquid interface with media infused with cigarette smoke for 9 days. After this short time point of acute smoke exposure there was a significant down regulation of Utx gene expression (p=0.03). We carried out UTX overexpression studies to assess the role of UTX in lung cancer cells lines and found up-regulation of UTX results in up-regulation of genes involved in lung development and differentiation, including Nrf2, FoxJ2 and FZD4. Our finding support the hypothesis that smoking causes a reduction in UTX that affects the proliferation and differentiation of airway epithelial cells contributing to lung cancer progression. Re-activation of UTX therefore may be a potential therapeutic target for the prevention of lung cancer progression. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 1042. doi:1538-7445.AM2012-1042

Comparative expression analysis of the H3K27 demethylases, JMJD3 and UTX, with the H3K27 methylase, EZH2, in Xenopus

The regulated removal of the gene-silencing epigenetic mark, trimethylation of lysine 27 of histone H3 (H3K27me3), has been shown to be critical for tissue-specific activation of developmental genes; however, the extent of embryonic expression of its demethylases, JMJD3 and UTX, has remained unclear. In this study, we investigated the expression of jmjd3 and utx genes in Xenopus embryos in parallel with that of the H3K27 methylase gene, ezh2. At the blastula stage, jmjd3, utx and ezh2 showed similar expression patterns in the animal cap and marginal zone that give rise to the ectoderm and mesoderm, respectively. The three genes maintained similar expression patterns in the neural plate, preplacodal ectoderm and axial mesoderm during the gastrula and neurula stages. Later, expression was maintained in the developing brain and cranial sensory tissues, such as the eye and ear, of tailbud embryos. These findings suggest that the H3K27 demethylases and methylase may function continuously for progressive switching of genetic programs during neural development, a model involving the simultaneous action of both of the demethylases for the de-repression of silent genes and the methylase for the silencing of active genes.

Mutational Spectrum In Chronic Myelomonocytic Leukemia Includes Genes Associated with Epigenetic Regulation Such as UTX and EZH2

AbstractAbstract 611Chronic myelomonocytic leukemia (CMML), a myelodysplastic/myeloproliferative overlap neoplasm, is characterized by monocytic proliferation, cytomorphologic dysplasia and frequent progression to acute myelogeneous leukemia (AML). The molecular basis of CMML is poorly defined, although somatic mutations in a number of genes have recently been identified in a proportion of patients. Single nucleotide polymorphisms array (SNP-A) technologies have improved the definition of shared regions of loss of heterozygosity (LOH), including uniparental disomy (UPD) and facilitated discovery of new mutations c-CBL, TET2, and EZH2 which can occur in a homozygous configuration in the areas of UPD. Other mutations such as ASXL1 have been found in heterozygous form. In myeloid malignancies we have also identified mutations in UTX, which like EZH2 and ASXL1, are involved in modification of histone methylation. Based on these findings we hypothesized that defining the mutational spectrum of CMML would help in the molecular characterization of this disease and have diagnostic and prognostic significance. Within this spectrum, we stipulated that various genes involved in epigenetic regulation may be especially affected by mutations in CMML.Here we present results of broad molecular screen in a group of 63 patients with CMML (32 CMML-1, 15 CMML-2 and 16 CMML-derived sAML) which included SNP-A karyotyping and mutational screen for IDH1/2, RAS, TET2, ASXL1, c-CBL, JAK2, UTX and EZH2. First, we aligned all lesions that were detected by SNP-A. In addition to microdeletions involving 4q24 and 11q23.3, we detected recurrent areas of somatic UPD involving chromosomes 1, 4, 7 and 11 and the corresponding homozygous mutations in RAS (UPD1p, N=1), EZH2 (UPD7q N=3), c-CBL (UPD11q, N=4), TET2 (UPD4q, N=6), and UTX genes (UPDXq, N=1). When all patients were sequenced, TET2, ASXL1, c-CBL, IDH1/2, RAS, JAK2, UTX and EZH2 mutations were found in 48%, 24%, 14%, 5%, 11%, 2%, 6% and 8% of patients, respectively. In 78% of patients, >1 mutation was found. Concomitant second and third mutations were found in 34% and 5% of patients, respectively. The most frequently observed combinations included TET2 and ASXL1 (14%) and TET2 and c-CBL (6%). Only 22% of patients had no alterations in analyzed genes. Novel UTX and EZH2 mutations were present either alone or in combination with other mutations. Study of potential functional consequences of the foregoing gene mutations revealed an association of TET2 mutations with consistently low levels of 5-hydroxymethylcytosine (5-hmC), quantitated by dot blot assay, while c-CBL mutations were associated with aberrant phospho-STAT5 staining. Loss of H3K27-me3 in cases with EZH2 mutations but not controls, and an increase in UTX mutant case was identified as measured by ELISA and western blot.When we tested for association of different mutations with pathomorphologic features, specific clinical features were not identified, except for an association of TET2 and c-CBL mutations with more advanced age (p=.0004 and p=.02, respectively), RAS mutation with increased blasts (p=.03) and UTX with dysplastic megakaryocytes (p=.03). Splenomegaly was noted more frequent in c-CBL mutants than any other patient group. No differences in OS and EFS were observed between mutant and wt cases. There is a trend toward better OS in TET2 mutants compared to WT in the good cytogenetic risk group (17 vs 8 mo, p=.07) but worse outcomes in TET2 mutants in the intermediate cytogenetic risk group (OS 2 vs. 16 mo, p=.001; EFS 2 vs. 9 mo, p=.04). As expected, patients who have accumulated more mutations have a trend toward inferior outcomes compared to those with single mutations but better than those who are WT (>1 mutations vs 1 mutation vs WT, 16 vs 18 vs 9 mo, p=.07 in low risk CMML).In summary, our study identified the presence of a wide spectrum of mutations in CMML with various combinations, including the newly discovered mutations in UTX and EZH2 genes. Our results suggest that molecular abnormalities affecting various pathways can lead to a clinically indistinguishable phenotype. It is possible that these mutations are secondary in nature but work in conjunction with a yet unidentified founder defect. The abundance of mutations in factors known or hypothesized to be involved in epigenetic regulation in CMML provide important implications for future research into the development of effective therapies for this disease. Disclosures:No relevant conflicts of interest to declare.

UTX Mutations and Epigenetic Changes In MDS/MPN and Related Myeloid Malignancies

AbstractAbstract 121Balanced and unbalanced chromosomal lesions and genetic mutations are hallmarks of myeloid malignancies. In addition, aberrant methylation of CpG islands, leading to epigenetic silencing appears to play a significant role in tumor suppressor gene inactivation and malignant progression. While various mechanisms of chromosomal instability have been identified, the pathogenesis of epigenetic instability remains unexplored. ASXL1, EZH2 and TET2 mutations, found in myeloid disorders provide a potential link between genetic and epigenetic events. UTX is a histone H3K27 demethylase belonging to the polycomb group of proteins. Methylation at lysine 27 correlates with gene silencing and repression. A recent mutational screen of cancer cell lines has identified mutations in UTX in AML THP-1 cells.We screened a series of myeloid disorders for UTX gene mutations and identified an index case in a CMML patient with a somatic UTX missense mutation. This sample demonstrated × chromosome copy neutral loss of heterozygosity (CN-LOH) that included UTX on a whole genome SNP-A array. UTX was then sequenced in 49 additional patients with MDS/MPN and 24 with secondary AML. Two of the MDS/MPN (2/49, 4%) and 3 of the sAML patients (3/24; 12%) showed UTX mutation. Three previously unreported missense polymorphisms were noted (present in paired CD3+ cells and detected with a frequency of <1/400 of controls). No mutations were found in MDS (N=15). The identified lesions were not associated with loss of sex chromosomes. Both homozygous (UPD) and heterozygous UTX mutations were identified and included frame shift or stop codons, as well as missense mutations. Inactivating or hypomorphic mutations may result in similar functional consequences with decreased expression of UTX. We quantitated UTX mRNA abundance in 15 hematopoietic cell lines using TaqMan PCR, and THP1 cells (UTX null) showed the lowest UTX mRNA expression. In primary patient samples, decreased UTX mRNA levels were found in the bone marrow of MDS/MPN patients (N=21) as compared to controls. To examine UTX expression in normal hematopoietic differentiation, CD34+ cells, along with myeloid CD33+ or monocytic CD14+ cell fractions (N=5) were tested and expressed easily quantifiable UTX transcripts, while the highest UTX expression was found in Gly-A+ erythroid precursors.UTX mutation and dysfunction might be predicted to lead to altered histone repression marks and alterations in epigenetic regulation. Consistent with this hypothesis, we tested for H3K27-Me3 methylation by ELISA and observed a 45% methylation increase in a UTX mutant patient sample.To further investigate the function of UTX, consequences and treatment implications of the corresponding mutations we transduced THP-1 cells with a lentiviral vector containing the UTX cDNA (UTX+) or an empty vector. Following clone selection, the resultant UTX+ cell line showed 1000-fold UTX mRNA increase and distinct protein overexpression by western blot (WB), as compared to the baseline UTX null cells. Proliferation kinetics up to 120h indicated that overexpression of UTX results in increased cell proliferation by 20% compared to controls. Treatment with decitabine (120h; 0.5μ M) resulted in a differential effect on UTX+ cells: proliferation increased 4.6-fold vs. 1.3-fold in UTX null cells.Based on the hypothesis that mutation in UTX may lead to epigenetic instability and accumulation of aberrant methylated CpG sites, we further investigated epigenetic changes in UTX+ and UTX null cells. WB showed comparable levels of global H3K27-Me3 and H3 acetylation levels in UTX+ and UTX null cells, and only modest decreases (9%) of trimethylation in a more precise ELISA assay. Similarly, when methylation array (14K genes; 27K CpG sites) was used to assess the effects of UTX overexpression, global methylation levels expressed by averaged β-values did not significantly differ. However, 153 specific CpG sites were found to be differentially methylated on the array: with 68 genes hypo- and 85 hypermethylated. In particular SSRP1, NCOR2 and DIRAS3 genes showed hypomethylated promoters in the UTX+ cells, suggesting that UTX is involved in gene specific demethylation.In sum, our results suggest that UTX gene may be involved in epigenetic regulation of promoters through site-specific histone demethylation function. UTX mutation may compromise this function, thereby promoting repression of tumor suppressor genes. Disclosures:No relevant conflicts of interest to declare.

Identification of the Chromatin Regions Coated by Non-coding Xist RNA

Xist non-coding RNA (ncRNA) is essential for X chromosome inactivation (XCI). Some genes can escape from XCI, but how this occurs is unknown. We developed a modified RNA tagging and recovery of associated proteins (TRAP) method to study the association between Xist RNA and its target genes. In mouse cells, Xist RNA was detected on the Uba1 gene, but not on Jarid1c and Utx genes, which escape from XCI. Using this technique we were able to show that the Xist RNA molecule is not present on active genes that escape from XCI, but is present on genes inactivated by XCI, suggesting that this method is a powerful tool for functional analysis of ncRNA.

The UTX gene escapes X inactivation in mice and humans.

We recently have identified a ubiquitously transcribed mouse Y chromosome gene, Uty , which encodes a tetratricopeptide repeat (TPR) protein. A peptide derived from the UTY protein confers H-Y antigenicity on male cells. Here we report the characterization of a widely transcribed X-linked homologue of Uty , called Utx , which maps to the proximal region of the mouse X chromosome and which detects a human X-linked homologue at Xp11.2. Given that Uty is ubiquitously transcribed, we assayed for Utx expression from the inactive X chromosome (Xi) in mice and found that Utx escapes X chromosome inactivation. Only Smcx and the pseudoautosomal Sts gene on the mouse X chromosome have been reported previously to escape inactivation. The human UTX gene was also found to be expressed from Xi. We discuss the significance of these data for our understanding of dosage compensation of X-Y homologous genes in humans and mice.