Expression of histone methyltransferases as novel biomarkers for renal cell tumor diagnosis and prognostication

Renal cell tumors (RCTs) are the most lethal of the common urological cancers. The widespread use of imaging entailed an increased detection of small renal masses, emphasizing the need for accurate distinction between benign and malignant RCTs, which is critical for adequate therapeutic management. Histone methylation has been implicated in renal tumorigenesis, but its potential clinical value as RCT biomarker remains mostly unexplored. Hence, the main goal of this study was to identify differentially expressed histone methyltransferases (HMTs) and histone demethylases (HDMs) that might prove useful for RCT diagnosis and prognostication, emphasizing the discrimination between oncocytoma (a benign tumor) and renal cell carcinoma (RCC), especially the chromophobe subtype (chRCC). We found that the expression levels of three genes-SMYD2, SETD3, and NO66-was significantly altered in a set of RCTs, which was further validated in a large independent cohort. Higher expression levels were found in RCTs compared to normal renal tissues (RNTs) and in chRCCs comparatively to oncocytomas. SMYD2 and SETD3 mRNA levels correlated with protein expression assessed by immunohistochemistry. SMYD2 transcript levels discriminated RCTs from RNT, with 82.1% sensitivity and 100% specificity (AUC=0.959), and distinguished chRCCs from oncocytomas, with 71.0% sensitivity and 73.3% specificity (AUC: 0.784). Low expression levels of SMYD2, SETD3, and NO66 were significantly associated with shorter disease-specific and disease-free survival, especially in patients with non-organ confined tumors. We conclude that expression of selected HMTs and HDMs might constitute novel biomarkers to assist in RCT diagnosis and assessment of tumor aggressiveness.

E2F1 mediates DNA damage and apoptosis through HCF-1 and the MLL family of histone methyltransferases.

E2F1 is a key positive regulator of human cell proliferation and its activity is altered in essentially all human cancers. Deregulation of E2F1 leads to oncogenic DNA damage and anti-oncogenic apoptosis. The molecular mechanisms by which E2F1 mediates these two processes are poorly understood but are important for understanding cancer progression. During the G1-to-S phase transition, E2F1 associates through a short DHQY sequence with the cell-cycle regulator HCF-1 together with the mixed-lineage leukaemia (MLL) family of histone H3 lysine 4 (H3K4) methyltransferases. We show here that the DHQY HCF-1-binding sequence permits E2F1 to stimulate both DNA damage and apoptosis, and that HCF-1 and the MLL family of H3K4 methyltransferases have important functions in these processes. Thus, HCF-1 has a broader role in E2F1 function than appreciated earlier. Indeed, sequence changes in the E2F1 HCF-1-binding site can modulate both up and down the ability of E2F1 to induce apoptosis indicating that HCF-1 association with E2F1 is a regulator of E2F1-induced apoptosis.

E2F activation of S phase promoters via association with HCF-1 and the MLL family of histone H3K4 methyltransferases.

E2F transcriptional regulators control human-cell proliferation by repressing and activating the transcription of genes required for cell-cycle progression, particularly the S phase. E2F proteins repress transcription in association with retinoblastoma pocket proteins, but less is known about how they activate transcription. Here, we show that the human G1 phase regulator HCF-1 associates with both activator (E2F1 and E2F3a) and repressor (E2F4) E2F proteins, properties that are conserved in insect cells. Human HCF-1-E2F interactions are versatile: their associations and binding to E2F-responsive promoters are cell-cycle selective, and HCF-1 displays coactivator properties when bound to the E2F1 activator and corepressor properties when bound to the E2F4 repressor. During the G1-to-S phase transition, HCF-1 recruits the mixed-lineage leukemia (MLL) and Set-1 histone H3 lysine 4 methyltransferases to E2F-responsive promoters and induces histone methylation and transcriptional activation. These results suggest that HCF-1 induces cell-cycle-specific transcriptional activation by E2F proteins to promote cell proliferation.

The γ-tocopherol-like family of N-methyltransferases: A taxonomically clustered gene family encoding enzymes responsible for N-methylation of monoterpene indole alkaloids

The plant family Apocynaceae accumulates thousands of monoterpene indole alkaloids (MIAs) which originate, biosynthetically, from the common secoiridoid intermediate, strictosidine, that is formed from the condensation of tryptophan and secologanin molecules. MIAs demonstrate remarkable structural diversity and have pharmaceutically valuable biological activities. For example; a subunit of the potent anti-neoplastic molecules vincristine and vinblastine is the aspidosperma alkaloid, vindoline. Vindoline accumulates to trace levels under natural conditions. Research programs have determined that there is significant developmental and light regulation involved in the biosynthesis of this MIA. Furthermore, the biosynthetic pathway leading to vindoline is split among at least five independent cell types. Little is known of how intermediates are shuttled between these cell types. The late stage events in vindoline biosynthesis involve six enzymatic steps from tabersonine. The fourth biochemical step, in this pathway, is an indole N-methylation performed by a recently identified N-methyltransfearse (NMT). For almost twenty years the gene encoding this NMT had eluded discovery; however, in 2010 Liscombe et al. reported the identification of a γ-tocopherol C-methyltransferase homologue capable of indole N-methylating 2,3-dihydrotabersonine and Virus Induced Gene Silencing (VIGS) suppression of the messenger has since proven its involvement in vindoline biosynthesis. Recent large scale sequencing initiatives, performed on non-model medicinal plant transcriptomes, has permitted identification of candidate genes, presumably involved, in MIA biosynthesis never seen before in plant specialized metabolism research. Probing the transcriptome assemblies of Catharanthus roseus (L.)G.Don, Vinca minor L., Rauwolfia serpentine (L.)Benth ex Kurz, Tabernaemontana elegans, and Amsonia hubrichtii, with the nucleotide sequence of the N-methyltransferase involved in vindoline biosynthesis, revealed eight new homologous methyltransferases. This thesis describes the identification, molecular cloning, recombinant expression and biochemical characterization of two picrinine NMTs, one from V. minor and one from R. serpentina, a perivine NMT from C. roseus, and an ajmaline NMT from R. serpentina. While these TLMTs were expressed and functional in planta, they were active at relatively low levels and their N-methylated alkaloid products were not apparent our from alkaloid isolates of the plants. It appears that, for the most part, these TLMTs, participate in apparently silent biochemical pathways, awaiting the appropriate developmental and environmental cues for activity.

Characterization of the Drosophila protein arginine methyltransferases DART1 and DART4

The role of arginine methylation in Drosophila melanogaster is unknown. We identified a family of nine PRMTs (protein arginine methyltransferases) by sequence homology with mammalian arginine methyltransferases, which we have named DART1 to DART9 ( Drosophila arginine methyltransferases 1-9). In keeping with the mammalian PRMT nomenclature, DART1, DART4, DART5 and DART7 are the putative homologues of PRMT1, PRMT4, PRMT5 and PRMT7. Other DART family members have a closer resemblance to PRMT1, but do not have identifiable homologues. All nine genes are expressed in Drosophila at various developmental stages. DART1 and DART4 have arginine methyltransferase activity towards substrates, including histones and RNA-binding proteins. Amino acid analysis of the methylated arginine residues confirmed that both DART1 and DART4 catalyse the formation of asymmetrical dimethylated arginine residues and they are type I arginine methyltransferases. The presence of PRMTs in D. melanogaster suggest that flies are a suitable genetic system to study arginine methylation.

DNA methyltransferases of the cyanobacterium Anabaena PCC 7120

From the characterization of enzyme activities and the analysis of genomic sequences, the complement of DNA methyltransferases (MTases) possessed by the cyanobacterium Anabaena PCC 7120 has been deduced. Anabaena has nine DNA MTases. Four are associated with Type II restriction enzymes (AvaI, AvaII, AvaIII and the newly recognized inactive AvaIV), and five are not. Of the latter, four may be classified as solitary MTases, those whose function lies outside of a restriction/modification system. The group is defined here based on biochemical and genetic characteristics. The four solitary MTases, DmtA/M.AvaVI, DmtB/M.AvaVII, DmtC/M.AvaVIII and DmtD/M.AvaIX, methylate at GATC, GGCC, CGATCG and rCCGGy, respectively. DmtB methylates cytosines at the N4 position, but its sequence is more similar to N6-adenine MTases than to cytosine-specific enzymes, indicating that it may have evolved from the former. The solitary MTases, appear to be of ancient origin within cyanobacteria, while the restriction MTases appear to have arrived by recent horizontal transfer as did five now inactive Type I restriction systems. One Mtase, M.AvaV, cannot reliably be classified as either a solitary or restriction MTase. It is structurally unusual and along with a few proteins of prokaryotic and eukaryotic origin defines a structural class of MTases distinct from all previously described.

Sequence-specific recognition of cytosine C5 and adenine N6 DNA methyltransferases requires different deformations of DNA.

DNA methyltransferases modify specific cytosines and adenines within 2-6 bp recognition sequences. We used scanning force microscopy and gel shift analysis to show that M.HhaI, a cytosine C-5 DNA methyltransferase, causes only a 2 degree bend upon binding its recognition site. Our results are consistent with prior crystallographic analysis showing that the enzyme stabilizes an extrahelical base while leaving the DNA duplex otherwise unperturbed. In contrast, similar analysis of M.EcoRI, an adenine N6 DNA methyltransferase, shows an average bend angle of approximately 52 degrees. This distortion of DNA conformation by M.EcoRI is shown to be important for sequence-specific binding.

Epigenetic regulation in neural crest development : role of DNA methyltransferases 3A and 3B

The neural crest is a group of migratory, multipotent stem cells that play a crucial role in many aspects of embryonic development. This uniquely vertebrate cell population forms within the dorsal neural tube but then emigrates out and migrates long distances to different regions of the body. These cells contribute to formation of many structures such as the peripheral nervous system, craniofacial skeleton, and pigmentation of the skin. Why some neural tube cells undergo a change from neural to neural crest cell fate is unknown as is the timing of both onset and cessation of their emigration from the neural tube. In recent years, growing evidence supports an important role for epigenetic regulation as a new mechanism for controlling aspects of neural crest development. In this thesis, I dissect the roles of the de novo DNA methyltransferases (DNMTs) 3A and 3B in neural crest specification, migration and differentiation. First, I show that DNMT3A limits the spatial boundary between neural crest versus neural tube progenitors within the neuroepithelium. DNMT3A promotes neural crest specification by directly mediating repression of neural genes, like Sox2 and Sox3. Its knockdown causes ectopic Sox2 and Sox3 expression at the expense of neural crest territory. Thus, DNMT3A functions as a molecular switch, repressing neural to favor neural crest cell fate. Second, I find that DNMT3B restricts the temporal window during which the neural crest cells emigrate from the dorsal neural tube. Knockdown of DNMT3B causes an excess of neural crest emigration, by extending the time that the neural tube is competent to generate emigrating neural crest cells. In older embryos, this resulted in premature neuronal differentiation. Thus, DNMT3B regulates the duration of neural crest production by the neural tube and the timing of their differentiation. My results in avian embryos suggest that de novo DNA methylation, exerted by both DNMT3A and DNMT3B, plays a dual role in neural crest development, with each individual paralogue apparently functioning during a distinct temporal window. The results suggest that de novo DNA methylation is a critical epigenetic mark used for cell fate restriction of progenitor cells during neural crest cell fate specification. Our discovery provides important insights into the mechanisms that determine whether a cell becomes part of the central nervous system or peripheral cell lineages.

Fitness cost and interference of Arm/Rmt aminoglycoside resistance with the RsmF housekeeping methyltransferases

Arm/Rmt methyltransferases have emerged recently in pathogenic bacteria as enzymes that confer high-level resistance to 4,6-disubstituted aminoglycosides through methylation of the G1405 residue in the 16S rRNA (like ArmA and RmtA to -E). In prokaryotes, nucleotide methylations are the most common type of rRNA modification, and they are introduced posttranscriptionally by a variety of site-specific housekeeping enzymes to optimize ribosomal function. Here we show that while the aminoglycoside resistance methyltransferase RmtC methylates G1405, it impedes methylation of the housekeeping methyltransferase RsmF at position C1407, a nucleotide that, like G1405, forms part of the aminoglycoside binding pocket of the 16S rRNA. To understand the origin and consequences of this phenomenon, we constructed a series of in-frame knockout and knock-in mutants of Escherichia coli, corresponding to the genotypes rsmF(+), ΔrsmF, rsmF(+) rmtC(+), and ΔrsmF rmtC(+). When analyzed for the antimicrobial resistance pattern, the ΔrsmF bacteria had a decreased susceptibility to aminoglycosides, including 4,6- and 4,5-deoxystreptamine aminoglycosides, showing that the housekeeping methylation at C1407 is involved in intrinsic aminoglycoside susceptibility in E. coli. Competition experiments between the isogenic E. coli strains showed that, contrary to expectation, acquisition of rmtC does not entail a fitness cost for the bacterium. Finally, matrix-assisted laser desorption ionization (MALDI) mass spectrometry allowed us to determine that RmtC methylates the G1405 residue not only in presence but also in the absence of aminoglycoside antibiotics. Thus, the coupling between housekeeping and acquired methyltransferases subverts the methylation architecture of the 16S rRNA but elicits Arm/Rmt methyltransferases to be selected and retained, posing an important threat to the usefulness of aminoglycosides worldwide.

Molecular And Biochemical Characterization Of Caffeine Synthase And Purine Alkaloid Concentration In Guarana Fruit.

Guarana seeds have the highest caffeine concentration among plants accumulating purine alkaloids, but in contrast with coffee and tea, practically nothing is known about caffeine metabolism in this Amazonian plant. In this study, the levels of purine alkaloids in tissues of five guarana cultivars were determined. Theobromine was the main alkaloid that accumulated in leaves, stems, inflorescences and pericarps of fruit, while caffeine accumulated in the seeds and reached levels from 3.3% to 5.8%. In all tissues analysed, the alkaloid concentration, whether theobromine or caffeine, was higher in young/immature tissues, then decreasing with plant development/maturation. Caffeine synthase activity was highest in seeds of immature fruit. A nucleotide sequence (PcCS) was assembled with sequences retrieved from the EST database REALGENE using sequences of caffeine synthase from coffee and tea, whose expression was also highest in seeds from immature fruit. The PcCS has 1083bp and the protein sequence has greater similarity and identity with the caffeine synthase from cocoa (BTS1) and tea (TCS1). A recombinant PcCS allowed functional characterization of the enzyme as a bifunctional CS, able to catalyse the methylation of 7-methylxanthine to theobromine (3,7-dimethylxanthine), and theobromine to caffeine (1,3,7-trimethylxanthine), respectively. Among several substrates tested, PcCS showed higher affinity for theobromine, differing from all other caffeine synthases described so far, which have higher affinity for paraxanthine. When compared to previous knowledge on the protein structure of coffee caffeine synthase, the unique substrate affinity of PcCS is probably explained by the amino acid residues found in the active site of the predicted protein.

The effects of dietary supplementation of methionine on genomic stability and p53 gene promoter methylation in rats

Methionine is a component of one-carbon metabolism and a precursor of S-adenosylmethionine (SAM), the methyl donor for DNA methylation. When methionine intake is high, an increase of S-adenosylmethionine (SAM) is expected. DNA methyltransferases convert SAM to S-adenosylhomocysteine (SAH). A high intracellular SAH concentration could inhibit the activity of DNA methyltransferases. Therefore, high methionine ingestion could induce DNA damage and change the methylation pattern of tumor suppressor genes. This study investigated the genotoxicity of a methionine-supplemented diet. It also investigated the diet`s effects on glutathione levels, SAM and SAH concentrations and the gene methylation pattern of p53. Wistar rats received either a methionine-supplemented diet (2% methionine) or a control diet (0.3% methionine) for six weeks. The methionine-supplemented diet was neither genotoxic nor antigenotoxic to kidney cells, as assessed by the comet assay. However, the methionine-supplemented diet restored the renal glutathione depletion induced by doxorubicin. This fact may be explained by the transsulfuration pathway, which converts methionine to glutathione in the kidney. Methionine supplementation increased the renal concentration of SAH without changing the SAM/SAH ratio. This unchanged profile was also observed for DNA methylation at the promoter region of the p53 gene. Further studies are necessary to elucidate this diet`s effects on genomic stability and DNA methylation. (C) 2011 Elsevier ay. All rights reserved.

Analysis of the genes flanking xabB: a methyltransferase gene is involved in albicidin biosynthesis in Xanthomonas albilineans

Transposon mutagenesis and complementation studies previously identified a gene (xabB) for a large (526 kDa) polyketide-peptide synthase required for biosynthesis of albicidin antibiotics and phytotoxins in the sugarcane leaf scald pathogen Xanthomonas albilineans. A cistron immediately downstream from xabB encodes a polypeptide of 343 aa containing three conserved motifs characteristic of a family of S-adenosyl-L-methionine (SAM)-dependent O-methyltransferases. Insertional mutagenesis and complementation indicate that the product of this cistron (designated xabC) is essential for albicidin production, and that there is no other required downstream cistron. The xab promoter region is bidirectional, and insertional mutagenesis of the first open reading frame (ORF) in the divergent gene also blocks albicidin biosynthesis. This divergent ORF (designated thp) encodes a protein of 239 aa displaying high similarity to several IS21-like transposition helper proteins. The thp cistron is not located in a recognizable transposon, and is probably a remnant from a past transposition event that may have contributed to the development of the albicidin biosynthetic gene cluster. Failure of 'in trans' complementation of rhp indicates that a downstream cistron transcribed with thp is required for albicidin biosynthesis. (C) 2000 Elsevier Science B.V. All rights reserved.

Functional association of human Ki-1/57 with pre-mRNA splicing events

The cytoplasmic and nuclear protein Ki- 1 / 57 was first identified in malignant cells from Hodgkin`s lymphoma. Despite studies showing its phosphorylation, arginine methylation, and interaction with several regulatory proteins, the functional role of Ki- 1 / 57 in human cells remains to be determined. Here, we investigated the relationship of Ki- 1 / 57 with RNA functions. Through immunoprecipitation assays, we verified the association of Ki- 1 / 57 with the endogenous splicing proteins hnRNPQ and SFRS9 in HeLa cell extracts. We also found that recombinant Ki- 1 / 57 was able to bind to a poly- U RNA probe in electrophoretic mobility shift assays. In a classic splicing test, we showed that Ki- 1 / 57 can modify the splicing site selection of the adenoviral E1A minigene in a dose- dependent manner. Further confocal and. uorescence microscopy analysis revealed the localization of enhanced green. uorescent protein - Ki- 1 / 57 to nuclear bodies involved in RNA processing and or small nuclear ribonucleoprotein assembly, depending on the cellular methylation status and its N- terminal region. In summary, our findings suggest that Ki- 1 / 57 is probably involved in cellular events related to RNA functions, such as pre- mRNA splicing.

Getting the adrenaline going: Crystal structure of the adrenaline-synthesizing enzyme PNMT

Background: Adrenaline is localized to specific regions of the central nervous system (CNS), but its role therein is unclear because of a lack of suitable pharmacologic agents. Ideally, a chemical is required that crosses the blood-brain barrier, potently inhibits the adrenaline-synthesizing enzyme PNMT, and does not affect other catecholamine processes. Currently available PNMT inhibitors do not meet these criteria. We aim to produce potent, selective, and CNS-active PNMT inhibitors by structure-based design methods. The first step is the structure determination of PNMT. Results: We have solved the crystal structure of human PNMT complexed with a cofactor product and a submicromolar inhibitor at a resolution of 2.4 Angstrom. The structure reveals a highly decorated methyltransferase fold, with an active site protected from solvent by an extensive cover formed from several discrete structural motifs. The structure of PNMT shows that the inhibitor interacts with the enzyme in a different mode from the (modeled) substrate noradrenaline. Specifically, the position and orientation of the amines is not equivalent. Conclusions: An unexpected finding is that the structure of PNMT provides independent evidence of both backward evolution and fold recruitment in the evolution of a complex enzyme from a simple fold. The proposed evolutionary pathway implies that adrenaline, the product of PNMT catalysis, is a relative newcomer in the catecholamine family. The PNMT structure reported here enables the design of potent and selective inhibitors with which to characterize the role of adrenaline in the CNS. Such chemical probes could potentially be useful as novel therapeutics.