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1.  Drosophila UTX Is a Histone H3 Lys27 Demethylase That Colocalizes with the Elongating Form of RNA Polymerase II▿  
Molecular and Cellular Biology  2007;28(3):1041-1046.
Histone H3 methylation at Lys27 (H3K27 methylation) is a hallmark of silent chromatin, while H3K4 methylation is associated with active chromatin regions. Here we report 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. Our 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.
PMCID: PMC2223382  PMID: 18039863
2.  UTX coordinates steroid hormone-mediated autophagy and cell death 
Nature communications  2013;4:2916.
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. Here we show 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. We demonstrate that 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. We further show that salivary gland degradation requires dUTX catalytic activity. Our 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.
PMCID: PMC3973156  PMID: 24336022
3.  UTX and UTY Demonstrate Histone Demethylase-Independent Function in Mouse Embryonic Development 
PLoS Genetics  2012;8(9):e1002964.
UTX (KDM6A) and UTY are homologous X and Y chromosome members of the Histone H3 Lysine 27 (H3K27) demethylase gene family. UTX can demethylate H3K27; however, in vitro assays suggest that human UTY has lost enzymatic activity due to sequence divergence. We produced mouse mutations in both Utx and Uty. Homozygous Utx mutant female embryos are mid-gestational lethal with defects in neural tube, yolk sac, and cardiac development. We demonstrate that mouse UTY is devoid of in vivo demethylase activity, so hemizygous XUtx− Y+ mutant male embryos should phenocopy homozygous XUtx− XUtx− females. However, XUtx− Y+ mutant male embryos develop to term; although runted, approximately 25% survive postnatally reaching adulthood. Hemizygous X+ YUty− mutant males are viable. In contrast, compound hemizygous XUtx− YUty− males phenocopy homozygous XUtx− XUtx− females. Therefore, despite divergence of UTX and UTY in catalyzing H3K27 demethylation, they maintain functional redundancy during embryonic development. Our data suggest that UTX and UTY are able to regulate gene activity through demethylase independent mechanisms. We conclude that UTX H3K27 demethylation is non-essential for embryonic viability.
Author Summary
Trimethylation at Lysine 27 of histone H3 (H3K27me3) establishes a repressive chromatin state in silencing an array of crucial developmental genes. Polycomb repressive complex 2 (PRC2) catalyzes this precise posttranslational modification and is required in several critical aspects of development including Hox gene repression, gastrulation, X-chromosome inactivation, mono-allelic gene expression and imprinting, stem cell maintenance, and oncogenesis. Removal of H3K27 trimethylation has been proposed to be a mechanistic switch to activate large sets of genes in differentiating cells. Mouse Utx is an X-linked H3K27 demethylase that is essential for embryonic development. We now demonstrate that Uty, the Y-chromosome homolog of Utx, has overlapping redundancy with Utx in embryonic development. Mouse UTY has a polymorphism in the JmjC demethylase domain that renders the protein incapable of H3K27 demethylation. Therefore, the overlapping function of UTX and UTY in embryonic development is due to H3K27 demethylase independent mechanism. Moreover, the presence of UTY allows UTX-deficient mouse embryos to survive until birth. Thus, UTX H3K27 demethylation is not essential for embryonic viability. These intriguing results raise new questions on how H3K27me3 repression is removed in the early embryo.
PMCID: PMC3459986  PMID: 23028370
4.  The histone H3-K27 demethylase Utx regulates HOX gene expression in Drosophila in a temporally restricted manner 
Development (Cambridge, England)  2013;140(16):3478-3485.
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.
PMCID: PMC3912917  PMID: 23900545
Polycomb; PcG; Trithorax; trxG; Utx; H3-K27me3
5.  The Drosophila Ortholog of MLL3 and MLL4, trithorax related, Functions as a Negative Regulator of Tissue Growth 
Molecular and Cellular Biology  2013;33(9):1702-1710.
The human MLL genes (MLL1 to MLL4) and their Drosophila orthologs, trithorax (trx) and trithorax related (trr), encode proteins capable of methylating histone H3 on lysine 4. MLL1 and MLL2 are most similar to trx, while MLL3 and MLL4 are more closely related to trr. Several MLL genes are mutated in human cancers, but how these proteins regulate cell proliferation is not known. Here we show that trr mutant cells have a growth advantage over their wild-type neighbors and display changes in the levels of multiple proteins that regulate growth and cell division, including Notch, Capicua, and cyclin B. trr mutant clones display markedly reduced levels of H3K4 monomethylation without obvious changes in the levels of H3K4 di- and trimethylation. The trr mutant phenotype resembles that of Utx, which encodes a H3K27 demethylase, consistent with the observation that Trr and Utx are found in the same protein complex. In contrast to the overgrowth displayed by trr mutant tissue, trx clones are underrepresented, express low levels of the antiapoptotic protein Diap1, and exhibit only modest changes in global levels of H3K4 methylation. Thus, in Drosophila eye imaginal discs, Trr, likely functioning together with Utx, restricts tissue growth. In contrast, Trx appears to promote cell survival.
PMCID: PMC3624179  PMID: 23459941
6.  KDM6 Demethylase Independent Loss of Histone H3 Lysine 27 Trimethylation during Early Embryonic Development 
PLoS Genetics  2014;10(8):e1004507.
The early mammalian embryo utilizes histone H3 lysine 27 trimethylation (H3K27me3) to maintain essential developmental genes in a repressive chromatin state. As differentiation progresses, H3K27me3 is removed in a distinct fashion to activate lineage specific patterns of developmental gene expression. These rapid changes in early embryonic chromatin environment are thought to be dependent on H3K27 demethylases. We have taken a mouse genetics approach to remove activity of both H3K27 demethylases of the Kdm6 gene family, Utx (Kdm6a, X-linked gene) and Jmjd3 (Kdm6b, autosomal gene). Male embryos null for active H3K27 demethylation by the Kdm6 gene family survive to term. At mid-gestation, embryos demonstrate proper patterning and activation of Hox genes. These male embryos retain the Y-chromosome UTX homolog, UTY, which cannot demethylate H3K27me3 due to mutations in catalytic site of the Jumonji-C domain. Embryonic stem (ES) cells lacking all enzymatic KDM6 demethylation exhibit a typical decrease in global H3K27me3 levels with differentiation. Retinoic acid differentiations of these ES cells demonstrate loss of H3K27me3 and gain of H3K4me3 to Hox promoters and other transcription factors, and induce expression similar to control cells. A small subset of genes exhibit decreased expression associated with reduction of promoter H3K4me3 and some low-level accumulation of H3K27me3. Finally, Utx and Jmjd3 mutant mouse embryonic fibroblasts (MEFs) demonstrate dramatic loss of H3K27me3 from promoters of several Hox genes and transcription factors. Our results indicate that early embryonic H3K27me3 repression can be alleviated in the absence of active demethylation by the Kdm6 gene family.
Author Summary
H3K27me3 represses developmental genes at initial embryonic stages. The KDM6 family, comprised of UTX and JMJD3, are the only known proteins that demethylate H3K27me3 and they are hypothesized to catalyze the rapid removal of repressive chromatin in early mammalian development. However, we report that male embryos carrying mutations in both Utx and Jmjd3 survive to term and appear phenotypically normal at mid-gestation. We utilize several cell culture models to demonstrate that H3K27me3 is lost from repressed promoters in the absence of active KDM6 demethylation. Our data indicate that KDM6 H3K27me3 demethylation is not essential in the early embryo and that H3K27me3 loss from developmental genes occurs via novel mechanisms.
PMCID: PMC4125042  PMID: 25101834
7.  Histone Demethylase UTX and Chromatin Remodeler BRM Bind Directly to CBP and Modulate Acetylation of Histone H3 Lysine 27 
Molecular and Cellular Biology  2012;32(12):2323-2334.
Trithorax group (TrxG) proteins antagonize Polycomb silencing and are required for maintenance of transcriptionally active states. We previously showed that the Drosophila melanogaster acetyltransferase CREB-binding protein (CBP) acetylates histone H3 lysine 27 (H3K27ac), thereby directly blocking its trimethylation (H3K27me3) by Polycomb repressive complex 2 (PRC2) in Polycomb target genes. Here, we show that H3K27ac levels also depend on other TrxG proteins, including the histone H3K27-specific demethylase UTX and the chromatin-remodeling ATPase Brahma (BRM). We show that UTX and BRM are physically associated with CBP in vivo and that 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. We propose that direct binding of UTX and BRM to CBP and their modulation of H3K27ac play an important role in antagonizing Polycomb silencing.
PMCID: PMC3372260  PMID: 22493065
8.  The C. elegans H3K27 Demethylase UTX-1 Is Essential for Normal Development, Independent of Its Enzymatic Activity 
PLoS Genetics  2012;8(5):e1002647.
Epigenetic modifications influence gene expression and provide a unique mechanism for fine-tuning cellular differentiation and development in multicellular organisms. Here we report on the biological functions of UTX-1, the Caenorhabditis elegans homologue of mammalian UTX, a histone demethylase specific for H3K27me2/3. We demonstrate that utx-1 is an essential gene that is required for correct embryonic and postembryonic development. Consistent with its homology to UTX, UTX-1 regulates global levels of H3K27me2/3 in C. elegans. Surprisingly, we found that the catalytic activity is not required for the developmental function of this protein. Biochemical analysis identified UTX-1 as a component of a complex that includes SET-16(MLL), and genetic analysis indicates that the defects associated with loss of UTX-1 are likely mediated by compromised SET-16/UTX-1 complex activity. Taken together, these results demonstrate that UTX-1 is required for many aspects of nematode development; but, unexpectedly, this function is independent of its enzymatic activity.
Author Summary
Chromatin organization influences gene expression, and its regulation is crucial to achieve correct cellular differentiation and development in multicellular organisms. Histone demethylases are among several factors responsible for regulating chromatin dynamics. Here we report on the biological functions of UTX-1, the C. elegans homologue of the mammalian histone demethylase UTX, which specifically catalyzes the demethylation of di- and tri-methylated lysine 27 of histone H3 (H3K27me2/3). Indeed, we demonstrate that UTX-1 regulates global levels of H3K27me2/3 in C. elegans, a mark generally associated with silencing of gene expression. We also show that utx-1 is an essential gene that is required for correct embryonic and postembryonic development. Specifically, the loss of utx-1 results in developmental defects, sterility, and embryonic lethality. Surprisingly, our data show that the catalytic activity of UTX-1 is not required for its developmental functions. Our biochemical and genetic analyses indicate that loss of UTX-1 compromises the activity of the SET-16(MLL) complex, which UTX-1 is an integral part of. Taken together, these results demonstrate that UTX-1 plays an essential role in development independent of its enzymatic activity.
PMCID: PMC3342935  PMID: 22570628
9.  UTX mediates demethylation of H3K27me3 at muscle-specific genes during myogenesis 
The EMBO Journal  2010;29(8):1401-1411.
Polycomb (PcG) and Trithorax (TrxG) group proteins act antagonistically to establish tissue-specific patterns of gene expression. The PcG protein Ezh2 facilitates repression by catalysing histone H3-Lys27 trimethylation (H3K27me3). For expression, H3K27me3 marks are removed and replaced by TrxG protein catalysed histone H3-Lys4 trimethylation (H3K4me3). Although H3K27 demethylases have been identified, the mechanism by which these enzymes are targeted to specific genomic regions to remove H3K27me3 marks has not been established. Here, we demonstrate a two-step mechanism for UTX-mediated demethylation at muscle-specific genes during myogenesis. Although the transactivator Six4 initially recruits UTX to the regulatory region of muscle genes, the resulting loss of H3K27me3 marks is limited to the region upstream of the transcriptional start site. Removal of the repressive H3K27me3 mark within the coding region then requires RNA Polymerase II (Pol II) elongation. Interestingly, blocking Pol II elongation on transcribed genes leads to increased H3K27me3 within the coding region, and formation of bivalent (H3K27me3/H3K4me3) chromatin domains. Thus, removal of repressive H3K27me3 marks by UTX occurs through targeted recruitment followed by spreading across the gene.
PMCID: PMC2868576  PMID: 20300060
chromatin; histone demethylase; myogenesis; transcription; UTX
10.  Drosophila UTX Coordinates with p53 to Regulate ku80 Expression in Response to DNA Damage 
PLoS ONE  2013;8(11):e78652.
UTX is known as a general factor that activates gene transcription during development. Here, we demonstrate 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. We further showed that 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, our findings further elucidate the molecular function of UTX.
PMCID: PMC3827076  PMID: 24265704
11.  Expression and significance of histone H3K27 demethylases in renal cell carcinoma 
BMC Cancer  2012;12:470.
The histone H3K27 demethylases UTX and JMJD3 are important regulatory factors that modulate gene expression by altering the physical state of chromatin. Previous studies have indicated an abnormal H3K27 methylation status in carcinogenesis. We therefore investigated the expression patterns of UTX and JMJD3 in renal cell carcinoma (RCC) and their roles in cancer development.
The mRNA expression levels of the UTX and JMJD3 genes were determined in cancer tissues and adjacent normal tissues in 36 patients with primary RCC, using quantitative real-time-polymerase chain reaction. The UTX and JMJD3 protein contents were measured by western blotting and immunohistochemical analysis.
UTX and JMJD3 transcripts were significantly increased in cancer tissues compared to normal tissues (P < 0.05). mRNA levels of the inhibitor of cyclin-dependent kinases 4 and 6 p16INK4a were also increased in cancer tissues (P < 0.001). Western blotting indicated that levels of both demethylases were increased in cancer tissues. The level of tri-methylated H3K27 (H3K27me3) was lower in cancer tissues compared to normal tissues, but expression of the H3K27 methyltransferase EZH2 was increased (P < 0.05). These results suggest that the two H3K27 demethylases may play critical roles in the regulation of H3K27 methylation status in RCC. Immunohistochemical analysis confirmed that UTX and JMJD3 expression were upregulated in cancer tissues compared to adjacent tissues.
This study demonstrated that UTX and JMJD3 were upregulated in cancer tissues, suggesting that they may be involved in the development of primary RCC. The potential roles of H3K27 demethylases as biomarkers in the early diagnosis of RCC need to be further explored.
PMCID: PMC3520868  PMID: 23057811
Renal cell carcinoma; Histone H3K27 demethylase; UTX; JMJD3; Epigenetics
12.  Utx Is Required for Proper Induction of Ectoderm and Mesoderm during Differentiation of Embryonic Stem Cells 
PLoS ONE  2013;8(4):e60020.
Embryonic development requires chromatin remodeling for dynamic regulation of gene expression patterns to ensure silencing of pluripotent transcription factors and activation of developmental regulators. Demethylation of H3K27me3 by the histone demethylases Utx and Jmjd3 is important for the activation of lineage choice genes in response to developmental signals. To further understand the function of Utx in pluripotency and differentiation we generated Utx knockout embryonic stem cells (ESCs). Here we show that Utx is not required for the proliferation of ESCs, however, Utx contributes to the establishment of ectoderm and mesoderm in vitro. Interestingly, this contribution is independent of the catalytic activity of Utx. Furthermore, we provide data showing that the Utx homologue, Uty, which is devoid of detectable demethylase activity, and Jmjd3 partly compensate for the loss of Utx. Taken together our results show that Utx is required for proper formation of ectoderm and mesoderm in vitro, and that Utx, similar to its C.elegans homologue, has demethylase dependent and independent functions.
PMCID: PMC3616089  PMID: 23573229
13.  UTX, a histone H3-lysine 27 demethylase, acts as a critical switch to activate the cardiac developmental program 
Developmental cell  2011;22(1):25-37.
The removal of histone H3 lysine27 (H3K27) trimethylation mark is important for the robust induction of many cell type-specific genes during differentiation. Here we show that UTX, a H3K27 demethylase, acts as a critical switch to promote a cardiac-specific gene program. UTX-deficient ESCs failed to develop heart-like rhythmic contractions under a cardiac differentiation condition. UTX deficient mice show severe defects in heart development and embryonic lethality. We found that UTX is recruited to cardiac-specific enhancers by associating with core cardiac transcription factors and demethylates H3K27 residues in cardiac genes. In addition, UTX facilitates the recruitment of Brg1 to the cardiac-specific enhancers. Together, our data reveal key roles for UTX in a timely transition from poised to active chromatin in cardiac genes during heart development and a fundamental mechanism by which a H3K27 demethylase triggers tissue-specific chromatin changes.
PMCID: PMC4111644  PMID: 22192413
UTX; KDM6A; histone demethylase; H3K27; histone; chromatin; transcription; heart; development
14.  H3K27 Demethylase UTX-1 Regulates C. elegans Lifespan in a Germline-Independent, Insulin-Dependent, Manner 
Aging cell  2011;10(6):980-990.
Aging is accompanied by alterations in epigenetic marks that control chromatin states, including histone acetylation and methylation. Enzymes that reversibly affect histone marks associated with active chromatin have recently been found to regulate aging in C. elegans. However, relatively little is known about the importance for aging of histone marks associated with repressed chromatin. Here we use a targeted RNAi screen in C. elegans to identify four histone demethylases that significantly regulate worm lifespan, UTX-1, RBR-2, LSD-1, and T26A5.5. Interestingly, UTX-1 belongs to a conserved family of histone demethylases specific for lysine 27 of histone H3 (H3K27me3), a mark associated with repressed chromatin. Both utx-1 knock-down and heterozygous mutation of utx-1 extend lifespan and increase the global levels of the H3K27me3 mark in worms. The H3K27me3 mark significantly drops in somatic cells during the normal aging process. UTX-1 regulates lifespan independently of the presence of the germline, but in a manner that depends on the insulin-FoxO signaling pathway. These findings identify the H3K27me3 histone demethylase UTX-1 as a novel regulator of worm lifespan in somatic cells.
PMCID: PMC3215905  PMID: 21834846
Histone demethylase; aging; lifespan; UTX; H3K27me3; epigenetic; Insulin pathway; FoxO transcription factor; germline; soma; C. elegans
15.  A Companion Cell–Dominant and Developmentally Regulated H3K4 Demethylase Controls Flowering Time in Arabidopsis via the Repression of FLC Expression 
PLoS Genetics  2012;8(4):e1002664.
Flowering time relies on the integration of intrinsic developmental cues and environmental signals. FLC and its downstream target FT are key players in the floral transition in Arabidopsis. Here, we characterized the expression pattern and function of JMJ18, a novel JmjC domain-containing histone H3K4 demethylase gene in Arabidopsis. JMJ18 was dominantly expressed in companion cells; its temporal expression pattern was negatively and positively correlated with that of FLC and FT, respectively, during vegetative development. Mutations in JMJ18 resulted in a weak late-flowering phenotype, while JMJ18 overexpressors exhibited an obvious early-flowering phenotype. JMJ18 displayed demethylase activity toward H3K4me3 and H3K4me2, and bound FLC chromatin directly. The levels of H3K4me3 and H3K4me2 in chromatins of FLC clade genes and the expression of FLC clade genes were reduced, whereas FT expression was induced and the protein expression of FT increased in JMJ18 overexpressor lines. The early-flowering phenotype caused by the overexpression of JMJ18 was mainly dependent on the functional FT. Our findings suggest that the companion cell–dominant and developmentally regulated JMJ18 binds directly to the FLC locus, reducing the level of H3K4 methylation in FLC chromatin and repressing the expression of FLC, thereby promoting the expression of FT in companion cells to stimulate flowering.
Author Summary
Flowering is an important developmental transition during plant life cycle and the key process for production of the next generation. Flowering time is controlled by both intrinsic developmental and environmental signals. FLC and its target FT work as repressor and activator, respectively, to regulate flowering time in Arabidopsis; thus the regulation of FLC and FT expression is the key for the control of floral transition. Epigenetic modifications are critical for transcription regulation. Here, we show that a novel JmjC domain-containing histone H3K4 demethylase, JMJ18, is a key regulator for the expression of FLC and FT in companion cells and flowering time. JMJ18 is dominantly expressed in vascular tissue; its temporal expression pattern was developmentally regulated, and negatively and positively correlated with FLC and FT, respectively. JMJ18 mutation leads to weak late-flowering, while JMJ18 overexpressor exhibited an obvious early-flowering phenotype. JMJ18 binds to chromatin of FLC, represses its expression, and releases expression of FT in companion cells. Our results suggest that JMJ18 is a developmentally regulated companion cell–dominantly expressed signal to control flowering time by binding to FLC—reducing level of H3K4 methylation in FLC and repressing expression of FLC—thereby promoting expression of FT in companion cells during vegetative development in Arabidopsis.
PMCID: PMC3334889  PMID: 22536163
16.  Structural Basis of a Histone H3 Lysine 4 Demethylase Required for Stem Elongation in Rice 
PLoS Genetics  2013;9(1):e1003239.
Histone lysine methylation is an important epigenetic modification in regulating chromatin structure and gene expression. Histone H3 lysine 4 methylation (H3K4me), which can be in a mono-, di-, or trimethylated state, has been shown to play an important role in gene expression involved in plant developmental control and stress adaptation. However, the resetting mechanism of this epigenetic modification is not yet fully understood. In this work, we identified a JmjC domain-containing protein, JMJ703, as a histone lysine demethylase that specifically reverses all three forms of H3K4me in rice. Loss-of-function mutation of the gene affected stem elongation and plant growth, which may be related to increased expression of cytokinin oxidase genes in the mutant. Analysis of crystal structure of the catalytic core domain (c-JMJ703) of the protein revealed a general structural similarity with mammalian and yeast JMJD2 proteins that are H3K9 and H3K36 demethylases. However, several specific features were observed in the structure of c-JMJ703. Key residues that interact with cofactors Fe(II) and N-oxalylglycine and the methylated H3K4 substrate peptide were identified and were shown to be essential for the demethylase activity in vivo. Several key residues are specifically conserved in known H3K4 demethylases, suggesting that they may be involved in the specificity for H3K4 demethylation.
Author Summary
Genomic DNA is associated with histone proteins to form the basic structure of chromatin. Lysine residues within the N-terminal end of histones H3 and H4 can be methylated, which may have a positive or a negative effect on the activity of associated DNA or genes, depending on the position of the lysines in the histones. Histone lysine methylation can be reversed by histone demethylases. However, it is not very clear how the specificity of histone demethylases to different histone lysines is determined. In this work we have identified a rice histone demethylase, namely JMJ703, which specifically demethylates methylated histone H3 lysine 4. We found that loss of the enzyme reduces cell division rate of the stem and the size of plant stature, indicating the importance of the protein in plant growth. The crystal structure of the catalytic domain of the protein shares a general similarity with that of mammalian and yeast proteins that demethylate methylated histone H3 lysine 9 and lysine 36, but displays several distinct structural features that are important for substrate and cofactor binding and enzymatic activity of the protein. We found that key amino acids involved in the specific structures are conserved within known H3 lysine 4 demethylases, which may be involved in the specificity to histone H3 lysine 4.
PMCID: PMC3554631  PMID: 23357881
17.  PTIP Associates with MLL3- and MLL4-containing Histone H3 Lysine 4 Methyltransferase Complex*…◆ 
The Journal of biological chemistry  2007;282(28):20395-20406.
PTIP, a protein with tandem BRCT domains, has been implicated in DNA damage response. However, its normal cellular functions remain unclear. Here we show that while ectopically expressed PTIP is capable of interacting with DNA damage response proteins including 53BP1, endogenous PTIP, and a novel protein PA1 are both components of a Set1-like histone methyltransferase (HMT) complex that also contains ASH2L, RBBP5, WDR5, hDPY-30, NCOA6, SET domain-containing HMTs MLL3 and MLL4, and substoichiometric amount of JmjC domain-containing putative histone demethylase UTX. PTIP complex carries robust HMT activity and specifically methylates lysine 4 (K4) on histone H3. Furthermore, PA1 binds PTIP directly and requires PTIP for interaction with the rest of the complex. Moreover, we show that hDPY-30 binds ASH2L directly. The evolutionarily conserved hDPY-30, ASH2L, RBBP5, and WDR5 likely constitute a subcomplex that is shared by all human Set1-like HMT complexes. In contrast, PTIP, PA1, and UTX specifically associate with the PTIP complex. Thus, in cells without DNA damage agent treatment, the endogenous PTIP associates with a Set1-like HMT complex of unique subunit composition. As histone H3 K4 methylation associates with active genes, our study suggests a potential role of PTIP in the regulation of gene expression.
PMCID: PMC2729684  PMID: 17500065
18.  Essential Functions of the Histone Demethylase Lid 
PLoS Genetics  2010;6(11):e1001221.
Drosophila Little imaginal discs (Lid) is a recently described member of the JmjC domain class of histone demethylases that specifically targets trimethylated histone H3 lysine 4 (H3K4me3). To understand its biological function, we have utilized a series of Lid deletions and point mutations to assess the role that each domain plays in histone demethylation, in animal viability, and in cell growth mediated by the transcription factor dMyc. Strikingly, we find that lid mutants are rescued to adulthood by either wildtype or enzymatically inactive Lid expressed under the control of its endogenous promoter, demonstrating that Lid's demethylase activity is not essential for development. In contrast, ubiquitous expression of UAS-Lid transgenes lacking its JmjN, C-terminal PHD domain, and C5HC2 zinc finger were unable to rescue lid homozygous mutants, indicating that these domains carry out Lid's essential developmental functions. Although Lid-dependent demethylase activity is not essential, dynamic removal of H3K4me3 may still be an important component of development, as we have observed a genetic interaction between lid and another H3K4me3 demethylase, dKDM2. We also show that Lid's essential C-terminal PHD finger binds specifically to di- and trimethylated H3K4 and that this activity is required for Lid to function in dMyc-induced cell growth. Taken together, our findings highlight the importance of Lid function in the regulated removal and recognition of H3K4me3 during development.
Author Summary
Correct spatial and temporal control of gene expression is essential for development. One of the many ways that gene expression is regulated is by the addition, recognition, and removal of methyl groups from the histone proteins around which DNA is wrapped within the nucleus. Here we describe a systematic analysis of Little imaginal discs (Lid), a protein that regulates transcription via a number of different mechanisms that involve regulated removal and recognition of histone methylation. We show that while Lid's histone demethylase activity is not essential for development, numerous other conserved domains of this protein are. Furthermore, we find a genetic interaction between lid and another histone demethylase, dKDM2, that suggests this enzyme can compensate for the loss of Lid's enzymatic activity. These findings have significance for our insight into how gene expression is normally regulated and have implications for our understanding of how this goes awry during disease progression.
PMCID: PMC2991268  PMID: 21124823
19.  Identification of Histone Demethylases in Saccharomyces cerevisiae*s 
The Journal of biological chemistry  2007;282(19):14262-14271.
Based on the prediction that histone lysine demethylases may contain the JmjC domain, we examined the methylation patterns of five knock-out strains (ecm5Δ, gis1Δ, rph1Δ, jhd1Δ, and jhd2Δ (yjr119cΔ)) of Saccharomyces cerevisiae. Mass spectrometry (MS) analyses of histone H3 showed increased modifications in all mutants except ecm5Δ. High-resolution MS was used to unequivocally differentiate trimethylation from acetylation in various tryptic fragments. The relative abundance of specific fragments indicated that histones K36me3 and K4me3 accumulate in rph1Δ and jhd2Δ strains, respectively, whereas both histone K36me2 and K36me accumulate in gis1Δ and jhd1Δ strains. Analyses performed with strains overexpressing the JmjC proteins yielded changes in methylation patterns that were the reverse of those obtained in the complementary knock-out strains. In vitro enzymatic assays confirmed that the JmjC domain of Rph1 specifically demethylates K36me3 primarily and K36me2 secondarily. Overexpression of RPH1 generated a growth defect in response to UV irradiation. The demethylase activity of Rph1 is responsible for the phenotype. Collectively, in addition to Jhd1, our results identified three novel JmjC domain-containing histone demethylases and their sites of action in budding yeast S. cerevisiae. Furthermore, the methodology described here will be useful for identifying histone demethylases and their target sites in other organisms.
PMCID: PMC2855503  PMID: 17369256
20.  Interleukin-4-Mediated 15-Lipoxygenase-1 Trans-Activation Requires UTX Recruitment and H3K27me3 Demethylation at the Promoter in A549 Cells 
PLoS ONE  2014;9(1):e85085.
Arachidonate 15-lipoxygenase-1 (ALOX15) oxygenates polyunsaturated fatty acids and bio-membranes, generating multiple lipid signalling mediators involved in inflammation. Several lines of evidence indicate that ALOX15 activation in the respiratory tract contributes to asthma progression. Recent experimental data reveals that histone modification at the promoter plays a critical role in ALOX15 gene transcription. In the present study, we examined the status of histone H3 trimethyl-lysine 27 (H3K27me3) at the ALOX15 promoter by chromatin immunoprecipitation assay in human lung epithelial carcinoma A549 cells incubated with or without interleukin (IL)-4. We identified demethylation of H3K27me3 at the ALOX15 promoter after IL-4 treatment. Furthermore, we found that the H3K27me2/3-specific demethylase, ubiquitously transcribed tetratricopeptide repeat, X chromosome (UTX), mediates the H3K27me3 demethylation during ALOX15 transcriptional activation. When UTX expression was knocked down using siRNA, IL-4-mediated H3K27me3 demethylation and ALOX15 induction were significantly attenuated. The critical role of UTX in ALOX15 expression was confirmed in human monocytes and the Hodgkin lymphoma (HL) cell line L1236, but was in these cells not related to H3K27me3-demethylase activity. These results demonstrate that UTX is implicated in IL-4 mediated transcriptional activation of the ALOX15 gene.
PMCID: PMC3896354  PMID: 24465480
21.  Sex-specific Differences in Expression of Histone Demethylases Utx and Uty in Mouse Brain and Neurons 
Although X inactivation is thought to balance gene expression between the sexes, some genes escape inactivation, potentially contributing to differences between males and females. Utx is an escapee gene that encodes a demethylase specific for lysine 27 of histone H3, a mark of repressed chromatin. We found Utx to be expressed higher in females than in males in developing and adult brains and in adult liver. XX mice had a higher level of Utx than XY mice regardless of whether they had testes or ovaries, indicating that the sexually dimorphic gene expression was a consequence of the sex chromosome complement. Females had significantly higher levels of Utx than males in most brain regions except in the amygdala. The regional expression of the Y-linked paralogue Uty was somewhat distinct from that of Utx, specifically in the paraventricular nucleus of the hypothalamus (high Uty) and the amygdala (high Utx), implying that the two paralogues may be differentially regulated. Higher expression of Utx compared to Uty was detected in P19 pluripotent embryonic carcinoma cells as well as in P19-derived neurons. This transcriptional divergence between the two paralogues was associated with high levels of histone H3 lysine 4 dimethylation at the Utx promoter and of histone H4 lysine 16 acetylation throughout the gene body, which suggests that epigenetic mechanisms control differential expression of paralogous genes.
PMCID: PMC2643472  PMID: 18434530
neuron; cognition; Turner syndrome; Klinefelter's syndrome; estrogen; androgen
22.  Functional Interactions between the erupted/tsg101 Growth Suppressor Gene and the DaPKC and rbf1 Genes in Drosophila Imaginal Disc Tumors 
PLoS ONE  2009;4(9):e7039.
The Drosophila gene erupted (ept) encodes the fly homolog of human Tumor Susceptibility Gene-101 (TSG101), which functions as part of the conserved ESCRT-1 complex to facilitate the movement of cargoes through the endolysosomal pathway. Loss of ept or other genes that encode components of the endocytic machinery (e.g. synatxin7/avalanche, rab5, and vps25) produces disorganized overgrowth of imaginal disc tissue. Excess cell division is postulated to be a primary cause of these ‘neoplastic’ phenotypes, but the autonomous effect of these mutations on cell cycle control has not been examined.
Principal Findings
Here we show that disc cells lacking ept function display an altered cell cycle profile indicative of deregulated progression through the G1-to-S phase transition and express reduced levels of the tumor suppressor ortholog and G1/S inhibitor Rbf1. Genetic reductions of the Drosophila aPKC kinase (DaPKC), which has been shown to promote tumor growth in other fly tumor models, prevent both the ept neoplastic phenotype and the reduction in Rbf1 levels that otherwise occurs in clones of ept mutant cells; this effect is coincident with changes in localization of Notch and Crumbs, two proteins whose sorting is altered in ept mutant cells. The effect on Rbf1 can also be blocked by removal of the γ-secretase component presenilin, suggesting that cleavage of a γ-secretase target influences Rbf1 levels in ept mutant cells. Expression of exogenous rbf1 completely ablates ept mutant eye tissues but only mildly affects the development of discs composed of cells with wild type ept.
Together, these data show that loss of ept alters nuclear cell cycle control in developing imaginal discs and identify the DaPKC, presenilin, and rbf1 genes as modifiers of molecular and cellular phenotypes that result from loss of ept.
PMCID: PMC2739425  PMID: 19855819
23.  A Shared Role for RBF1 and dCAP-D3 in the Regulation of Transcription with Consequences for Innate Immunity 
PLoS Genetics  2012;8(4):e1002618.
Previously, we discovered a conserved interaction between RB proteins and the Condensin II protein CAP-D3 that is important for ensuring uniform chromatin condensation during mitotic prophase. The Drosophila melanogaster homologs RBF1 and dCAP-D3 co-localize on non-dividing polytene chromatin, suggesting the existence of a shared, non-mitotic role for these two proteins. Here, we show that the absence of RBF1 and dCAP-D3 alters the expression of many of the same genes in larvae and adult flies. Strikingly, most of the genes affected by the loss of RBF1 and dCAP-D3 are not classic cell cycle genes but are developmentally regulated genes with tissue-specific functions and these genes tend to be located in gene clusters. Our data reveal that RBF1 and dCAP-D3 are needed in fat body cells to activate transcription of clusters of antimicrobial peptide (AMP) genes. AMPs are important for innate immunity, and loss of either dCAP-D3 or RBF1 regulation results in a decrease in the ability to clear bacteria. Interestingly, in the adult fat body, RBF1 and dCAP-D3 bind to regions flanking an AMP gene cluster both prior to and following bacterial infection. These results describe a novel, non-mitotic role for the RBF1 and dCAP-D3 proteins in activation of the Drosophila immune system and suggest dCAP-D3 has an important role at specific subsets of RBF1-dependent genes.
Author Summary
The retinoblastoma protein (pRB) is a tumor suppressor protein known for its ability to repress transcription of E2F-dependent genes and induce cell cycle arrest. We have previously shown that RB proteins in Drosophila and human cells interact with the Condensin II subunit, CAP-D3, in an E2F-independent manner. Condensins promote condensation of chomosomes in mitosis. Our previous studies suggested that the Drosophila pRB and CAP-D3 homologs, RBF1 and dCAP-D3, co-localize on DNA and may share a function in cells that never undergo mitosis. In this study, we show that one non-mitotic function shared between RBF1 and dCAP-D3 is the regulation of many non-cell-cycle-related, clustered, and cell-type-specific transcripts including a conserved family of genes that are important for the immune response in the fly. In fact, results show that normal levels of dCAP-D3 and RBF1 expression are necessary for the ability of the fly to clear infection with human bacterial pathogens. This work demonstrates that dCAP-D3 proteins can regulate a unique subset of RBF1-dependent transcripts in vivo and identifies a novel role for both RBF1 and dCAP-D3 protein in activation of innate immune genes, which may be conserved in human cells.
PMCID: PMC3320600  PMID: 22496667
24.  PHF8 Targets Histone Methylation and RNA Polymerase II To Activate Transcription▿ †  
Molecular and Cellular Biology  2010;30(13):3286-3298.
Mutations in PHF8 are associated with X-linked mental retardation and cleft lip/cleft palate. PHF8 contains a plant homeodomain (PHD) in its N terminus and is a member of a family of JmjC domain-containing proteins. While PHDs can act as methyl lysine recognition motifs, JmjC domains can catalyze lysine demethylation. Here, we show that PHF8 is a histone demethylase that removes repressive histone H3 dimethyl lysine 9 marks. Our biochemical analysis revealed specific association of the PHF8 PHD with histone H3 trimethylated at lysine 4 (H3K4me3). Chromatin immunoprecipitation followed by high-throughput sequencing indicated that PHF8 is enriched at the transcription start sites of many active or poised genes, mirroring the presence of RNA polymerase II (RNAPII) and of H3K4me3-bearing nucleosomes. We show that PHF8 can act as a transcriptional coactivator and that its activation function largely depends on binding of the PHD to H3K4me3. Furthermore, we present evidence for direct interaction of PHF8 with the C-terminal domain of RNAPII. Importantly, a PHF8 disease mutant was defective in demethylation and in coactivation. This is the first demonstration of a chromatin-modifying enzyme that is globally recruited to promoters through its association with H3K4me3 and RNAPII.
PMCID: PMC2897584  PMID: 20421419
25.  Crystal Structure and Functional Analysis of JMJD5 Indicate an Alternate Specificity and Function 
Molecular and Cellular Biology  2012;32(19):4044-4052.
JMJD5 is a Jumonji C (JmjC) protein that has been implicated in breast cancer tumorigenesis, circadian rhythm regulation, embryological development, and osteoclastogenesis. Recently, JMJD5 (also called KDM8) has been reported to demethylate dimethylated Lys-36 in histone H3 (H3K36me2), regulating genes that control cell cycle progression. Here, we report high-resolution crystal structures of the human JMJD5 catalytic domain in complex with the substrate 2-oxoglutarate (2-OG) and the inhibitor N-oxalylglycine (NOG). The structures reveal a β-barrel fold that is conserved in the JmjC family and a long shallow cleft that opens into the enzyme's active site. A comparison with other JmjC enzymes illustrates that JMJD5 shares sequence and structural homology with the asparaginyl and histidinyl hydroxylase FIH-1 (factor inhibiting hypoxia-inducible factor 1 [HIF-1]), the lysyl hydroxylase JMJD6, and the RNA hydroxylase TYW5 but displays limited homology to JmjC lysine demethylases (KDMs). Contrary to previous findings, biochemical assays indicate that JMJD5 does not display demethylase activity toward methylated H3K36 nor toward the other methyllysines in the N-terminal tails of histones H3 and H4. Together, these results imply that JMJD5 participates in roles independent of histone demethylation and may function as a protein hydroxylase given its structural homology with FIH-1 and JMJD6.
PMCID: PMC3457536  PMID: 22851697

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