Methyltransferase Activity Assay Research Articles

Label-free and immobilization-free photoelectrochemical biosensing strategy using methylene blue in homogeneous solution as signal probe for facile DNA methyltransferase activity assay

Photoelectrochemical (PEC) methods have recently witnessed ever expanding application in bioanalysis, but it is still desirable to further simplify the sensing procedures and develop simple and reliable PEC biosensing approaches. Herein, we proposed a truly label-free and immobilization-free PEC sensing platform, utilizing solution-phase methylene blue (MB) as the signal probe, and bare indium tin oxide (ITO) glass as the photoelectrode. Based on the diffusivity difference between free MB molecules and MB intercalated in DNA G-quadruplex, the activity and inhibition of DNA adenine methyltransferase (Dam), a proof-of-concept methyltransferase (MTase), is quantitatively analyzed. By taking advantage of the endonuclease-catalyzed cleavage of the Dam-methylated hairpin DNA probe, as well as the KF polymerase/Nt.AlwI endonuclease-aided signal amplification, highly sensitive and specific PEC detection of Dam activity has been achieved. Moreover, this approach can be easily extended to assay other types of MTase by choosing the appropriate methylation-sensitive endonucleases. The as-proposed strategy has also been successfully applied to analyze Dam spiked in human serum samples and to assess the inhibitory effects of antibiotics on Dam activity. More importantly, this label-free and truly immobilization-free PEC biosensing strategy shows additional merits of simplicity and satisfactory repeatability, due to the elimination of both labelling and immobilization procedures, making it a promising candidate for the application in highly sensitive, facile and reliable bioanalysis and drug screening.

Terminal Deoxynucleotidyl Transferase and Rolling Circle Amplification Induced G-triplex Formation: A Label-free Fluorescent Strategy for DNA Methyltransferase Activity Assay

It is of great significance to sensitively detect DNA methyltransferase (MTase) activity and screen its inhibitory drugs in the field of human cancer diagnosis and disease therapeutics. In this work, based on terminal deoxynucleotidyl transferase (TdT)-induced rolling circle amplification (RCA), we construct a label-free and highly sensitive fluorescence biosensing strategy for DNA MTase activity analysis. The hairpin substrate is methylated by Dam MTase and cleaved by Dpnl to generate a DNA fragment with a free 3′-hydroxy (3′−OH) terminus that is extended by TdT to obtain a long adenine (poly-A) tail. Subsequently, the resulting poly-A work as primer triggers RCA reaction and generate lots of G-triplexes which can combine with Thioflavin T (ThT) to achieve the apparent fluorescence signal. Integrate TdT-induced RCA with G-triplex@ThT, Dam MTase can be detected down to 0.058 U/mL with a wide linear range from 0.1 U/mL to 40 U/mL. In addition, the method exhibits high selectivity to other MTases. We also successfully demonstrate this work can be used in complex matrixes assay and screen MTase inhibitors. Thus, in the area of DNA MTase-related cancer diagnosis and disease therapeutics, the developed method may be a promising tool for the detection of Dam MTase.

Histone H3 tail binds a unique sensing pocket in EZH2 to activate the PRC2 methyltransferase

Enhancer of Zeste Homolog 2 (EZH2) is the catalytic subunit of Polycomb Repressor Complex 2 (PRC2), the enzyme that catalyzes monomethylation, dimethylation, and trimethylation of lysine 27 on histone H3 (H3K27). Trimethylation at H3K27 (H3K27me3) is associated with transcriptional silencing of developmentally important genes. Intriguingly, H3K27me3 is mutually exclusive with H3K36 trimethylation on the same histone tail. Disruptions in this cross-talk result in aberrant H3K27/H3K36 methylation patterns and altered transcriptional profiles that have been implicated in tumorigenesis and other disease states. Despite their importance, the molecular details of how PRC2 "senses" H3K36 methylation are unclear. We demonstrate that PRC2 is activated in cis by the unmodified side chain of H3K36, and that this activation results in a fivefold increase in the kcat of its enzymatic activity catalyzing H3K27 methylation compared with activity on a substrate methylated at H3K36. Using a photo-cross-linking MS strategy and histone methyltransferase activity assays on PRC2 mutants, we find that EZH2 contains a specific sensing pocket for the H3K36 methylation state that allows the complex to distinguish between modified and unmodified H3K36 residues, altering enzymatic activity accordingly to preferentially methylate the unmodified nucleosome substrate. We also present evidence that this process may be disrupted in some cases of Weaver syndrome.

Structural characterization of B. subtilis m1A22 tRNA methyltransferase TrmK: insights into tRNA recognition.

1-Methyladenosine (m1A) is a modified nucleoside found at positions 9, 14, 22 and 58 of tRNAs, which arises from the transfer of a methyl group onto the N1-atom of adenosine. The yqfN gene of Bacillus subtilis encodes the methyltransferase TrmK (BsTrmK) responsible for the formation of m1A22 in tRNA. Here, we show that BsTrmK displays a broad substrate specificity, and methylates seven out of eight tRNA isoacceptor families of B. subtilis bearing an A22. In addition to a non-Watson–Crick base-pair between the target A22 and a purine at position 13, the formation of m1A22 by BsTrmK requires a full-length tRNA with intact tRNA elbow and anticodon stem. We solved the crystal structure of BsTrmK showing an N-terminal catalytic domain harbouring the typical Rossmann-like fold of Class-I methyltransferases and a C-terminal coiled-coil domain. We used NMR chemical shift mapping to drive the docking of BstRNASer to BsTrmK in complex with its methyl-donor cofactor S-adenosyl-L-methionine (SAM). In this model, validated by methyltransferase activity assays on BsTrmK mutants, both domains of BsTrmK participate in tRNA binding. BsTrmK recognises tRNA with very few structural changes in both partner, the non-Watson–Crick R13–A22 base-pair positioning the A22 N1-atom close to the SAM methyl group.

Mechanism of Bacterial Ribosome 30S and 50S Subunit Recognition and Modification by M. tuberculosis TlyA

RNA (rRNA) modification is important for correct ribosome assembly, can alter ribosome function, and can confer resistance to clinically important ribosome‐targeting antibiotics. While the activity of most ribosome‐targeting antibiotics is blocked by rRNA methylation, the tuberactinomycin antibiotic capreomycin requires ribose 2′‐OH methylation at C1409 of the 16S rRNA within the small ribosome subunit (30S) and C1920 of the 23S rRNA within the large ribosome subunit (50S). TlyA is the 2′‐O‐methyltransferase that incorporates both modifications using S‐adenosyl‐L‐methionine (SAM) as a methyl group donor. The X‐ray crystal structure of the C‐terminal domain (CTD) of TlyA showed that the domain adopts a Class I methyltransferase fold, while homology modeling suggests the N‐terminal domain (NTD) adopts an S4 ribosomal protein fold. Additionally, the TlyA CTD structural studies revealed that the short interdomain linker was able to adopt two different conformations and was unexpectedly critical for SAM binding within the CTD. This project will test the resulting hypothesis that the interdomain linker acts a “molecular switch,” altering the interaction between the NTD and the CTD to control TlyA activity upon correct recognition of its two structurally distinct substrates. Using a sequence alignment and structure‐guided mutagenesis approach coupled with fluorescence polarization (FP) TlyA‐30S binding and methyltransferase activity assays, the role of the NTD and individual residues in this domain in 30S and 50S binding will be defined. Using complementary studies of TlyA protein dynamics (using HDX‐MS) and structure (using X‐ray crystallography and cryo‐EM), the long‐term goal of this project is to determine TlyA's molecular mechanism of site‐specific ribosome subunit recognition and modification. Defining these molecular details will help complete the picture of TlyA activity and its role in bacterial drug sensitivity or resistance, as well as furthering our general understanding of rRNA modification processes that are prevalent in all domains of life.Support or Funding InformationThis work was supported in part by the Graduate Division of Biological and Biomedical Sciences (GDBBS) of the Laney Graduate School (LGS) of Emory University, NIH grant R01‐AI088025, and the Antibiotic Resistance and Therapeutic Discovery Training Program (ARTDTP) T32‐AI 106699.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Molecular regulation of lysine trimethylation by the P. aeruginosa methyltransferase EftM

Pseudomonas aeruginosa is an opportunistic pathogen and a leading cause of severe life‐threatening lung and other infections in individuals with cystic fibrosis, compromised immune systems or severe burns. P. aeruginosa is also intrinsically resistant to many antibiotics making infections difficult to treat once established. We previously showed that the S‐adenosyl‐L‐methionine‐dependent methyltransferase EftM is responsible for trimethylation of lysine 5 of the translation factor EF‐Tu and that this modification enhances P. aeruginosa adhesion to host epithelial cells. Additionally, methyltransferase activity, and thus EF‐Tu modification, is temperature regulated via the intrinsic thermolability of EftM. Understanding EftM's complete mechanism of action may reveal new strategies for developing effective therapeutics targeting this pathogen. As the next step in this process, we now aim to define the substrate recognition and catalytic mechanisms of EftM. A phylogenetic reconstruction of the larger EftM family was generated to identify conservation patterns of putative key residues involved in interaction with EF‐Tu and facilitating catalysis. This analysis was supported by generation of an EftM homology model and protein‐protein docking studies to propose a EftM:EF‐Tu binding model. This model and phylogentic insights were used to guide site‐directed mutagenesis of residues in EftM and EF‐Tu to test their role in protein‐protein recognition using methyltransferase activity and binding assays. Finally, to begin investigating the role of protein dynamics and potential allosteric effects of SAM cosubstrate binding, other computational methods such as normal mode analysis were used to propose a coarse‐grain model of dynamics of EftM in parallel with experimental hydrogen‐deuterium exchange coupled with mass spectrometry (HDX‐MS) analysis of EftM and its complexes with SAM and EF‐Tu. In conclusion, our results provide the first structural insight into the EftM:EF‐Tu recognition, which paves the way for further understanding EftM's substrate recognition and catalytic mechanisms.Support or Funding InformationCystic Fibrosis Foundation Postdoctoral Research FellowshipThis abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

A sensitive photoelectrochemical methyltransferase activity assay based on a novel "Z-scheme" CdSe QD/afGQD heterojunction and multiple signal amplification strategies.

Here, based on a newly developed "Z-scheme" CdSe QD/afGQD heterojunction and multiple signal amplification strategies, a sensitive photoelectrochemical methyltransferase activity assay was carried out and a low detection limit of 0.046 U mL-1 was obtained.

Highly Sensitive Assay of Methyltransferase Activity Based on an Autonomous Concatenated DNA Circuit

Methyltransferase-involved DNA methylation is one of the most important epigenetic processes, making the ultrasensitive MTase assay highly desirable in clinical diagnosis as well as biomedical research. Traditional single-stage amplification means often achieve linear amplification that might not fulfill the increasing demands for detecting trace amount of target. It is desirable to construct multistage cascaded amplifiers that allow for enhanced signal amplifications. Herein, a powerful nonenzymatic MTase-sensing platform is successfully engineered based on a two-layered DNA circuit, in which the upstream catalytic hairpin assembly (CHA) circuit successively generates DNA product that could be used to activate the downstream hybridization chain reaction (HCR) circuit, resulting in the generation of a dramatically amplified fluorescence signal. In the absence of M.SssI MTase, HpaII endonuclease could specifically recognize the auxiliary hairpin substrate and then catalytically cleave the corresponding recognition site, releasing a DNA fragment that triggers the CHA-HCR-mediated FRET transduction. Yet the M.SssI-methylated hairpin substrate could not be cleaved by HpaII enzyme, and thus prohibits the CHA-HCR-mediated FRET generation, providing a substantial signal difference with that of MTase-absent system. Taking advantage of the high specificity of multiple-guaranteed recognitions of MTase/endonuclease and the synergistic amplification features of concatenated CHA-HCR circuit, this method enables an ultrasensitive detection of MTase and its inhibitors in serum and E. coli cells. Furthermore, the rationally assembled CHA-HCR also allows for probing other different biotransformations through a facile design of the corresponding substrates. It is anticipated that the infinite layer of multilayered DNA circuit could further improve the signal gain of the system for accurately detecting other important biomarkers, and thus holds great promise for cancerous treatment and biomedical research.

A microchip electrophoretic assay for DNA methyltransferase activity based on methylation-sensitive endonuclease DpnⅡ.

DNA methylation is a significant epigenetic modification and the methods for the detection of DNA methyltransferase (MTase) activity are important due to aberrant methylation closely related to the occurrence of cancer. In this study, a simple and rapid microchip electrophoresis (ME) coupled with LED-induced fluorescence (LEDIF) method was presented for the detection of Dam MTase activity. This strategy was based on methylation-sensitive endonuclease DpnⅡ which could recognize the same specific site 5'-GATC-3' with Dam MTase in double-stranded DNA (dsDNA). The adenines in the specific site could be methylated by Dam MTase, then the special site could not be digested by DpnⅡ. Both methylated dsDNA and unmethylated dsDNA could be analyzed by ME-LEDIF after stained by SYBR gold. The results showed the fluorescence intensities of methylated dsDNA were directly proportional to Dam MTase activities in the range of 0.5-20U/mL with a detection limit of 0.12U/mL. Furthermore, the method could successfully be applied to evaluation experiments of Dam MTase inhibitors. The results confirmed the ME-LEDIF method is a promising approach for inhibitors screening of DNA MTase and development of anticancer drugs.

A colorimetric assay of DNA methyltransferase activity based on peroxidase mimicking of DNA template Ag/Pt bimetallic nanoclusters.

DNA methylation catalyzed by DNA methyl transferase (MTase) is a significant epigenetic process for modulating gene expression. Abnormal levels of DNA MTase enzyme have been regarded as a cancer biomarker or a sign of bacterial diseases. We developed a novel colorimetric method to assay M.SssI MTase activity employing peroxidase-like activity of DNA template Ag/Pt NCs without using restriction enzymes. Based on inhibiting the peroxidase reaction that occurred in the TMB-H2O2 system, in the presence of MTase, a highly sensitive and selective colorimetric biosensor was fabricated with a detection limit (LOD) of 0.05 U/mL and a linear range from 0.5 to 10 U/mL. The changes in absorption intensity were monitored to quantify the M.SssI activity. This strategy had a high selectivity over other proteins. Furthermore, it is also demonstrated that this method can be used for the evaluation and screening of inhibitors for DNA MTase.

A simple and universal electrochemical assay for sensitive detection of DNA methylation, methyltransferase activity and screening of inhibitors

Many studies have confirmed that DNA methylation is highly correlated with the occurrence and development of various diseases including cancers. In this work, we developed a simple, sensitive, selective, and universal electrochemical biosensor for detection of DNA methylation and assay of DNA methyltransferase (MTase) activity using M.SssI MTase as an example. The thiolated single-stranded DNA S1 was self-assembled on the surface of gold nanoparticles deposition modified glassy carbon electrode via AuS bonding, then hybridization between the DNA S1 and its complementary DNA S2 formed a double-stranded target sequence for both M.SssI MTase and restriction endonuclease HpaII. HpaII could not cleave the target sequence after it was methylated by M.SssI MTase, while the sequence without methylation could be cleaved. Here, we used methylene blue (MB) as electrochemical indicator. The electrochemical signal of MB increased linearly with increasing M.SssI MTase concentration from 0.5 to 25 U/mL and from 25 to 400 U/mL with a detection limit of 0.04 U/mL. Moreover, screening of M.SssI MTase inhibitors 5-azacytidine (5-Aza) and 5-Aza-2′-deoxycytidine (5-Aza-dC) were successfully investigated using the fabricated electrochemical biosensor and showed that the two classic drugs could both inhibit the M.SssI MTase activity with the IC50 of 2.8 μM and 0.37 μM, respectively, indicating potential application in discovery of new anticancer drugs.

Biochemical validation of EHMT1 missense mutations in Kleefstra syndrome.

Kleefstra syndrome (KS) (9q34 deletion syndrome) is a rare autosomal dominant disorder characterized by intellectual disability, frequently coupled with a spectrum of complex physical and clinical manifestations. As the euchromatic histone methyltransferase-1 gene (EHMT1, GLP, or KMT1D) within the 9q34 region is deleted or mutated in most of the individuals with KS, its absence or defect in one allele is speculated to cause the major symptoms of the syndrome. Most of the EHMT1 mutations are frameshift or nonsense mutations, but two individuals with KS were reported to possess EHMT1 missense mutations. These two mutations have been predicted to cause a defective enzymatic function, but precise biochemical validation was not conducted. Therefore, we validated these two mutations by performing in vitro histone methyltransferase (HMT) activity assay and found that C1073Y and R1197W mutations severely affected the HMT activity. Additionally, the same amino-acid substitutions in mouse GLP induced impairment of in vivo GLP function. Furthermore, these two EHMT1 mutants showed defective heterocomplex formation with G9a (partner HMT) which is essential for their in vivo HMT function. Conclusively, our biochemical characterization clearly demonstrates that the previously reported two missense mutations of EHMT1 deteriorate HMT activity and GLP function, which presumably cause KS.

Nanopore-Based, Label-Free, and Real-Time Monitoring Assay for DNA Methyltransferase Activity and Inhibition.

DNA methylation catalyzed by DNA methyltransferase plays an important role in many biological processes. However, conventional assays proposed for DNA methyltransferase activity are laborious and discontinuous. We have proposed a novel method for real-time monitoring of the activity and kinetics of Escherichia coli DNA adenine methyltransferase (Dam) using nanopore technique coupled with enzyme-linkage reactions. A double-stranded DNA probe AB having a recognition sequence 5'-GATC-3' for both Dam and MboI restriction endonuclease was prepared. Dam catalyzed the methylation of substrate probe AB, which blocked the cleavage reaction of MboI, while the absence of Dam resulted in cleavage of nonmethylated probe AB into four ssDNA fragments by MboI. When tested with nanopore, double-stranded methylated probe AB generated long-lived events, distinguished clearly from MboI-cleavage-mediated ssDNA fragments that generated only spikelike events. The proposed method has a detection limit of 0.03 U/mL for Dam in a short assay time of about 150 min. This sensing system is easy to perform, simple to design and circumvents the use of radioactive substances, resulting in efficient detection of the activity of Dam even in complex matrixes like human serum sample. Furthermore, it has the potential to screen Dam-targeted inhibitor drugs which may assist in the discovery of new anticancer medicines. This method is general and could be extended easily for monitoring activity of a wide variety of methyltransferases by coupling with their corresponding methylation-sensitive endonucleases.

Ultrasensitive and Accurate Assay of Human Methyltransferase Activity at the Single-Cell Level Based on a Single Integrated Magnetic Microprobe.

Human DNA methyltransferase (MTase) activity expression patterns and inhibition response are linked to related cancer initiation, progression, and therapeutic responses. Sensitive and accurate human MTase activity assay in cancer cells, especially at the single-cell level, is essential for biological study, clinical diagnosis, and therapy. Here, we developed an ultrasensitive and accurate DNA (cytosine-5)-methyltransferase 1 (Dnmt1) activity assay at the single-cell level based on a single integrated magnetic microprobe of functionalized double-stranded DNA (dsDNA) anchored to a single magnetic microbead surface. Functionalized dsDNA is designed with a hemimethylated DNA site for Dnmt1 recognition and a single-stranded tail to trigger in situ rolling circle amplification (RCA). Under the action of Dnmt1, hemimethylated dsDNA could be recognized and catalyzed to fully methylated dsDNA, which would protect them from the cleavage of BssHII. However, the dsDNA without full methylation would be cut by BssHII, making single-stranded tail separated from the single integrated microprobe. Subsequently, full methylation-protected in situ RCA could be performed, and multiple signal probes were hybridized to the single integrated microprobe for amplified signal accumulation. Finally, Dnmt1 activity could be evaluated by reading the fluorescence of the single integrated microprobe. Meanwhile, to minimize matrix interferences, magnetic separation was performed in the process. In this strategy, the single integrated magnetic microprobe was provided with integrated capacities of target recognition, signal amplification, signal accumulation, and matrix isolation. Therefore, an ultralow detection limit of 0.007 U/mL Dnmt1 was obtained, and accurate Dnmt1 activity assays in multiple cell lysates at the single-cell level were achieved. Furthermore, the inhibition effect of RG108 was evaluated conveniently. These results indicate that the single integrated magnetic microprobe-based strategy is an excellent candidate for sensitive monitoring of Dnmt1 activity and screening of anticancer drugs.

Photoelectrochemical immunosensing platform for M. SssI methyltransferase activity analysis and inhibitor screening based on g-C3N4 and CdS quantum dots

DNA methylation is one of the important epigenetics events, and it is responsible for transcription, genomic imprinting and cellular differentiation. Aberrant DNA methylation is always contacted with various diseases. Herein, a simple and low-cost photoelectrochemical (PEC) biosensing method is proposed for detection of DNA methylation, assay of DNA methyltransferase (MTase) activity and screening of MTase inhibitor, which is based on M. SssI MTase-HpaII endonuclease system. The graphite-like C3N4 (g-C3N4) and CdS quantum dots (CdS QDs) are used as photoactive materials. After the double-stranded DNA (dsDNA) was treated with M. SssI MTase in the presence of S-adenosylmethionine, the methylated dsDNA cannot be digested by HpaII endonuclease and dithiol group at the terminal of dsDNA can be retained. As a result, CdS QDs could be coupled successfully with methylated dsDNA between the reaction of CdS QDs with dithiol of dsDNA, achieving the amplification of PEC response. Thus, the photocurrent of PEC biosensor is consistent with the M. SssI activity. The increased photocurrent was in proportion to M. SssI activity in the linear range from 1 to 80U/mL with a detection limit of 0.316U/mL. Furthermore, the effects of atrazine and azamethipos on M. SssI MTase activity were investigated, which might provide useful information on carcinogenic mechanism of pesticide. The PEC biosensor has potential to screen of DNA MTase inhibitor, providing valuable information for anti-cancer drug research and also for cancer therapy.

Use of Molecular Beacons in a Simple and Sensitive Fluorescence Assay for CG Methyltransferase Activity

Use of Molecular Beacons in a Simple and Sensitive Fluorescence Assay for CG Methyltransferase Activity

The colorimetric assay of DNA methyltransferase activity based on strand displacement amplification

In this paper, we present a colorimetric method for the assay of DNA methyltransferase (MTase) activity based on strand displacement amplification (SDA). In our study, a well-designed hairpin DNA I (HPI) containing the sequence of 5′-CCGG-3′ is specifically recognized by CpG methyltransferase (M.SssI) and HpaII endonuclease. The methylated HPI is able to coexist with all the DNA and enzymes in the solution while the unmethylated HPI can be cleaved into single-stranded DNA (ssDNA) fragments. The amplification can be triggered by the HpaII digestion products hybridization with another hairpin structure DNA II (HPII) to form a duplex, which would be replaced by probe DNA, leading to the aggregation of gold nanoparticles (AuNPs). Simultaneously, ssDNA fragments released from the duplex, and triggered the cycle anew. Varying concentrations of M.SssI in the solution therefore would lead to differences of absorption and color changed from red to pale. A linear response was obtained when the M.SssI concentration ranging from 0.2 to 50UmL−1 with a detection limit of 0.08UmL−1. In addition, the developed assay in this study can also be applied to screen the inhibitors of M.SssI.

Abstract 3596: KLLN protects genomic stability by maintaining H3K9 trimethylation (H3K9me3) at the pericentric heterochromatin

Abstract Dysregulation in the maintenance of chromatin organization resulting in genomic instability is a major driving force for inappropriate development and carcinogenesis. Tumor suppressor genes play a critical role in this regulation through the maintenance of epigenetic marks. Our study focuses on the role of KLLN in the maintenance of pericentric H3K9 trimethylation (H3K9me3) and genomic stability. Germline hypermethylation of KLLN resulting in decreased KLLN expression has been linked to Cowden cancer-predisposition syndrome (CS) in PTEN mutation negative patients. KLLN is a tumor suppressor gene necessary for p53-mediated apoptosis. The protein mediates S-phase arrest and is known to have DNA binding ability. Here, we first used chromatin immunoprecipitation-based sequencing (ChIP-seq) to investigate regions of KLLN binding on the genome and compared it to regions of H3K9me3 enrichment. We used H3K9 specific histone methyltransferase (HMT) activity assay and immunoblotting to measure levels of H3K9me3. Immunostaining was used to study the localization of KLLN, and micronuclei frequency and aberrations in chromosome number was used to assess chromosomal instability. Analysis of ChIP-seq shows enrichment of KLLN binding in regions of H3K9me3. Overexpression of KLLN using plasmid-based transfection correlates with increased H3K9 methyltransferase activity and increased global H3K9me3, while loss of KLLN expression through siRNA-mediated knockdown had an opposite effect. We also established that KLLN localizes to pericentric regions, and loss of KLLN results in dysregulation of pericentric heterochromatin, with consequent chromosomal instability manifested by increased micronuclei frequency and numerical chromosomal aberrations. KLLN regulation of H3K9me3 could be correlated with its interaction with deleted in breast cancer (DBC1). DBC1 is a known inhibitor of SUV39H1, a H3K9 specific histone methyltransferase responsible for maintenance of pericentric heterochromatin. We hypothesize that KLLN sequesters DBC1 through their interaction, thereby abrogating DBC1 inhibition of SUV39H1, resulting in maintenance of pericentric heterochromatin and genomic stability. Our study therefore suggests a critical role for KLLN in the deterrence of tumor initiation and progression. SIGNIFICANCE: Our study demonstrates a clear role for KLLN in maintenance of chromatin organization thereby maintaining genomic stability, which begins to uncover the basis of KLLN as a tumor suppressor and as a susceptibility gene for inherited cancers. Citation Format: Madhav Sankunny, Emily Nizialek, Farshad Niazi, Charis Eng. KLLN protects genomic stability by maintaining H3K9 trimethylation (H3K9me3) at the pericentric heterochromatin. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3596.

Abstract 4534: Taxol therapy-induced catalytic EZH2 limits therapeutic responses in PTEN null prostate cancer

Abstract Prostate cancer (PCa) is the most common cancer and a major cause of cancer related deaths in men in North America. The phosphatase and tensin homolog gene (PTEN) is frequently inactivated in PCa. Approximately 40% of primary and 70% of metastatic PCa's have genomic alterations in the PI3K-signaling, mostly through the loss of PTEN. Majority of metastatic PCa associated with PTEN loss are treated with docetaxel; an FDA approved therapy for recurrent PCa. However, in PTEN null PCa docetaxel treatment enhances senescence and does not significantly impact tumor volume. These findings were further confirmed in PCa patients with decreased levels of PTEN. In order to understand the global impact of docetaxel in PTEN null PCa we performed an unbiased RNA-seq of prostate tissues harvested from docetaxel treated prostate specific PTEN null mice. Data analysis showed strong enrichment of chromatin modifying enzymes reactome, mainly PRC2 as one of the top ranked categories. Among them EZH2, an oncogenic methyltransferase was found to be significantly upregulated. Quantitative PCR analysis confirmed statistically significant upregulation of EZH2 in docetaxel treated mice compared to vehicle control. EZH2 upregulation was independent of androgen levels as both androgen dependent and independent cells showed striking upregulation of EZH2 when treated with docetaxel. Next, we confirmed the nature of the upregulated EZH2 (catalytic or non-catalytic) by docetaxel. EZH2 methyltransferase activity assay data showed significant induction of methyltransferase activity by docetaxel in both androgen dependent and independent conditions and in PTEN null mouse tissues. Further, we measured other methyltransferase marks i.e. H3K27me1&2, H3K4me3, H3K9me3 in the presence of docetaxel and found only specific upregulation of H3K27me3, confirming upregulation of only catalytic EZH2 by docetaxel. These data suggested a need for co-targeting EZH2 during docetaxel treatment in PTEN null PCa conditions. In order to provide a proof-of-principle we performed a tumor xenograft study in nude mice implanted with PTEN null PC3 cells. Combination of DZNeP (an EZH2 inhibitor) and docetaxel significantly inhibited tumor growth compared to each agent alone. Results obtained from the xenograft studies prompted us to perform a preclinical study in prostate specific PTEN null mice. Data obtained from this study clearly showed a significant reduction in number of high grade lesions in PTEN null tumors in combination group when compared to either docetaxel or DZNeP treated mice. We also observed clearance of senescent cells and accumulation of CD8+ T cells and classical dendritic (CD8+ Clec9a+ positive) cells. Our data suggests the necessity of designing a combination therapy of targeting EZH2 during docetaxel treatment specifically, which will likely increase the efficacy of docetaxel in PTEN null PCa. Citation Format: Mohammad Imran Khan, Abid Hamid, Vaqar Mustafa Adhami, Chloe Lang, Hasan Mukhtar. Taxol therapy-induced catalytic EZH2 limits therapeutic responses in PTEN null prostate cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4534.

SDR-recycling signal amplification for highly sensitive methyltransferase activity assay

In this work, a new electrochemical method for rapid and sensitive evaluation of DNA methyltransferase (MTase) activity based on DNA strand displacement reaction (SDR) recycling signal amplification strategy was developed. Briefly, DNA adenine methyltransferase (Dam MTase) can catalyze the methylation of adenine into N6-methyladenine adenine (m6A) in the specific 5′-GATC-3′ of the hairpin DNA probe 1 (H1). Methylated H1 can be specifically recognized and cleaved by the restriction endonuclease DpnI, which releases a unstable loop DNA and quickly converted to a single-stranded DNA (S1). Then S1 can hybridize with hairpin DNA probe 2 (H2) on the electrode and open the hairpin structure of H2, and hairpin DNA probe 3 (H3) in the solution would hybridize with H2 to form a double-strand DNA (dsDNA), freeing S1 to trigger another reaction cycle and generating a significant response signal. The results demonstrated that the developed method could be used in highly sensitive detection of Dam MTase with a low detection limit of 0.03UmL−1. Furthermore, the proposed method suggested that 5-Fluorouracil could inhibit the Dam MTase activity with the half maximal inhibitory concentration (IC50) of 0.6μM, implying its promising application in screening of suitable inhibitors for Dam MTase.