Imaging Histone Methylations in Living Animals

  • Thillai V. Sekar
  • Ramasamy PaulmuruganEmail author
Part of the Methods in Molecular Biology book series (MIMB, volume 1461)


Histone modifications (methylation, acetylation, phosphorylation, sumoylation, etc.,) are at the heart of cellular regulatory mechanisms, which control expression of genes in an orderly fashion and control the entire cellular regulatory networks. Histone lysine methylation has been identified as one of the several posttranslational histone modifications that plays crucial role in regulating gene expressions in facultative heterochromatic DNA regions while maintaining structural integrity in constitutive heterochromatic DNA regions. Since histone methylation is dysregulated in various cellular diseases, it has been considered a potential therapeutic target for drug development. Currently there is no simple method available to screen and preclinically evaluate drugs modulating this cellular process, we recently developed two different methods by adopting reporter gene technology to screen drugs and to preclinically evaluate them in living animals. Method detects and quantitatively monitors the level of histone methylations in intact cells, is of a prerequisite to screen small molecules that modulate histone lysine methylation. Here, we describe two independent optical imaging sensors developed to image histone methylations in cells and in living animals. Since we used standard PCR-based cloning strategies to construct different plasmid vectors shown in this chapter, we are not providing any details regarding the construction methods, instead, we focus on detailing various methods used for measuring histone methylation-assisted luciferase quantitation in cells and imaging in living animals.

Key words

Histone methylation Optical imaging Reporter genes Luciferase Split reporters Protease Degron In vivo imaging 



The funding support by National Institutes of Health (NIH grant R01 CA161091 and R21 CA185805 to R.P) and Department of Radiology, Stanford University is gratefully acknowledged. We also thank Dr. Sanjiv Sam Gambhir, Chairman, Department of Radiology, Stanford University, for his constant support. We gratefully acknowledge the use of the SCi3 Core Facility and Canary Center, Stanford University.


  1. 1.
    Kouzarides T (2007) SnapShot: histone-modifying enzymes. Cell 128:802CrossRefPubMedGoogle Scholar
  2. 2.
    Kouzarides T (2007) Chromatin modifications and their function. Cell 128:693–705CrossRefPubMedGoogle Scholar
  3. 3.
    Guenther MG, Levine SS, Boyer LA, Jaenisch R, Young RA (2007) A chromatin landmark and transcription initiation at most promoters in human cells. Cell 130:77–88CrossRefPubMedPubMedCentralGoogle Scholar
  4. 4.
    Rivera C, Gurard-Levin ZA, Almouzni G, Loyola A (2014) Histone lysine methylation and chromatin replication. Biochim Biophys Acta 1839:1433–1439CrossRefPubMedGoogle Scholar
  5. 5.
    Holm K, Grabau D, Lovgren K, Aradottir S, Gruvberger-Saal S, Howlin J, Saal LH, Ethier SP, Bendahl PO, Stal O, Malmstrom P, Ferno M, Ryden L, Hegardt C, Borg A, Ringner M (2012) Global H3K27 trimethylation and EZH2 abundance in breast tumor subtypes. Mol Oncol 6:494–506CrossRefPubMedGoogle Scholar
  6. 6.
    Seligson DB, Horvath S, McBrian MA, Mah V, Yu H, Tze S, Wang Q, Chia D, Goodglick L, Kurdistani SK (2009) Global levels of histone modifications predict prognosis in different cancers. Am J Pathol 174:1619–1628CrossRefPubMedPubMedCentralGoogle Scholar
  7. 7.
    Seligson DB, Horvath S, Shi T, Yu H, Tze S, Grunstein M, Kurdistani SK (2005) Global histone modification patterns predict risk of prostate cancer recurrence. Nature 435:1262–1266CrossRefPubMedGoogle Scholar
  8. 8.
    Shinkai Y, Tachibana M (2011) H3K9 methyltransferase G9a and the related molecule GLP. Genes Dev 25:781–788CrossRefPubMedPubMedCentralGoogle Scholar
  9. 9.
    Vedadi M, Barsyte-Lovejoy D, Liu F, Rival-Gervier S, Allali-Hassani A, Labrie V, Wigle TJ, Dimaggio PA, Wasney GA, Siarheyeva A, Dong A, Tempel W, Wang SC, Chen X, Chau I, Mangano TJ, Huang XP, Simpson CD, Pattenden SG, Norris JL, Kireev DB, Tripathy A, Edwards A, Roth BL, Janzen WP, Garcia BA, Petronis A, Ellis J, Brown PJ, Frye SV, Arrowsmith CH, Jin J (2011) A chemical probe selectively inhibits G9a and GLP methyltransferase activity in cells. Nat Chem Biol 7:566–574CrossRefPubMedPubMedCentralGoogle Scholar
  10. 10.
    Wang L, Chang J, Varghese D, Dellinger M, Kumar S, Best AM, Ruiz J, Bruick R, Pena-Llopis S, Xu J, Babinski DJ, Frantz DE, Brekken RA, Quinn AM, Simeonov A, Easmon J, Martinez ED (2013) A small molecule modulates Jumonji histone demethylase activity and selectively inhibits cancer growth. Nat Commun 4:2035PubMedPubMedCentralGoogle Scholar
  11. 11.
    Sekar TV, Foygel K, Devulapally R, Paulmurugan R (2015) Degron protease blockade sensor to image epigenetic histone protein methylation in cells and living animals. ACS Chem Biol 10:165–174CrossRefPubMedGoogle Scholar
  12. 12.
    Sekar TV, Foygel K, Gelovani JG, Paulmurugan R (2015) Genetically encoded molecular biosensors to image histone methylation in living animals. Anal Chem 87:892–899CrossRefPubMedGoogle Scholar
  13. 13.
    Paulmurugan R, Gambhir SS (2003) Monitoring protein-protein interactions using split synthetic Renilla luciferase protein-fragment-assisted complementation. Anal Chem 75:1584–1589CrossRefPubMedPubMedCentralGoogle Scholar
  14. 14.
    Paulmurugan R, Gambhir SS (2005) Firefly luciferase enzyme fragment complementation for imaging in cells and living animals. Anal Chem 77:1295–1302CrossRefPubMedPubMedCentralGoogle Scholar
  15. 15.
    Paulmurugan R, Gambhir SS (2006) An intramolecular folding sensor for imaging estrogen receptor-ligand interactions. Proc Natl Acad Sci U S A 103:15883–15888CrossRefPubMedPubMedCentralGoogle Scholar
  16. 16.
    Paulmurugan R, Gambhir SS (2007) Combinatorial library screening for developing an improved split-firefly luciferase fragment-assisted complementation system for studying protein-protein interactions. Anal Chem 79:2346–2353CrossRefPubMedPubMedCentralGoogle Scholar
  17. 17.
    Paulmurugan R, Ray P, De A, Chan CT, Gambhir SS (2006) Split luciferase complementation assay for studying interaction of proteins x and y in cells. CSH Protoc 2006; doi: 10.1101/pdb.prot4596 Google Scholar
  18. 18.
    Paulmurugan R, Ray P, De A, Chan CT, Gambhir SS (2006) Split luciferase complementation assay for studying interaction of proteins X and Y in living mice. CSH Protoc 2006; doi: 10.1101/pdb.prot4595 Google Scholar
  19. 19.
    Paulmurugan R, Tamrazi A, Massoud TF, Katzenellenbogen JA, Gambhir SS (2011) In vitro and in vivo molecular imaging of estrogen receptor α and β homo- and heterodimerization: exploration of new modes of receptor regulation. Mol Endocrinol 26:2029–2040CrossRefGoogle Scholar

Copyright information

© Springer Science+Business Media New York 2016

Authors and Affiliations

  1. 1.Molecular Imaging Program at Stanford, Bio-X ProgramStanford University School of MedicineStanfordUSA
  2. 2.Department of RadiologyStanford University School of MedicinePalo AltoUSA

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