DOCSLIB.ORG

Whole Transcriptome Sequencing Reveals Extensive Unspliced Mrna in Metastatic Castration-Resistant Prostate Cancer Adam G

Total Page：16

File Type：pdf, Size：1020Kb

Download full-text PDF Read full-text
Whole Transcriptome Sequencing Reveals Extensive Unspliced Mrna in Metastatic Castration-Resistant Prostate Cancer Adam G Published OnlineFirst September 4, 2014; DOI: 10.1158/1541-7786.MCR-14-0273 Chromatin, Gene, and RNA Regulation Molecular Cancer Research Whole Transcriptome Sequencing Reveals Extensive Unspliced mRNA in Metastatic Castration-Resistant Prostate Cancer Adam G. Sowalsky1, Zheng Xia2, Liguo Wang2, Hao Zhao2, Shaoyong Chen1, Glenn J. Bubley1, Steven P. Balk1, and Wei Li2 Abstract Men with metastatic prostate cancer who are treated with be the result of incomplete splicing at canonical splice junctions. androgen deprivation therapies (ADT) usually relapse within 2 Using quantitative PCR (qPCR), a series of genes (AR, KLK2, to 3 years with disease that is termed castration-resistant prostate KLK3, STEAP2, CPSF6, and CDK19) were confirmed to have a cancer (CRPC). To identify the mechanism that drives these greater proportion of unspliced RNA in CRPC specimens than in advanced tumors, paired-end RNA-sequencing (RNA-seq) was normal prostate epithelium, untreated primary prostate cancer, performed on a panel of CRPC bone marrow biopsy specimens. and cultured prostate cancer cells. This inefficient coupling of From this genome-wide approach, mutations were found in a transcription and mRNA splicing suggests an overall increase in series of genes with prostate cancer relevance, including AR, transcription or defect in splicing. NCOR1, KDM3A, KDM4A, CHD1, SETD5, SETD7, INPP4B, RASGRP3, RASA1, TP53BP1, and CDH1, and a novel SND1:BRAF Implications: Inefficient splicing in advanced prostate can- gene fusion. Among the most highly expressed transcripts were 10 cer provides a selective advantage through effects on microRNA noncoding RNAs (ncRNAs), including MALAT1 and PABPC1, networks but may render tumors vulnerable to agents that which are involved in RNA processing. Notably, a high percentage suppress rate-limiting steps in splicing. Mol Cancer Res; 13(1); of sequence reads mapped to introns, which were determined to 98–106. Ó2014 AACR. Introduction androgen receptor (AR) is substantially reactivated in a large proportion of these relapsed tumors through increased intratu- With more than 230,000 new patients and nearly 30,000 moral androgen synthesis, in conjunction with other mechanisms deaths annually, prostate cancer is the second most common that may enhance AR expression and activity, and many of these cause of cancer-related deaths in men in the United States (1). tumors will respond to agents that further suppress androgen Although greater than 75% of patients with early-stage prostate synthesis (CYP17A1 inhibitors such as abiraterone) or new AR cancer can be cured with surgical and/or radiation treatment, the antagonists (such as enzalutamide). Unfortunately, these men remainder ultimately recur with metastatic disease. Androgen generally relapse within 1 to 2 years, and increasing serum PSA deprivation therapy (surgical castration or the administration of in most cases suggests that AR is again active in these resistant luteinizing hormone–releasing hormone agonists) is the standard tumors. treatment for metastatic prostate cancer (2), but most tumors We reported previously on an analysis of gene expression in eventually relapse despite castrate androgen levels (castration- CRPC bone marrow metastases using Affymetrix oligonucleotide resistant prostate cancer, CRPC). It has now become clear that microarrays and immunohistochemistry, which showed increased expression of enzymes mediating androgen synthesis 1Division of Hematology and Oncology, Department of Medicine, Beth and alterations in the expression of additional genes linked to Israel Deaconess Medical Center and Harvard Medical School, Boston, tumor progression (3). We hypothesize that additional mechan- Massachusetts. 2Division of Biostatistics, Dan L Duncan Cancer Center isms mediating progression to CRPC will also contribute to tumor and Department of Molecular and Cellular Biology, Baylor College of progression after treatment with new hormonal agents including Medicine, Houston, Texas. abiraterone and enzalutamide. Therefore, in this study, we have Note: Supplementary data for this article are available at Molecular Cancer used paired-end RNA-sequencing (RNA-seq) to assess more com- Research Online (http://mcr.aacrjournals.org/). prehensively the transcriptome of 8 CRPC bone marrow metas- A.G. Sowalsky and Z. Xia contributed equally to this article. tases that had been examined previously on Affymetrix U133A Corresponding Authors: Steven P. Balk, Beth Israel Deaconess Medical Center, microarrays. 330 Brookline Avenue, Boston, MA 02215. Phone: 617-735-2065; Fax: 1-617-735- 2050; E-mail: [email protected]; and Wei Li, Baylor College of Medicine, 1 Baylor Plaza, Mail Stop BCM305, Cullen Building, Suite 529D, Houston, TX Materials and Methods 77030. Phone: 713-798-7854; E-mail: [email protected] Tissue samples doi: 10.1158/1541-7786.MCR-14-0273 All tissue samples in this study were obtained with consent Ó2014 American Association for Cancer Research. from patients with prostate cancer in compliance with the Beth 98 Mol Cancer Res; 13(1) January 2015 Downloaded from mcr.aacrjournals.org on September 25, 2021. © 2015 American Association for Cancer Research. Published OnlineFirst September 4, 2014; DOI: 10.1158/1541-7786.MCR-14-0273 Extensive Unspliced RNA in Advanced Prostate Cancer Israel Deaconess Medical Center Institutional Review Board. Results CRPC biopsies were obtained from the posterior iliac crest and RNA-seq gene expression analysis is concordant with previous snap frozen as previously described (3–5). Frozen sections stained microarray analysis with hematoxylin and eosin (H&E) were examined histologically We had previously analyzed on Affymetrix U133A microarrays and four to six 6-mm ribbons with >90% tumor and minimal bone a panel of 33 CRPC bone marrow biopsies in comparison with a marrow elements were treated with TRIzol (Invitrogen) for puri- series of primary prostate cancer (3). However, the additional fication of total RNA. information that can be gained by paired-end RNA-seq led us to To obtain nonneoplastic prostate epithelium, we examined re-analyze a subset of these CRPC samples, which were selected on snap-frozen samples from radical prostatectomies in patients the basis of very low contaminating hematopoietic or stromal cell with low volume prostate cancer and collected sections with content (>90% tumor by H&E) and availability of adequate RNA. 20% to 80% normal prostate epithelium and no evident For each of the 8 samples selected, 50 ng of total RNA was tumor on histology. DNase-treated RNA was extracted from amplified into double-stranded cDNA and Illumina paired-end ten 6-mm ribbons using the RNeasy Plus Micro Kit (Qiagen). adaptors were ligated onto the library for 76 cycles of paired-end To obtain primary prostate cancer, samples from radical sequencing (samples 49 and 66) or 101 cycles of paired-end prostatectomies were fixed in PaxGene (Qiagen), processed sequencing (samples 24, 28, 39, 55, 71, and 74; see Supplemen- into paraffin, and sectioned at 5 mm onto Arcturus polyeth- tary Methods). ylene naphthalate metal-framed slides (Molecular Machines Although RNA from the previously analyzed primary prostate & Industries). Approximately 50,000 cells in Gleason pattern cancer was not available, we were still interested in whether gene 3and4glandsidentified by a board-certified pathologist expression data from the RNA-seq and the previous Affymetrix were captured onto caps using 20-mm infrared pulses and U133A microarrays were consistent. Therefore, we re-analyzed the excised from the adjacent tissue using the ultraviolet laser on Affymetrix raw data to perform a transcript-level normalization an ArcturusXT Nikon Eclipse Ti-E microdissection system. and performed a correlation analysis between the intensity values DNase-treated RNA was extracted using the PaxGene Tissue of these arrays with the RPKM from our RNA-seq data (see RNA Kit (Qiagen). Supplementary Methods). Considering approximately 13,000 transcripts (Supplementary Table S1), our analysis showed a Library preparation and data analysis statistically significant, positive correlation between gene expres- Fifty nanograms of RNA from CRPC samples was prepared for sion values measured from the same CRPC sample on both Illumina paired-end sequencing using the Ovation RNA-Seq platforms (Supplementary Fig. S1). Our observation of r values System (NuGEN), and FastQ files were aligned to the human less than 0.7 may be attributed to the 3-prime bias intrinsic in the genome (version Hg19). Complete descriptions of library prep- U133A microarray, whereas our random priming, whole tran- aration methods and sequencing data analysis are provided as scriptomic RNA-seq approach resulted in consistent coverage Supplementary Material. across transcripts (8) and better detection of low-abundance transcripts (9). Spearman r values increased when only the last Cell lines exon RPKM was used for correlation analysis (data not shown). VCaP and LNCaP cells were obtained from ATCC and pas- Nonetheless, this result indicated that gene expression values were saged for fewer than 6 months after receipt. VCS2 (6) and C4-2 not platform-dependent and supported our previous conclusions (7) cells were derived from VCaP and LNCaP cells, respectively. regarding gene expression differences between the primary pros- Subconfluent cultures of VCaP, LNCaP, VCS2, and C4-2 cells tate cancer and CRPC samples (3). grown in the presence of androgen (5%–10% FBS) were used as a source of control RNA. Cell lines' identities were routinely
Load more

Recommended publications
  • Interplay Between Epigenetics and Metabolism in Oncogenesis: Mechanisms and Therapeutic Approaches
    OPEN Oncogene (2017) 36, 3359–3374 www.nature.com/onc REVIEW Interplay between epigenetics and metabolism in oncogenesis: mechanisms and therapeutic approaches CC Wong1, Y Qian2,3 and J Yu1 Epigenetic and metabolic alterations in cancer cells are highly intertwined. Oncogene-driven metabolic rewiring modifies the epigenetic landscape via modulating the activities of DNA and histone modification enzymes at the metabolite level. Conversely, epigenetic mechanisms regulate the expression of metabolic genes, thereby altering the metabolome. Epigenetic-metabolomic interplay has a critical role in tumourigenesis by coordinately sustaining cell proliferation, metastasis and pluripotency. Understanding the link between epigenetics and metabolism could unravel novel molecular targets, whose intervention may lead to improvements in cancer treatment. In this review, we summarized the recent discoveries linking epigenetics and metabolism and their underlying roles in tumorigenesis; and highlighted the promising molecular targets, with an update on the development of small molecule or biologic inhibitors against these abnormalities in cancer. Oncogene (2017) 36, 3359–3374; doi:10.1038/onc.2016.485; published online 16 January 2017 INTRODUCTION metabolic genes have also been identified as driver genes It has been appreciated since the early days of cancer research mutated in some cancers, such as isocitrate dehydrogenase 1 16 17 that the metabolic profiles of tumor cells differ significantly from and 2 (IDH1/2) in gliomas and acute myeloid leukemia (AML), 18 normal cells. Cancer cells have high metabolic demands and they succinate dehydrogenase (SDH) in paragangliomas and fuma- utilize nutrients with an altered metabolic program to support rate hydratase (FH) in hereditary leiomyomatosis and renal cell 19 their high proliferative rates and adapt to the hostile tumor cancer (HLRCC).
    [Show full text]
  • Catalysis by the Jmjc Histone Demethylase KDM4A Integrates Substrate Dynamics, Correlated Motions Cite This: Chem
    Chemical Science View Article Online EDGE ARTICLE View Journal | View Issue Catalysis by the JmjC histone demethylase KDM4A integrates substrate dynamics, correlated motions Cite this: Chem. Sci., 2020, 11, 9950 † All publication charges for this article and molecular orbital control have been paid for by the Royal Society a a b of Chemistry Rajeev Ramanan, Shobhit S. Chaturvedi, Nicolai Lehnert, Christopher J. Schofield, c Tatyana G. Karabencheva-Christova *a and Christo Z. Christov *a 3 The N -methyl lysine status of histones is important in the regulation of eukaryotic transcription. The Fe(II) and 2-oxoglutarate (2OG) -dependent JmjC domain enzymes are the largest family of histone N3-methyl lysine demethylases (KDMs). The human KDM4 subfamily of JmjC KDMs is linked with multiple cancers and some of its members are medicinal chemistry targets. We describe the use of combined molecular dynamics (MD) and Quantum Mechanical/Molecular Mechanical (QM/MM) methods to study the mechanism of KDM4A, which catalyzes demethylation of both tri- and di-methylated forms of histone Creative Commons Attribution-NonCommercial 3.0 Unported Licence. H3 at K9 and K36. The results show that the oxygen activation at the active site of KDM4A is optimized towards the generation of the reactive Fe(IV)-oxo intermediate. Factors including the substrate binding mode, correlated motions of the protein and histone substrates, and molecular orbital control synergistically contribute to the reactivity of the Fe(IV)-oxo intermediate. In silico substitutions were performed to investigate the roles of residues (Lys241, Tyr177, and Asn290) in substrate orientation. The Lys241Ala substitution abolishes activity due to altered substrate orientation consistent with reported experimental studies.
    [Show full text]
  • Mutant IDH, (R)-2-Hydroxyglutarate, and Cancer
    Downloaded from genesdev.cshlp.org on October 1, 2021 - Published by Cold Spring Harbor Laboratory Press REVIEW What a difference a hydroxyl makes: mutant IDH, (R)-2-hydroxyglutarate, and cancer Julie-Aurore Losman1 and William G. Kaelin Jr.1,2,3 1Department of Medical Oncology, Dana-Farber Cancer Institute, Brigham and Women’s Hospital, Harvard Medical School, Boston, Massachusetts 02215, USA; 2Howard Hughes Medical Institute, Chevy Chase, Maryland 20815, USA Mutations in metabolic enzymes, including isocitrate whether altered cellular metabolism is a cause of cancer dehydrogenase 1 (IDH1) and IDH2, in cancer strongly or merely an adaptive response of cancer cells in the face implicate altered metabolism in tumorigenesis. IDH1 of accelerated cell proliferation is still a topic of some and IDH2 catalyze the interconversion of isocitrate and debate. 2-oxoglutarate (2OG). 2OG is a TCA cycle intermediate The recent identification of cancer-associated muta- and an essential cofactor for many enzymes, including tions in three metabolic enzymes suggests that altered JmjC domain-containing histone demethylases, TET cellular metabolism can indeed be a cause of some 5-methylcytosine hydroxylases, and EglN prolyl-4-hydrox- cancers (Pollard et al. 2003; King et al. 2006; Raimundo ylases. Cancer-associated IDH mutations alter the enzymes et al. 2011). Two of these enzymes, fumarate hydratase such that they reduce 2OG to the structurally similar (FH) and succinate dehydrogenase (SDH), are bone fide metabolite (R)-2-hydroxyglutarate [(R)-2HG]. Here we tumor suppressors, and loss-of-function mutations in FH review what is known about the molecular mechanisms and SDH have been identified in various cancers, in- of transformation by mutant IDH and discuss their im- cluding renal cell carcinomas and paragangliomas.
    [Show full text]
  • 1714 Gene Comprehensive Cancer Panel Enriched for Clinically Actionable Genes with Additional Biologically Relevant Genes 400-500X Average Coverage on Tumor
    xO GENE PANEL 1714 gene comprehensive cancer panel enriched for clinically actionable genes with additional biologically relevant genes 400-500x average coverage on tumor Genes A-C Genes D-F Genes G-I Genes J-L AATK ATAD2B BTG1 CDH7 CREM DACH1 EPHA1 FES G6PC3 HGF IL18RAP JADE1 LMO1 ABCA1 ATF1 BTG2 CDK1 CRHR1 DACH2 EPHA2 FEV G6PD HIF1A IL1R1 JAK1 LMO2 ABCB1 ATM BTG3 CDK10 CRK DAXX EPHA3 FGF1 GAB1 HIF1AN IL1R2 JAK2 LMO7 ABCB11 ATR BTK CDK11A CRKL DBH EPHA4 FGF10 GAB2 HIST1H1E IL1RAP JAK3 LMTK2 ABCB4 ATRX BTRC CDK11B CRLF2 DCC EPHA5 FGF11 GABPA HIST1H3B IL20RA JARID2 LMTK3 ABCC1 AURKA BUB1 CDK12 CRTC1 DCUN1D1 EPHA6 FGF12 GALNT12 HIST1H4E IL20RB JAZF1 LPHN2 ABCC2 AURKB BUB1B CDK13 CRTC2 DCUN1D2 EPHA7 FGF13 GATA1 HLA-A IL21R JMJD1C LPHN3 ABCG1 AURKC BUB3 CDK14 CRTC3 DDB2 EPHA8 FGF14 GATA2 HLA-B IL22RA1 JMJD4 LPP ABCG2 AXIN1 C11orf30 CDK15 CSF1 DDIT3 EPHB1 FGF16 GATA3 HLF IL22RA2 JMJD6 LRP1B ABI1 AXIN2 CACNA1C CDK16 CSF1R DDR1 EPHB2 FGF17 GATA5 HLTF IL23R JMJD7 LRP5 ABL1 AXL CACNA1S CDK17 CSF2RA DDR2 EPHB3 FGF18 GATA6 HMGA1 IL2RA JMJD8 LRP6 ABL2 B2M CACNB2 CDK18 CSF2RB DDX3X EPHB4 FGF19 GDNF HMGA2 IL2RB JUN LRRK2 ACE BABAM1 CADM2 CDK19 CSF3R DDX5 EPHB6 FGF2 GFI1 HMGCR IL2RG JUNB LSM1 ACSL6 BACH1 CALR CDK2 CSK DDX6 EPOR FGF20 GFI1B HNF1A IL3 JUND LTK ACTA2 BACH2 CAMTA1 CDK20 CSNK1D DEK ERBB2 FGF21 GFRA4 HNF1B IL3RA JUP LYL1 ACTC1 BAG4 CAPRIN2 CDK3 CSNK1E DHFR ERBB3 FGF22 GGCX HNRNPA3 IL4R KAT2A LYN ACVR1 BAI3 CARD10 CDK4 CTCF DHH ERBB4 FGF23 GHR HOXA10 IL5RA KAT2B LZTR1 ACVR1B BAP1 CARD11 CDK5 CTCFL DIAPH1 ERCC1 FGF3 GID4 HOXA11 IL6R KAT5 ACVR2A
    [Show full text]
  • Maternal Expression of the Histone Demethylase Kdm4a Is Crucial For
    © 2017. Published by The Company of Biologists Ltd | Development (2017) 144, 3264-3277 doi:10.1242/dev.155473 RESEARCH ARTICLE Maternal expression of the histone demethylase Kdm4a is crucial for pre-implantation development Aditya Sankar1,2,3,*,§, Susanne Marije Kooistra1,2,‡,§, Javier Martin Gonzalez4, Claes Ohlsson6, Matti Poutanen5,6 and Kristian Helin1,2,3,¶ ABSTRACT residue, as well as type and degree of modification (Greer and Shi, Regulation of chromatin composition through post-translational 2012; Kooistra and Helin, 2012; Dimitrova et al., 2015). modifications of histones contributes to transcriptional regulation Similar to the other members of the JMJD2/KDM4 family of and is essential for many cellular processes, including differentiation histone demethylases, KDM4A is capable of removing di- and tri- and development. KDM4A (JMJD2A) is a lysine demethylase with methylation from lysines 9 and 36 of histone H3 (Cloos et al., 2006; specificity towards di- and tri-methylated lysine 9 and lysine 36 of Klose et al., 2006; Whetstine et al., 2006; Chen et al., 2006, 2007; histone H3 (H3K9me2/me3 and H3K36me2/me3). Here, we report Ng et al., 2007). KDM4A, like KDM4C, binds to tri-methylated that Kdm4a as a maternal factor plays a key role in embryo survival lysine 4 on histone H3 (H3K4me3) through its double Tudor and is vital for female fertility. Kdm4a−/− female mice ovulate normally domain (Huang et al., 2006; Pedersen et al., 2016, 2014). In this with comparable fertilization but poor implantation rates, and cannot way, the KDM4 proteins provide a failsafe mechanism to prevent support healthy transplanted embryos to term. This is due to a role for spurious accumulation of H3K9me3 at active promotors, thereby Kdm4a in uterine function, where its loss causes reduced expression maintaining transcriptional competence (Pedersen et al., 2016).
    [Show full text]
  • Electronic Supplementary Material (ESI) for Metallomics
    Electronic Supplementary Material (ESI) for Metallomics. This journal is © The Royal Society of Chemistry 2018 Uniprot Entry name Gene names Protein names Predicted Pattern Number of Iron role EC number Subcellular Membrane Involvement in disease Gene ontology (biological process) Id iron ions location associated 1 P46952 3HAO_HUMAN HAAO 3-hydroxyanthranilate 3,4- H47-E53-H91 1 Fe cation Catalytic 1.13.11.6 Cytoplasm No NAD biosynthetic process [GO:0009435]; neuron cellular homeostasis dioxygenase (EC 1.13.11.6) (3- [GO:0070050]; quinolinate biosynthetic process [GO:0019805]; response to hydroxyanthranilate oxygenase) cadmium ion [GO:0046686]; response to zinc ion [GO:0010043]; tryptophan (3-HAO) (3-hydroxyanthranilic catabolic process [GO:0006569] acid dioxygenase) (HAD) 2 O00767 ACOD_HUMAN SCD Acyl-CoA desaturase (EC H120-H125-H157-H161; 2 Fe cations Catalytic 1.14.19.1 Endoplasmic Yes long-chain fatty-acyl-CoA biosynthetic process [GO:0035338]; unsaturated fatty 1.14.19.1) (Delta(9)-desaturase) H160-H269-H298-H302 reticulum acid biosynthetic process [GO:0006636] (Delta-9 desaturase) (Fatty acid desaturase) (Stearoyl-CoA desaturase) (hSCD1) 3 Q6ZNF0 ACP7_HUMAN ACP7 PAPL PAPL1 Acid phosphatase type 7 (EC D141-D170-Y173-H335 1 Fe cation Catalytic 3.1.3.2 Extracellular No 3.1.3.2) (Purple acid space phosphatase long form) 4 Q96SZ5 AEDO_HUMAN ADO C10orf22 2-aminoethanethiol dioxygenase H112-H114-H193 1 Fe cation Catalytic 1.13.11.19 Unknown No oxidation-reduction process [GO:0055114]; sulfur amino acid catabolic process (EC 1.13.11.19) (Cysteamine
    [Show full text]
  • Figure S1. Reverse Transcription‑Quantitative PCR Analysis of ETV5 Mrna Expression Levels in Parental and ETV5 Stable Transfectants
    Figure S1. Reverse transcription‑quantitative PCR analysis of ETV5 mRNA expression levels in parental and ETV5 stable transfectants. (A) Hec1a and Hec1a‑ETV5 EC cell lines; (B) Ishikawa and Ishikawa‑ETV5 EC cell lines. **P<0.005, unpaired Student's t‑test. EC, endometrial cancer; ETV5, ETS variant transcription factor 5. Figure S2. Survival analysis of sample clusters 1‑4. Kaplan Meier graphs for (A) recurrence‑free and (B) overall survival. Survival curves were constructed using the Kaplan‑Meier method, and differences between sample cluster curves were analyzed by log‑rank test. Figure S3. ROC analysis of hub genes. For each gene, ROC curve (left) and mRNA expression levels (right) in control (n=35) and tumor (n=545) samples from The Cancer Genome Atlas Uterine Corpus Endometrioid Cancer cohort are shown. mRNA levels are expressed as Log2(x+1), where ‘x’ is the RSEM normalized expression value. ROC, receiver operating characteristic. Table SI. Clinicopathological characteristics of the GSE17025 dataset. Characteristic n % Atrophic endometrium 12 (postmenopausal) (Control group) Tumor stage I 91 100 Histology Endometrioid adenocarcinoma 79 86.81 Papillary serous 12 13.19 Histological grade Grade 1 30 32.97 Grade 2 36 39.56 Grade 3 25 27.47 Myometrial invasiona Superficial (<50%) 67 74.44 Deep (>50%) 23 25.56 aMyometrial invasion information was available for 90 of 91 tumor samples. Table SII. Clinicopathological characteristics of The Cancer Genome Atlas Uterine Corpus Endometrioid Cancer dataset. Characteristic n % Solid tissue normal 16 Tumor samples Stagea I 226 68.278 II 19 5.740 III 70 21.148 IV 16 4.834 Histology Endometrioid 271 81.381 Mixed 10 3.003 Serous 52 15.616 Histological grade Grade 1 78 23.423 Grade 2 91 27.327 Grade 3 164 49.249 Molecular subtypeb POLE 17 7.328 MSI 65 28.017 CN Low 90 38.793 CN High 60 25.862 CN, copy number; MSI, microsatellite instability; POLE, DNA polymerase ε.
    [Show full text]
  • Active Motif Technical Data Sheet (TDS)
    Recombinant JMJD2A / KDM4A protein Catalog No: 31457, 31857 Quantity: 20, 1000 µg Expressed In: Baculovirus Concentration: 0.6 µg/µl Source: Human Buffer Contents: Full length recombinant JMJD2A / KDM4A protein was expressed in Sf9 cells and is supplied in 25 mM Hepes pH 7.5, 300 mM NaCl, 5% Glycerol, 0.04% Triton X-100, 0.2 mM TCEP. Background: KDM4A (lysine (K)-specific demethylase 4A), also known as JMJD2A (Jumonji Domain Containing 2A) is a nuclear protein that functions as a histone demethylase that preferentially demethylates di- and trimethylated lysine 9 (K9me3) and lysine 36 (K36me3) residues of histone H3, converting these trimethylated histone residues to mono- or dimethylated form. JMJD2A / KDM4A has no activity for monomethylated H3K9 and H3K36. KDM4A functions as a transcriptional repressor, participating in transcriptional repression of ASCL2 and E2F-responsive promoters via the recruitment of histone deacetylases and NCOR1, respectively. An additional KDM4A isoform, KDM4A Isoform 2, that lacks the N-terminal demethylase domain is crucial for muscle differentiation in promoting transcriptional activation of the MyoG gene by directing the removal of repressive chromatin marks at its promoter. Protein Details: Recombinant JMJD2A / KDM4A (accession number NP_055478.2) was expressed in Sf9 cells and contains an N-terminal FLAG-Tag with an observed molecular weight of 125.3 kDa. The recombinant protein is >85% pure by SDS-PAGE. Application Notes: Recombinant JMJD2A / KDM4A is suitable for use in the study of enzyme kinetics, inhibitor screening, and selectivity profiling. Histone Demethylase Assay Conditions: 50 mM HEPES pH 7.5, 0.02% Triton X100, 100 μM 2OG, 100 μM Ascorbate, 50 μM (NH4)2Fe(SO4)2·6H2O, 1 mM TCEP, 100 nM Recombinant JMJD2A / KDM4A protein, and 3.3 μM H3K9me3 (aa 1-21) peptide at 2 hours at room temperature.
    [Show full text]
  • NIH Public Access Author Manuscript Curr Cancer Drug Targets
    NIH Public Access Author Manuscript Curr Cancer Drug Targets. Author manuscript; available in PMC 2013 July 06. NIH-PA Author ManuscriptPublished NIH-PA Author Manuscript in final edited NIH-PA Author Manuscript form as: Curr Cancer Drug Targets. 2013 June 10; 13(5): 558–579. Histone Lysine-Specific Methyltransferases and Demethylases in Carcinogenesis: New Targets for Cancer Therapy and Prevention Xuejiao Tian1,2,3, Saiyang Zhang4, Hong-Min Liu4, Yan-Bing Zhang4, Christopher A Blair1,2, Dan Mercola5, Paolo Sassone-Corsi6, and Xiaolin Zi1,2,3,* 1Department of Urology, University of California, Irvine, Orange, CA 92868, USA 2Department of Pharmaceutical Sciences, University of California, Irvine, Orange, CA 92868, USA 3Department of Pharmacology, University of California, Irvine, Orange, CA 92868, USA 4School of Pharmaceutical Sciences, Zhengzhou University, No.100, Science Avenue, Zhengzhou 450001, Henan, China 5Department of Pathology and Laboratory medicine, University of California, Irvine, Orange, CA 92868, USA 6Department of Biological Chemistry, University of California, Irvine, Orange, CA 92868, USA Abstract Aberrant histone lysine methylation that is controlled by histone lysine methyltransferases (KMTs) and demethylases (KDMs) plays significant roles in carcinogenesis. Infections by tumor viruses or parasites and exposures to chemical carcinogens can modify the process of histone lysine methylation. Many KMTs and KDMs contribute to malignant transformation by regulating the expression of human telomerase reverse transcriptase (hTERT), forming a fused gene, interacting with proto-oncogenes or being up-regulated in cancer cells. In addition, histone lysine methylation participates in tumor suppressor gene inactivation during the early stages of carcinogenesis by regulating DNA methylation and/or by other DNA methylation independent mechanisms.
    [Show full text]
  • Epigenetic Changes in Fibroblasts Drive Cancer Metabolism and Differentiation
    26 12 Endocrine-Related R Mishra et al. TME based epigenetic targets 26:12 R673–R688 Cancer in cancer REVIEW Epigenetic changes in fibroblasts drive cancer metabolism and differentiation Rajeev Mishra1, Subhash Haldar2, Surabhi Suchanti1 and Neil A Bhowmick3,4 1Department of Biosciences, Manipal University Jaipur, Jaipur, Rajasthan, India 2Department of Biotechnology, Brainware University, Kolkata, India 3Department of Medicine, Cedars-Sinai Medical Center, Los Angeles, California, USA 4Department of Research, Greater Los Angeles Veterans Administration, Los Angeles, California, USA Correspondence should be addressed to N A Bhowmick: [email protected] Abstract Genomic changes that drive cancer initiation and progression contribute to the Key Words co-evolution of the adjacent stroma. The nature of the stromal reprogramming involves f endocrine therapy differential DNA methylation patterns and levels that change in response to the tumor resistance and systemic therapeutic intervention. Epigenetic reprogramming in carcinoma- f prostate associated fibroblasts are robust biomarkers for cancer progression and have a f neuroendocrine tumors transcriptional impact that support cancer epithelial progression in a paracrine manner. For prostate cancer, promoter hypermethylation and silencing of the RasGAP, RASAL3 that resulted in the activation of Ras signaling in carcinoma-associated fibroblasts. Stromal Ras activity initiated a process of macropinocytosis that provided prostate cancer epithelia with abundant glutamine for metabolic conversion
    [Show full text]
  • The Biological Axis of Protein Arginine Methylation and Asymmetric Dimethylarginine
    International Journal of Molecular Sciences Review The Biological Axis of Protein Arginine Methylation and Asymmetric Dimethylarginine Melody D. Fulton , Tyler Brown and Y. George Zheng * Department of Pharmaceutical and Biomedical Sciences, College of Pharmacy, University of Georgia, Athens, GA 30602, USA * Correspondence: [email protected]; Tel.: +1-706-542-0277 Received: 17 June 2019; Accepted: 4 July 2019; Published: 6 July 2019 Abstract: Protein post-translational modifications (PTMs) in eukaryotic cells play important roles in the regulation of functionalities of the proteome and in the tempo-spatial control of cellular processes. Most PTMs enact their regulatory functions by affecting the biochemical properties of substrate proteins such as altering structural conformation, protein–protein interaction, and protein–nucleic acid interaction. Amid various PTMs, arginine methylation is widespread in all eukaryotic organisms, from yeasts to humans. Arginine methylation in many situations can drastically or subtly affect the interactions of substrate proteins with their partnering proteins or nucleic acids, thus impacting major cellular programs. Recently, arginine methylation has become an important regulator of the formation of membrane-less organelles inside cells, a phenomenon of liquid–liquid phase separation (LLPS), through altering π-cation interactions. Another unique feature of arginine methylation lies in its impact on cellular physiology through its downstream amino acid product, asymmetric dimethylarginine (ADMA). Accumulation of ADMA in cells and in the circulating bloodstream is connected with endothelial dysfunction and a variety of syndromes of cardiovascular diseases. Herein, we review the current knowledge and understanding of protein arginine methylation in regards to its canonical function in direct protein regulation, as well as the biological axis of protein arginine methylation and ADMA biology.
    [Show full text]
  • Preconceptional Paternal Glycidamide Exposure Affects Embryonic Gene
    Reproductive Toxicology 32 (2011) 463–471 Contents lists available at SciVerse ScienceDirect Reproductive Toxicology jo urnal homepage: www.elsevier.com/locate/reprotox Preconceptional paternal glycidamide exposure affects embryonic gene expression: Single embryo gene expression study following in vitro fertilization a,∗ b a a a Asgeir Brevik , Vendula Rusnakova , Nur Duale , Hege Holte Slagsvold , Ann-Karin Olsen , c d a a Ritsa Storeng , Mikael Kubista , Gunnar Brunborg , Birgitte Lindeman a Norwegian Institute of Public Health, Division of Environmental Medicine, Department of Chemical Toxicology, P.O. Box 4404, Nydalen, N-0403 Oslo, Norway b Dept. of Gene Expression, Institute of Biotechnology AS CR, v.v.i., Videnska 1083, Prague 4, 142 20, Czech Republic c Oslo University Hospital - Rikshospitalet, 0027 Oslo, Norway d Tataa Biocenter, Odinsgatan 28, 411 03 Göteborg, Sweden a r t i c l e i n f o a b s t r a c t Article history: Recognition of early determinants of disease onset has sparked an interest in paternally transmitted Received 2 March 2011 factors and their impact on the developing embryo. Acrylamide (AA), a widely distributed xenobiotic Received in revised form 10 August 2011 compound, is converted to its active metabolite glycidamide (GA) by the CYP2E1 enzyme. Based on its Accepted 17 September 2011 capacity to induce dominant lethal mutations, we hypothesized that paternal GA exposure would have Available online 29 September 2011 a negative impact on embryonic genome activation, via GA-DNA and protamine adducts persisting in the fertilizing sperm. Using a combination of in vitro fertilization (IVF) techniques and RT-qPCR single Keywords: embryo gene expression (SEGE), we studied the expression of key DNA repair genes and genes important Single-cell gene expression Glycidamide for embryo development, at the 1-, 2-, 4- and 8-cell stage of the developing mouse embryo.
    [Show full text]