The Recruitment And Dynamics Of Transcription Factors At The Hsp70 Loci In Living Cells
Chromatin Immunoprecipitation (ChIP) provides snapshots of the localization of transcription factors on chromatin in cell populations. However, through the development of fluorescent proteins and subsequent live cell imaging techniques, it has become possible to address protein localization and dynamics in single living cells. Furthermore, the application of these techniques to the naturally amplified Drosophila polytene chromosomes allows the resolution of specific chromosomal loci within the natural threedimensional state of living nuclei. Therefore, we applied live cell imaging methods to address the recruitment, dynamics and retention at the inducible Hsp70 loci in individual Drosophila salivary gland nuclei for (1) a number of transcription factors, including HSF, Pol II, P-TEFb, Spt6, and Topo I; (2) H2B and Poly ADP Ribose (PAR) Polymerase (PARP); and (3) a previously uncharacterized Drosophila ortholog of SET1, dSet1. We observed that the recruitment of the master regulator, HSF, is first detected within 20 seconds of gene activation, and that the timing of its recruitment resolves from RNA polymerase II and P-TEFb, and these factors resolve from Spt6 and Topo I. Remarkably, the recruitment of each factor is highly synchronous between different cells. Fluorescence Recovery after Photobleaching (FRAP) analyses show that the entry and exit of multiple factors are progressively constrained upon gene activation, suggesting the gradual formation of a transcription compartment at the Hsp70 loci. Using live cell imaging methods and perfusion of PJ34, a specific inhibitor of PARP catalytic activity, we were able to show that the maintenance of the transcription compartment and subsequent retention of transcription factors is dependent on the activity of PARP, which is indicative of the role of PAR in the transcription compartment. Furthermore, we observed the retention of H2B and PARP at the Hsp70 loci before and after HS, even though mean fluorescence intensity of these factors decreases after activation. Finally, we applied these live cell imaging methods to characterize dSet1. In yeast, SET1 is responsible for the trimethylation of Histone H3 Lysine 4 (H3K4me3) a histone mark that is required for active elongation. Therefore, we addressed the timing of recruitment and dynamics of dSet1 at the Hsp70 loci and observed recruitment of dSet1 occurs slightly before Pol II, indicating that H3K4me3 occurs before elongation of Pol II at the Hsp70 loci. Together these live cell imaging assays provide an important assay which can be used to distinguish possible mechanisms for the role of transcription factors, histones, PARP and dSet1 in transcription activity.
Regulation of Transcription; Live Cell Imaging; gfp; Recruitment and Dynamics
Lis, John T
Barbash, Daniel A.; Roberts, Jeffrey Warren
Ph. D., Biochemistry
Doctor of Philosophy
dissertation or thesis