QUANTITATIVE EVALUATION OF INTERACTIONS OF TRANSCRIPTION FACTOR KAP1 AND HETEROCHROMATIN PROTEIN HP1 IN VIVO
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Authors
A.T. Kulyyassov
Republican State Enterprise “National Center for Biotechnology” under the Science Committee of Ministry of Education and Science of the Republic of Kazakhstan, 13/1, Valikhanov str., Astana, 010000, Kazakhstan
G.S. Zhubanova
Republican State Enterprise “National Center for Biotechnology” under the Science Committee of Ministry of Education and Science of the Republic of Kazakhstan, 13/1, Valikhanov str., Astana, 010000, Kazakhstan
E.M. Ramanculov
Republican State Enterprise “National Center for Biotechnology” under the Science Committee of Ministry of Education and Science of the Republic of Kazakhstan, 13/1, Valikhanov str., Astana, 010000, Kazakhstan
V.V. Ogryzko
Institut Gustave Roussy, CNRS UMR8126, 94805, Villejuif, France, 39 Rue Camilles Desmoulin
Abstract
Protein-protein interactions (PPI) play a key role in many processes within the cell, and their affinity and specificity are fine-tuned with respect to the functions they perform. Disturbances in the signal transduction pathways associated with PPI lead to the development and progression of cancer. Study of protein-protein interactions not only improve our understanding the mechanism of the processes inside the cell, but also help in the search of small molecule modulators of PPI for the treatment of various types of tumors.
We have used method, called the Proximity Utilizing Biotinylation (PUB), based on co-expression within a single cell of the recombinant proteins - the protein of interest fused with biotin ligase BirA and its partner with the biotin acceptor peptide BAP, which allows an accurate quantitative assessment of the extent of their interaction in vivo.
The aim of this work is to develop a method for quantifying interactions in vivo of oncomarker proteins HP1a and transcription factor KAP1.
In experiments on protein expression of BAP and BirA fusions of HP1a and KAP1 in HEK293T cells we found elevated levels of biotinylation due to in vivo interaction of proteins BAP-HP1a and BirA-wtKAP1. The ratio of biotinylation between BAP-HP1a in samples with BirA-mutKap1 and BirA-wtKap1 was 0,43±0,086.
In reciprocal experiments, the level of biotinylation was higher in case of interaction between proteins BAP-wtKap1 and BirA-HP1a. The ratio of biotinylation of BAP-mutKap1 and BAP-wtKap1 in the presence of BirA-HP1 was 0,2±0,04.
Keywords
protein-protein interactions, biotinylation, oncomarkers, biotin ligase, biotin acceptor peptide, plasmids, transient transfection, western blot
Article Details
References
Nero T.L., Morton C.J., Holien J.K., Wielens J., Parker M.W. Oncogenic protein interfaces: small molecules, big challenges. Nature Rev.Cancer, 2014, vol. 14, pp. 248-262. doi: 10.1038/nrc3690.
Ivanov A.A., Khuri F.R., Fu H. Targeting protein-protein interactions as an anticancer strategy. Trends in Pharm. Sci., 2013, vol. 34, no. 7, pp. 393-400. dx.doi.org/10.1016/j.tips.2013.04.007.
Wells J.A., McClendon Ch.L. Reaching for high-hanging fruit in drug discovery at protein-protein interfaces. Nature, 2007, vol. 450, no. 13, pp. 1001-1009. doi: 10.1038/nature06526.
Hanahan D., Weinberg R.A. Hallmarks of cancer: The next generation. Cell, 2011, vol. 144, pp. 646-674. doi 10.1016/j.cell.2011.02.013.
Hoe K.K., Verma Ch.S., Lane D.P. Drugging the p53 pathway: understanding the route to clinical efficacy. Nature Rev.Drug Discovery, 2014, vol. 13, pp. 217-236. doi: 10.1038/nrd4236.
Westermarck J., Ivaska J., Corthals G.L. Identification of protein interactions involved in cellular signaling. Mol. Cell. Proteomics, 2013, vol. 12, no. 7, pp. 1752-1763. doi 10.1074/mcp.R113.027771.
Aeluri M., Chamakuri S., Dasari B., Guduru Sh.K.R., Jimmidi R., Jogula S., Arya P. Small molecule modulators of protein-protein interactions: Selected case studies. Chem. Rev., 2014, vol. 114, issue 9, pp. 4640-4694. dx.doi.org/10.1021/cr4004049.
Milroy L.-G., Grossmann T.N., Hennig S., Brunsveld L., Ottmann Ch. Modulators of protein-protein interactions. Chem. Rev., 2014, vol. 114, issue 9, pp. 4695-4748. dx.doi.org/10.1021/cr400698c.
Arkin M.R., Tang Y., Wells J.A. Small-molecule inhibitors of protein-protein interactions: Progressing toward the reality. Chem. & Biol., 2014, vol. 21, issue 9, pp. 1102-1114. dx.doi.org/10.1016/j.chembiol. 2014.09.001.
Britten R.J., and Davidson E.H. Gene regulation for higher cells: a theory. Science, 1969, vol. 165, pp. 349-357. doi:10.1126/science.165.3891.349.
Albagli O., Dhordain P., Deweindt C., Lecocq G., and Leprince D. The BTB/POZ domain: a new protein-protein interaction motif common to DNA- and actin-binding proteins. Cell Growth Differ, 1995, vol. 6, no. 9, pp. 1193-1198. PMID:8519696.
Bellefroid E.J., Poncelet D.A., Lecocq P.J., Revelant O., and Martial J.A. The evolutionarily conserved Kruppel-associated box domain defines a subfamily of eukaryotic multifingered proteins. Proc. Natl. Acad. Sci. USA, 1991, vol. 88, no. 9, pp. 3608-3612. doi: 10.1073/pnas.88.9.3608.
Dawson S.R., Turner D.L., Weintraub H., and Parkhurst S. M. Specificity for the hairy/enhancer of split basic helix-loop-helix (bHLH) proteins maps outside the bHLH domain and suggests two separable modes of transcriptional repression. Mol. Cell. Biol., 1995, vol. 15, no. 12, pp. 6923-6931. PMID:8524259.
Grimes H.L., Gilks C.B., Chan T.O., Porter S., and Tsichlis P.N. The Gfi-1 protooncoprotein represses Bax expression and inhibits T-cell death. Proc. Natl. Acad. Sci. USA, 1996, vol. 93, no. 25, pp. 14569-14573. PMID:8962093.
Friedman J.R., Fredericks W.J., Jensen D.E., Speicher D.W., Huang X.P., Neilson E.G., and F.J. Rauscher III. KAP-1, a novel corepressor for the highly conserved KRAB repression domain. Genes Dev., 1996, vol. 10, pp. 2067-2078. PMID:8769649.
Margolin J.F., Friedman J.R., Meyer W.K., Vissing H., Thiesen H.J., and Rauscher F.J. III. Kruppel-associated boxes are potent transcriptional repression domains. Proc. Natl. Acad. Sci. USA, 1994, vol. 91, no. 10, pp. 4509-4513. PMID:8183939.
Klug A., and Schwabe J.W. Protein motifs 5. Zinc fingers. FASEB J., 1995, vol. 9, no. 8, pp. 597-604. PMID:7768350.
Lechner M.S., Begg G.E., Speicher D.W., Rauscher III F.J. Molecular determinants for targeting heterochromatin protein 1-mediated gene silencing: direct chromoshadow domain KAP-1 corepressor interaction is essential. Mol. Cell. Biol., 2000, vol. 20, no. 17, pp. 6449-6465. PMID:10938122.
Collins T., Stone J.R., Williams A.J. All in the Family: the BTB/POZ, KRAB, and SCAN Domains. Mol. Cell. Biol., 2001, vol. 21, no. 11, pp. 3609-3615. PMID:11340155.
Wang G., Ma A., Cheok-Man W., Horsley D., Brown N.R., Cowell I.G., Singh P.B. Conservation of Heterochromatin Protein 1 Function. Mol. Cell. Biol., 2000, vol. 20, no. 18, pp. 6970-6983. PMID:10958692.
Yokoe T., Toiyama Y., Okugawa Y., Tanaka K., Ohi M., Inoue Y., Mohri Y., Miki C., and Kusunoki M. KAP1 is associated with peritoneal carcinomatosis in gastric cancer. Ann. Surg. Oncol., 2010, vol. 17, issue 3, pp. 821-828. doi: 10.1245/s10434-009-0795-8.
Nielsen A.L., Ortiz J.A., You J., Oulad-Abdelghani M., Khechumian R., Gansmuller A., Chambon P., Losson R. Interaction with members of the heterochromatin protein 1 (HP1) family and histone deacetylation are differentially involved in transcriptional silencing by members of the TIF1 family. EMBO J., 1999, vol. 18, no. 22, pp. 6385-6395. PMID:10562550.
Schultz D.C., Ayyanathan D.K., Negorev G.G., and Rauscher III F.J. SETDB1: a novel KAP-1-associated histone H3, lysine 9-specific methyltransferase that contributes to HP1-mediated silencing of euchromatic genes by KRAB zinc-finger proteins. Genes Dev., 2002, vol. 16, pp. 919-932. PMID:11959841.
Schultz D.C., Friedman J.R., and Rauscher III F.J. Targeting histone deacetylase complexes via KRAB-zinc finger proteins: the PHD and bromodomains of KAP-1 form a cooperative unit that recruits a novel isoform of the Mi-2alpha subunit of NuRD. Genes Dev., 2001, vol. 15, pp. 428-443. PMID:11230151.
Ryan R.F., Schultz D.C., Ayyanathan K., Singh P.B., Friedman J.R., Fredericks W.J., Rauscher III F.J. KAP-1 corepressor protein interacts and colocalizes with heterochromatic and euchromatic HP1 proteins: a potential role for Kruppel-associated box-zinc finger proteins in heterochromatin-mediated gene silencing. Mol. Cell. Biol., 1999, vol. 19, no. 6, pp. 4366-4378. PMID:10330177.
Iyengar S., Ivanov A.V., Jin V.X., Rauscher III F.J., and Farnham P.J. Functional Analysis of KAP1 Genomic Recruitment. Molecular and cellular biology, 2011, vol. 31, no. 9, pp. 1833-1847. doi: 10.1128/MCB.01331-10.
Hatakeyama Sh. TRIM proteins and cancer. Nature Reviews. Cancer, 2011, vol. 11, pp. 792-804. doi:10.1038/nrc3139.
Reymond A. Meroni G., Fantozzi A., Merla G., Cairo S., Luzi L., Riganelli D., Zanaria E., Messali S., Cainarca S., Guffanti A., Minucci S., Pelicci P. and Ballabio A. The tripartite motif family identifies cell compartments. EMBO J., 2001, vol. 20, no. 9, pp. 2140-2151. doi 10.1093/emboj/20.9.2140.
Short K.M., Cox T.C. Subclassification of the RBCC/TRIM superfamily reveals a novel motif necessary for microtubule binding. J. Biol. Chem., 2006, vol. 281, no. 13, pp. 8970-8980. PMID:16434393.
Ozato K., Shin D.M., Chang T.H., Morse H.C. TRIM family proteins and their emerging roles in innate immunity. Nature Rev. Immunol., 2008, vol. 8, pp. 849-860. doi:10.1038/nri2413.
McNab F.W., Rajsbaum R., Stoye J.P., O’Garra A. Tripartite-motif proteins and innate immune regulation. Curr. Opin. Immunol., 2011, vol. 23, issue 1, pp. 46-56. doi: 10.1016/j.coi.2010.10.021.
Meroni G., Diez-Roux G. TRIM/RBCC, a novel class of ‘single protein RING finger’ E3 ubiquitin ligases. Bioessays, 2005, vol. 27, issue11, pp. 1147-1157. PMID:16237670.
Borden K.L. RING domains: master builders of molecular scaffolds?. J. Mol. Biol., 2000, vol. 295, issue 5, pp. 1103-1112. PMID:10653689.
Peng H., Feldman I., Rauscher F.J. Hetero- oligomerization among the TIF family of RBCC/TRIM domain-containing nuclear cofactors: a potential mechanism for regulating the switch between coactivation and corepression. J. Mol. Biol., 2002, vol. 320, issue 3, pp. 629-644. PMID:12096914.
Le Douarin B., Nielsen A.L., Garnier J.M., Ichinose H., Jeanmougin F., Losson R., Chambon P. A possible involvement of TIF1 α and TIF1 β in the epigenetic control of transcription by nuclear receptors. EMBO J., 1996, vol. 15, no. 23, pp. 6701-6715. PMID:8978696.
Aasland R., Gibson T.J., Stewart A.F. The PHD finger: implications for chromatin-mediated transcriptional regulation. Trends. Biochem. Sci., 1995, vol. 20, issue 2, pp. 56-59. PMID:7701562.
Jeanmougin F., Wurtz J.M., Le Douarin B., Chambon P., Losson R. The bromodomain revisited. Trends. Biochem. Sci., 1997, vol. 22, issue 5, pp. 151-153. PMID:9175470.
Dyson M.H., Rose S., Mahadevan L.C. Acetyllysine-binding and function of bromodomain-containing proteins in chromatin. Front. Biosci., 2001, vol. 6, pp. 853-865. PMID:11487465.
Teyssier C., Ou C.Y., Khetchoumian K., Losson R., Stallcup M.R. Transcriptional intermediary factor 1α mediates physical interaction and functional synergy between the coactivator-associated arginine methyltransferase 1 and glucocorticoid receptor- interacting protein 1 nuclear receptor coactivators. Mol. Endocrinol., 2006, vol. 20, no. 6, pp. 1276-1286. dx.doi.org/10.1210/me.2005-0393.
Remboutsika E., Yamamoto K., Harbers M., Schmutz M. The bromodomain mediates transcriptional intermediary factor 1α -nucleosome interactions. J. Biol. Chem., 2002, vol. 277, no. 52, pp. 50318-50325. PMID:12384511.
Eissenberg J.C., Elgin S.C. The HP1 protein family: getting a grip on chromatin. Curr. Opin. Genet. Dev., 2000, vol. 10, issue 2, pp. 204-210. PMID:10753776.
Li Y., Kirschmann D.A., Wallrath L.L. Does heterochromatin protein 1 always follow code? Proc. Natl Acad. Sci. USA, 2002, vol. 99, no. 4, pp. 16462-16469. PMID:12151603.
Bartova E., Pachernik J., Kozubik A., Kozubek S. Differentiation-specific association of HP1α and HP1β with chromocentres is correlated with clustering of TIF1β at these sites. Histochem. Cell Biol. 2007, vol. 127, issue 4, pp. 375-388. PMID:17205308.
Cammas F., Oulad-Abdelghani M., Vonesch J.L., Huss-Garcia Y., Chambon P., Losson R. Cell differentiation induces TIF1β association with centromeric heterochromatin via an HP1 interaction. J. Cell Sci., 2002, vol. 115, no. 17, pp. 3439-3448. PMID:12154074.
Klugbauer S., Rabes H.M. The transcription coactivator HTIF1 and a related protein are fused to the RET receptor tyrosine kinase in childhood papillary thyroid carcinomas. Oncogene, 1999, vol. 18, no. 30, pp. 4388-4393. PMID:10439047.
Belloni E., Trubia M., Gasparini P., Micucci C., Tapinassi C., Confalonieri S., Nuciforo P., Martino B., Lo-Coco F., Pelicci P., Di Fiore P. 8p11 myeloproliferative syndrome with a novel t(7;8) translocation leading to fusion of the FGFR1 and TIF1 genes. Genes Chromosomes Cancer, 2005, vol. 42, issue3, pp. 320-325. doi:10.1002/gcc.20144.
Le Douarin B., Le Douarin B., Zechel C., Garnier J.M., Lutz Y., Tora L., Pierrat P., Heery D., Gronemeyer H., Chambon P., Losson R. The N-terminal part of TIF1, a putative mediator of the ligand-dependent activation function (AF-2) of nuclear receptors, is fused to B-raf in the oncogenic protein T18. EMBO J., 1995, vol. 14, no. 9, pp. 2020-2033. PMID:7744009.
Gandini D., De Angeli C., Aguiari G., Manzati E., Lanza F., Pandolfi P.P., Cuneo A., Castoldi G.L., del Senno L. Preferential expression of the transcription coactivator HTIF1α gene in acute myeloid leukemia and MDS-related AML. Leukemia, 2002, vol. 16, no. 5, pp. 886-893. PMID:11986951.
Tsai W.W., Wang Z., Yiu T.T., Akdemir K.C., Xia W., Winter S., Tsai C.Y., Shi X., Schwarzer D., Plunkett W., Aronow B., Gozani O., Fischle W., Hung M.C., Patel D.J., Barton M.C. TRIM24 links a non-canonical histone signature to breast cancer. Nature, 2010, vol. 468, pp. 927-932. doi: 10.1038/nature09542.
Chambon M., Orsetti B., Berthe M.L., Bascoul-Mollevi C., Rodriguez C., Duong V., Gleizes M., Thénot S., Bibeau F., Theillet C., Cavaillès V. Prognostic significance of TRIM24/ TIF-1α gene expression in breast cancer. Am. J. Pathol., 2011, vol. 178, issue 4, pp. 1461-1469. doi: 10.1016/j.ajpath.2010.12.026.
Aucagne R., Droin N., Paggetti J., Lagrange B., Largeot A., Hammann A., Bataille A., Martin L., Yan K.P., Fenaux P., Losson R., Solary E., Bastie J.N., Delva L. Transcription intermediary factor 1γ is a tumor suppressor in mouse and human chronic myelomonocytic leukemia. J. Clin. Invest., 2011, vol. 121, issue 6, pp. 2361-2370. doi: 10.1172/JCI45213.
Maniatis T., Fritsch E.F., Sambrook J. Molecular cloning: A laboratory manual. New York, Cold Spring Harbor Laboratory Press, 1989, 480 p.
Higgins S.J., Hames B.D. Protein Expression. A practical approach. Oxford University Press, 1999, 282 p.
Kulyyassov A., Shoaib M., Ogryzko V. Use of in vivo biotinylation for chromatin immunoprecipitation. Curr. Protoc. Cell Biol., 2011, chapter 17, unit 17.12, pp. 17.12.1-17.12.22. doi: 10.1002/0471143030.cb1712s51.
Kulyyassov A., Shoaib M., Pichugin A., Kannouche P., Ramanculov E., Lipinski M., Ogryzko V. PUB-MS: a mass spectrometry-based method to monitor protein-protein proximity in vivo. J. Proteome Res., 2011, vol. 10, no. 10, pp. 4416-4427. doi: 10.1021/pr200189p.
Shoaib M., Kulyyassov A., Robin C., Winczura K., Tarlykov P., Despas E., Kannouche P., Ramanculov E., Lipinski M., Ogryzko V. PUB-NChIP – “in vivo biotinylation” approach to study chromatin in proximity to a protein of interest. Genome Research, 2013, vol. 23, no. 2, pp. 331-340. doi:10.1101/gr.134874.111.
Kulyyassov A.T., Zhubanova G.S., Ramanculov E.M., Ogryzko V.V. Metod kolichestvennoj ocenki vzaimodejstvij geterohromatinovogo belka NR1 in vivo [Method of quantitative evaluation of heterochromatin protein HP1 interactions in vivo]. Biotehnologija. Teorija i praktika - Biotechnology. Theory and practice, 2014, no. 1, pp. 17-27.