Proteins move in the nucleus and transiently interact with binding sites there, but in most cases we do not know why they are so mobile or what they are bound to. Our work has focused on using fluorescence recovery after photobleaching and fluorescence correlation spectroscopy to investigate the mobility of transcription factors both at specific promoter sites and also at other generic sites throughout the nucleus. We have previously shown in a mouse cell line that the GFP-tagged glucocorticoid receptor is bound at a specific promoter for at most 60 seconds, even though transcription persists for several hours. To obtain a precise estimate of how long the glucocorticoid receptor remains bound to the promoter, we have developed mathematical models to analyze the diffusion and binding interactions of the receptor that occur during the fluorescent recovery after photobleaching experiment. Our model predicts that individual glucocorticoid receptors are bound at the promoter for less than a second. This very transient binding raises new questions about how the transcription complex can be assembled with such short residence times of the transcription factor. At the same time, we have shown that different analysis procedures for fluorescence recovery after photobleaching can yield different estimates of residence times. This shows that there are still uncertainties in the estimation of live cell binding parameters that will require developing alternate measurement procedures to arrive at consensus estimates. Towards this end, we have now developed a mathematical model to extract binding estimates from fluorescence correlation spectroscopy data, and used this to compare binding estimates obtained for the same molecule by fluorescence recovery after photobleaching and by flourescence correlation spectroscopy. We have shown that the two approaches agree, but only if a correction is made in the standard approach for fluorescence correlation spectroscopy. The correction must account for the bleaching which occurs during the measurement process. Thus, our cross validation procedure has helped to identify an error in one of the procedures, and at the same time has helped increase our confidence in our current live cell binding estimates. We have also extended these live cell binding procedures to examine cooperative interactions of a single molecule inside of a live cell. For this purpose, we used the linker histone H1 and analyzed its binding to chromatin by using our analysis procedures to estimate the fraction of bound H1 molecules. We did this for the wild type H1 molecule as well as for a series of mutants that lack various key binding domains. By comparing the fraction of molecules bound in the different mutants, we could determine which domains interact cooperatively in the binding process. In simple terms, cooperatively interacting domains are those for which a much higher fraction is bound when both are present compared to the sum of the bound fractions when either is present by itself. This procedure will be a generally useful one for investigating cooperative binding of molecules in live cells. Finally, we have developed single molecule tracking techniques to monitor transcription factor movement inside of live cell nuclei. We are using this procedure to estimate the binding residence times of transcription factors by measuring how long they remain immobile. This reflects the time that they are bound to chromatin. Preliminary data indicate that these times are somewhat faster by a factor of 2-5 than the residence times measured by fluorescence correlation spectroscopy and fluorescence recovery after photobleaching. The difference may be due to complex diffusion processes revealed by single molecule tracking which are not incorporated into the models used to analyze fluorescence correlation spectroscopy or fluorescence recovery after photobleaching.

Agency
National Institute of Health (NIH)
Institute
National Cancer Institute (NCI)
Type
Investigator-Initiated Intramural Research Projects (ZIA)
Project #
1ZIABC010561-08
Application #
8349048
Study Section
Project Start
Project End
Budget Start
Budget End
Support Year
8
Fiscal Year
2011
Total Cost
$840,427
Indirect Cost
Name
National Cancer Institute Division of Basic Sciences
Department
Type
DUNS #
City
State
Country
Zip Code
Mazza, Davide; Mueller, Florian; Stasevich, Timothy J et al. (2013) Convergence of chromatin binding estimates in live cells. Nat Methods 10:691-2
Lickwar, Colin R; Mueller, Florian; Hanlon, Sean E et al. (2012) Genome-wide protein-DNA binding dynamics suggest a molecular clutch for transcription factor function. Nature 484:251-5
Mazza, Davide; Stasevich, Timothy J; Karpova, Tatiana S et al. (2012) Monitoring dynamic binding of chromatin proteins in vivo by fluorescence correlation spectroscopy and temporal image correlation spectroscopy. Methods Mol Biol 833:177-200
Mazza, Davide; Abernathy, Alice; Golob, Nicole et al. (2012) A benchmark for chromatin binding measurements in live cells. Nucleic Acids Res 40:e119
Rieder, Dietmar; Trajanoski, Zlatko; McNally, James G (2012) Transcription factories. Front Genet 3:221
Mueller, Florian; Morisaki, Tatsuya; Mazza, Davide et al. (2012) Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis. Biophys J 102:1656-65
Quénet, Delphine; McNally, James G; Dalal, Yamini (2012) Through thick and thin: the conundrum of chromatin fibre folding in vivo. EMBO Rep 13:943-4
Mueller, Florian; Karpova, Tatiana S; Mazza, Davide et al. (2012) Monitoring dynamic binding of chromatin proteins in vivo by fluorescence recovery after photobleaching. Methods Mol Biol 833:153-76
McNally, James G (2011) Foreword. Biophysics in chromatin structure and nuclear dynamics. Chromosome Res 19:1-3
Maiuri, Paolo; Knezevich, Anna; De Marco, Alex et al. (2011) Fast transcription rates of RNA polymerase II in human cells. EMBO Rep 12:1280-5

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