METHOD OF QUANTITATIVE EVALUATION OF HETEROCHROMATIN PROTEIN HP1 INTERACTIONS IN VIVO
Main Article Content
Authors
A.T. Kulyyassov
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
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. Ramankulov
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
The development and progression of cancer is accompanied by changes in gene expression which are often can be caused by both genetic and epigenetic alterations and therefore proteins НР1a, b и g are potential oncomarkers.
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 his 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 HP1b.
In experiments on protein expression of BAP and BirA fusions of HP1a, HP1b and Tap54b in HEK293T cells we found elevated levels of biotinylation due to in vivo interaction of homologous proteins BAP-HP1 and BirA-HP1 (BAP-Tap54b and BirA-Tap54b). Signal ratio of heterologous to homologous interaction in all repeated experiments was 0,4 ± 0,14 (for samples containing BAP-HP1a) and 0,32 ± 0,08 (for samples with BAP-HP1b). Qualitative analysis by LC-MS/MS method of the gel fragments corresponding to the immunoblot bands of expressed proteins allowed to identify peptides relevant to BAP, Tap54b, HP1a and HP1b.
Keywords
heterochromatin, euchromatin, protein-protein interactions, biotinylation, oncomarkers, biotin ligase, biotin acceptor peptide, plasmids, transient transfection, western blot, mass spectrometry
Article Details
References
Berggerd T., Linse S., James P. Methods for the detection and analysis of protein-protein interactions. Proteomics, 2007, vol. 7, issue 16, pp. 2833-2842. doi:10.1002/pmic.200700131
Wodak Sh.J., Vlasblom J., Turinsky A.L., Pu Sh. Protein-protein interaction networks: the puzzling riches. Curr. Opinion in Struct. Biol., 2013, vol. 23, issue 6, pp. 941-953. doi:10.1016/j.sbi.2013.08.002
Navlakha S. and Kingsford C. The power of protein interaction networks for associating genes with diseases. Bioinformatics, 2010, vol. 26, no. 8, pp. 1057–1063. doi:10.1093/bioinformatics/btq076
Vidal M., Cusik M.E., Barabasi A.-L. Interactome networks and human disease. Cell, 2011, vol. 144, issue 6, pp. 986-998. Available at: 21414488. doi: 10.1016/j.cell.2011.02.016
Gonzalez M.W., Kann M.G. Chapter 4: Protein Interactions and Disease. PLOS Computational Biology, 2012, vol. 8, issue 12, e1002819. doi: 10.1371/journal.pcbi.1002819
Grewal S.I., Elgin S.C. Heterochromatin: new possibilities for the inheritance of structure. Curr. Opin. Genet. Dev, 2002, vol. 12, issue 2, pp.178–187. Available at: 11893491.
doi: 10.1016/S0959-437X(02)00284-8
Chevillard C., Reik W., McDermott M., Fontes M., Mattei M.G., Singh P.B. Chromosomal localization of human homologs of the Drosophila heterochromatin protein 1 (HP1) gene. Mamm. Genome, 1993, vol. 4, issue 2, pp. 124–126. Available at: 8431637. doi: 10.1007/BF00290438
Eissenberg J.C., Morris G.D., Reuter G., Hartnett T. The heterochromatinassociated protein HP-1 is an essential protein in Drosophila with dosage-dependent effects on position-effect variegation. Genetics, 1992, vol. 131, no. 2, pp. 345–352. Available at: 1644277
Horsley D., Hutchings A., Butcher G.W., Singh P.B. M32, amurine homologue of Drosophila heterochromatin protein 1 (HP1), localises to euchromatin within interphase nuclei and is largely excluded from constitutive heterochromatin. Cytogenet. Cell Genet, 1996, vol. 73, no. 4, pp. 308–331. Available at: 8751383. doi:10.1159/000134363
James T.C., Eissenberg J.C., Craig C., Dietrich V., Hobson A., Elgin S.C. Distribution patterns of HP1, a heterochromatin-associated nonhistone chromosomal protein of Drosophila. Eur. J. Cell Biol., 1989, vol. 50, pp. 170–180. Available at: 2515059
Mateescu B., Bourachot B., Rachez C., Ogryzko V., Muchardt C. Regulation of an inducible promoter by an HP1beta-HP1gamma switch. EMBO Rep., 2008, vol. 9, no. 3, pp. 267–272. Available at: 18239689. doi: 10.1038/embor.2008.1
Lomberk G., Wallrath L., Urrutia R. The Heterochromatin Protein 1 family. Genome Biol., 2006, vol. 7, issue 7, pp. 228.1-228.8. Available at: 17224041. doi:10.1186/gb-2006-7-7-228
Vermaak D., Henikoff S., Malik H.S. Positive selection drives the evolution of rhino, a member of the heterochromatin protein 1 family in Drosophila. PLoS Genet, 2005, vol. 1, issue 1, pp. 96–108. Available at: 16103923. doi: 10.1371/journal.pgen.0010009.
Singh P.B., Miller J.R., Pearce J., Kothary R., Burton R.D., Paro R., James T.C., Gaunt S.J. A sequence motif found in a Drosophila heterochromatin protein is conserved in animals and plants. Nucleic Acids Res., 1991, vol. 19, no. 4, pp. 789–794. Available at: 1708124. doi: 10.1093/nar/19.4.789
Minc E., Allory Y., Courvalin J.C., Buendia B. Immunolocalization of HP1 proteins in metaphasic mammalian chromosomes. Methods Cell Sci., 2001, vol. 23, issue 1-3, pp. 173–176. Available at: 11741155. doi: 10.1023/A:1013168323754
Minc E., Allory Y., Worman H.J., Courvalin J.C., Buendia B. Localization and phosphorylation of HP1 proteins during the cell cycle in mammalian cells. Chromosoma, 1999, vol. 108, no. 4, pp. 220–234. Available at: 10460410
Minc E., Courvalin J.C., Buendia B. HP1gamma associates with euchromatin and heterochromatin in mammalian nuclei and chromosomes. Cytogenet. Cell Genet, 2000, vol. 90, pp. 279–284. Available at: 11124534. doi: 10.1159/000056789
Obuse C., Iwasaki O., Kiyomitsu T., Goshima G., Toyoda Y., Yanagida M. A conserved Mis12 centromere complex is linked to heterochromatic HP1 and outer kinetochore protein Zwint-1. Nat. Cell Biol., 2004, vol. 6, no. 11, pp. 1135–1141. Available at: 15502821. doi:10.1038/ncb1187
Dialynas G. K., Vitalini M.W., Wallrath L.L. Linking Heterochromatin Protein 1 (HP1) to cancer progression. Mutation Research, 2008, vol.647, issue 1-2, pp. 13–20. Available at: 18926834. doi:10.1016/j.mrfmmm.2008.09.007
Maison Chr. And Almouzni G. HP1 and the dynamics of heterochromatin maintenance. Nature reviews/Molecular cell biology, 2004, vol. 5, pp. 296-304. Available at: 15071554. doi:10.1038/nrm1355
De Koning L., Savignoni A., Boumendil Ch., Rehman H., Asselain B.,Sastre-Garau X., Almouzni G. Heterochromatin protein 1a: a hallmark of cell proliferation relevant to clinical oncology. EMBO Mol. Med., 2009, vol. 1, issue3, pp. 178-191. Available at: 20049717.doi: 10.1002/emmm.200900022
Kirschmann D.A., Lininger R.A., Gardner L.M., Seftor E.A., Odero V.A., Ainsztein A.M., Earnshaw W.C., Wallrath L.L., Hendrix M.J. Down-regulation of HP1Hsalpha expression is associated with the metastatic phenotype in breast cancer. Cancer Res., 2000, vol. 60, no. 13, pp. 3359-3363. Available at: 10910038
Harel B.A., Mechold U., Viens A., Gilbert C., Lehrman H., Ogryzko V. Vectors for expression of biotinylated proteins in mammalian cells, and their use for identification of protein-nucleic acid interactions in vivo, EPO Patent 1367125-A1, 03.12.2003, 31 p.
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.
Westermeier R., Naven T., Höpker H.-R. Proteomics in practice. Weinheim:Wiley-VCH Verlag-GmbH, 2008, 482 p.
Von Hagen J. Proteomics sample preparation. Weinheim:Wiley-VCH Verlag-GmbH, 2008, 453 p.
Viens A., Mechold U., Lehrmann H., Harel-Bellan A., Ogryzko V. Use of protein biotinylation in vivo for chromatin immunoprecipitation. Anal. Biochem., 2004, vol. 325, issue 1, pp. 68–76. Available at: 14715286. doi: 10.1016/j.ab.2003.10.015
Viens A., Harper F., Pichard E., Comisso M., Pierron G., Ogryzko V. Use of protein biotinylation in vivo for immunoelectron microscopic localization of a specific protein isoform. J. Histochem. Cytochem., 2008, vol. 56, no. 10, pp. 911–919. Available at: 18574249. doi: 10.1369/jhc.2008.951624.
Kulyyassov A., Shoaib M., Ogryzko V. Use of in vivo biotinylation for chromatin immunoprecipitation. Curr. Protoc. Cell Biol., 2011, Chapter 17, Unit17.12, pp. 17.12.1-17.12.22. Available at: 21688254. 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. Available at: 21842862. 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. Available at: 23038767. doi:10.1101/gr.134874.111
Eugene V. Koonin. Orthologs, Paralogs, and Evolutionary Genomics. Annu. Rev. Genet., 2005, vol. 39, pp. 309–338. Available at: 16285863. doi:10.1146/annurev.genet.39.073003.114725
Zeng W., Ball A.R.Jr and Yokomori K. HP1 Heterochromatin binding proteins working the genome. Epigenetics, 2010, vol. 5, issue 4, pp. 287-292. Available at: 20421743. doi: 10.4161/epi.5.4.11683
Ruthenburg A.J., Li H., Patel D.J., Allis C.D. Multivalent engagement of chromatin modifications by linked binding modules. Nature reviews. Molecular cell biology, 2007, vol. 8, pp. 983-994. Available at: 18037899. doi: 10.1038/nrm2298
Canzio D., Liao M., Naber N., Pate E., Larson A., Wu S., Marina D. B., Garcia J. F., Madhani H. D., Cooke R., Schuck P., Cheng Y., Narlikar G. J. A conformational switch in HP1 releases auto-inhibition to drive heterochromatin assembly. Nature, 2013, vol. 496, issue 7445, pp. 377-381. Available at: 23485968. doi: 10.1038/nature12032
De Koning L., Almouzni G. HP1alpha as a prognostic marker in human cancer. US Patent US2012/0046190 A1, 23.02.2012, 30 p.
Sridharan R., Gonzales-Cope M., Chronis C., Bonora G., McKee R., Huang Ch., Patel S., Lopez D., Mishra N., Pellegrini M., Carey M., Garcia B.A., Plath K. Proteomic and genomic approaches reveal critical functions of H3K9 methylation and heterochromatin protein-1 in reprogramming to pluripotency. Nature Cell Biology, 2013, vol. 15, no. 7, pp. 872-882. Available at: 23748610. doi: 10.1038/ncb2768
Gaspar-Maia A., Alajem A., Meshorer E., Ramalho-Santos M. Open chromatin in pluripotency and reprogramming. Nature Reviews. Molecular Cell Biology, 2011, vol. 12, pp. 36-47. Available at: 21179060. doi: 10.1038/nrm3036
Matias P.M., Gorynia S., Donner P., Carrondo A. Crystal structure of the human AAA+ protein RuvBL1. J. Biol. Chem., 2006, vol. 281, no. 50, pp. 38918–38929. Available at: 17060327. doi: 10.1074/jbc.M605625200
Puri T., Wendler P., Sigala B., Saibil H., Tsaneva I.R. Dodecameric structure and ATPase activity of the human Tip48/Tip49 complex. J.Mol.Biol., 2007, vol. 366, issue 1, pp. 179-192. Available at: 17157868. doi: 10.1016/j.jmb.2006.11.030
Ikura T., Ogryzko V., Grigoriev M., Groisman R., Wang J., Horikoshi M., Scully R., Qin J., Nakatani Y. Involvement of the Tip60 histone acetylase complex in DNA repair and apoptosis, Cell, 2000, vol. 102, issue 4, pp. 463-473. Available at: 10966108. doi:10.1016/S0092-8674(00)00051-9
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. Available at: 10753776. doi: 10.1016/S0959-437X(00)00058-7