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 some uncertainties in the estimation of live cell binding parameters that will require developing alternate measurement procedures to arrive at consensus estimates. To enable visualization of glucocorticoid receptor binding in live cells, the preceding analyses of glucocorticoid receptor binding were performed using a synthetic tandem array of 200 promoters and reporter genes comprising 2Mb of DNA This has raised the question of whether the transient binding detected might be an artifact of this artificial system. To address this, we have developed a completely natural system in a completely different organism. We are using a small (2 kb) array of ten genes that arises naturally in common yeast strains. By tagging the associated transcription factor with GFP, we have been able to visualize this array and perform FRAP on the transcription factor. We find that this transcription factor is also bound transiently to its promoter, suggesting that such transient interactions are not an artifact and may be rather common.
| 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; 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 |
| 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 |
| 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; Morisaki, Tatsuya; Mazza, Davide et al. (2012) Minimizing the impact of photoswitching of fluorescent proteins on FRAP analysis. Biophys J 102:1656-65 |
| 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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