Imaging in situ protein-DNA interactions in the cell nucleus using FRET-FLIM.
Although the distribution of DNA-binding proteins inside the cell nucleus can be analyzed by immunolabeling or by tagging proteins with GFP, we cannot establish whether the protein is bound to DNA or not. Here, we describe a novel approach that allows imaging of the in situ interaction between a GFP-fusion protein and DNA in the cell nucleus, using fluorescence resonance energy transfer (FRET). We used fluorescence lifetime imaging microscopy (FLIM) as a reliable tool to detect protein in contact with DNA. The method was successfully applied to the DNA-binding proteins histone H2B and the glucocorticoid receptor and to the heterochromatin-associated proteins HP1alpha and HP1beta.
Cremazy
Frédéric G E
FG
Swammerdam Institute for Life Sciences, BioCentrum Amsterdam, University of Amsterdam, Kruislaan 318, 1098 SM Amsterdam, The Netherlands.
Manders
Erik M M
EM
Bastiaens
Philippe I H
PI
Kramer
Gertjan
G
Hager
Gordon L
GL
van Munster
Erik B
EB
Verschure
Pernette J
PJ
Gadella
Theodorusw J
TJ
van Driel
Roel
R
D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D000090266:Chromobox Protein Homolog 5; D004247:DNA / Q000378:metabolism*; D031541:Fluorescence Resonance Energy Transfer / Q000379:methods*; D049452:Green Fluorescent Proteins / Q000378:metabolism; D006367:HeLa Cells; D006657:Histones / Q000378:metabolism; D006801:Humans; D008856:Microscopy, Fluorescence / Q000379:methods; D009687:Nuclear Proteins / Q000378:metabolism*; D011479:Protamines / Q000378:metabolism; D011485:Protein Binding / Q000502:physiology
C000715593:CBX1 protein, human; C000715594:CBX5 protein, human; D006657:Histones; D009687:Nuclear Proteins; D011479:Protamines; C068621:protamine 2; D000090266:Chromobox Protein Homolog 5; D049452:Green Fluorescent Proteins; D004247:DNA