title: Imaging Histone Methylations in Living Animals.
abstract: 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.
authors.affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Stanford, CA, 94305, USA.
authors.forename: Thillai V
authors.initials: TV
authors.lastname: Sekar
authors.affiliation: Molecular Imaging Program at Stanford, Bio-X Program, Stanford University School of Medicine, Stanford, CA, 94305, USA. paulmur8@stanford.edu.|Department of Radiology, Stanford University School of Medicine, 3155, Porter Drive, #2236, Palo Alto, CA, 94304, USA. paulmur8@stanford.edu.
authors.forename: Ramasamy
authors.initials: R
authors.lastname: Paulmurugan
mesh_terms: D000818:Animals; D015374:Biosensing Techniques; D015870:Gene Expression; D023061:Gene Order; D017930:Genes, Reporter; D005822:Genetic Vectors / Q000235:genetics; D057809:HEK293 Cells; D006657:Histones / Q000235:genetics / Q000378:metabolism*; D006801:Humans; D049410:Luciferases, Renilla / Q000235:genetics / Q000378:metabolism; D008163:Luminescent Measurements / Q000379:methods; D008745:Methylation; D051379:Mice; D057054:Molecular Imaging / Q000379:methods*; D009154:Mutation; D061848:Optical Imaging / Q000379:methods; D011993:Recombinant Fusion Proteins / Q000235:genetics / Q000378:metabolism
chemical_list: D006657:Histones; D011993:Recombinant Fusion Proteins; D049410:Luciferases, Renilla
keywords: Degron; Histone methylation; In vivo imaging; Luciferase; Optical imaging; Protease; Reporter genes; Split reporters