Visualization of the epigenetic states of cells in complex tissues is important since many cell types cannot be cultured in vitro and since the epigenetic states of cells can change during isolation and culture. Many tissues are composed of heterogeneous cell types and differences in epigenetic states among superficially identical cells affect their developmental and pathological potentials. We propose to develop technologies that enable visualization of the epigenetic states of individual cells in complex tissues of living animals. Epigenetic states consist of combinations of molecular marks in conjunction with multiprotein complexes that act on those marks. The consequences of different combinations of epigenetic marks are determined by combinatorial mechanisms. The functions of epigenetic regulatory proteins are determined by the combinations of interaction partners that are present in complexes formed by these proteins. We propose to develop technologies that enable visualization of combinatorial epigenetic marks, chromatin binding by epigenetic regulatory proteins, and epigenetic regulatory complexes formed by different combinations of interaction partners in animals. The technologies for visualization of combinatorial epigenetic marks and of epigenetic regulatory protein complexes in animals are to be based on fluorescent protein fragment complementation. Fragments of fluorescent proteins can associate with each other to produce a fluorescent complex when they are tethered in molecular proximity to each other. Technologies based on this principle are well suited for imaging epigenetic states in animals because (1) the direct fluorescence read-out is robust under challenging imaging conditions in animal tissues, (2) the high spatial resolution enables analysis of subnuclear localization, and (3) the lack of fluorescence of the individual fluorescent protein fragments produces a high signal to background ratio.
Project Narrative Animal cells use combinations of invisible tags to label genes that should be on or off in different kinds of cells growing in different environments. In this project, we develop molecules that enable researchers to see the combinations of tags in individual cells in living animals.
|Burns, Veronica Elizabeth; Kerppola, Tom Klaus (2017) ATR-101 inhibits cholesterol efflux and cortisol secretion by ATP-binding cassette transporters, causing cytotoxic cholesterol accumulation in adrenocortical carcinoma cells. Br J Pharmacol 174:3315-3332|
|Deng, Huai; Kerppola, Tom K (2017) Visualization of the Genomic Loci That Are Bound by Specific Multiprotein Complexes by Bimolecular Fluorescence Complementation Analysis on Drosophila Polytene Chromosomes. Methods Enzymol 589:429-455|
|Cheng, Yunhui; Kerppola, Raili Emilia; Kerppola, Tom Klaus (2016) ATR-101 disrupts mitochondrial functions in adrenocortical carcinoma cells and in vivo. Endocr Relat Cancer 23:1-19|
|Deng, Huai; Kerppola, Tom K (2014) Visualization of the Drosophila dKeap1-CncC interaction on chromatin illumines cooperative, xenobiotic-specific gene activation. Development 141:3277-88|
|Cheng, Bo; Ren, Xiaojun; Kerppola, Tom K (2014) KAP1 represses differentiation-inducible genes in embryonic stem cells through cooperative binding with PRC1 and derepresses pluripotency-associated genes. Mol Cell Biol 34:2075-91|
|Deng, Huai (2014) Multiple roles of Nrf2-Keap1 signaling: regulation of development and xenobiotic response using distinct mechanisms. Fly (Austin) 8:7-12|
|Kerppola, Tom K (2013) Bimolecular fluorescence complementation (BiFC) analysis of protein interactions in live cells. Cold Spring Harb Protoc 2013:727-31|
|Deng, Huai; Kerppola, Tom K (2013) Regulation of Drosophila metamorphosis by xenobiotic response regulators. PLoS Genet 9:e1003263|
|Burns, Veronica; Kerppola, Tom Klaus (2012) Opposite orientations of a transcription factor heterodimer bind DNA cooperatively with interaction partners but have different effects on interferon-? gene transcription. J Biol Chem 287:31833-44|
|Bai, Shoumei; Kerppola, Tom K (2011) Opposing roles of FoxP1 and Nfat3 in transcriptional control of cardiomyocyte hypertrophy. Mol Cell Biol 31:3068-80|
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