Our Science – McNally Website
James G. McNally, Ph.D.
In Vivo Imaging Analysis of Transcriptional Regulation
Transcription is a fundamental life process, but it has not been easy to study in live cells. We have developed techniques to accomplish this, using the mouse mammary tumor virus promoter (MMTV) as a model system for studying transcriptional control by steroid hormones. To analyze transcriptional regulation at the MMTV, we are using a cell line containing a GFP-tagged glucocorticoid receptor (GR) and an ~200-fold tandem array of the MMTV promoter, which provides a target site large enough to be visualized by fluorescence light microscopy. The target appears in the nucleus as a single fluorescent spot approximately 0.5 microns in diameter, or as a more extended string-like structure. To assess the dynamics of GR binding to the promoter, we have used fluorescence recovery after photobleaching (FRAP). After complete bleaching of the target site, fluorescence at the target recovers in a matter of seconds, demonstrating rapid exchange of GFP-GR molecules at this site. This rapid exchange appears to be coupled to transcription: faster exchange rates are typically correlated with less transcriptional output, while slower exchange rates with more transcriptional output. This suggests that longer GR residence times on the promoter increase the odds of polymerase loading and escape to elongation. As might be expected for a transcriptional regulatory mechanism, exchange rate appears to be tightly regulated. Both chaperone and proteasome function are required to tune the exchange rate, with chaperones acting to stabilize GR binding at the promoter and proteasomes destabilizing it.
In a second project, we are analyzing higher order chromatin structure on the scale of several hundred kilobases. Using the same GFP-GR MMTV array cell line to study large-scale chromatin structure, we find that upon hormone addition, the array undergoes dramatic morphological changes that correlate with transcriptional activity. Within 3 hrs after hormone addition, arrays visualized by GFP-GR or DNA FISH decondense to varying degrees (in the most pronounced cases from a ~0.5 microns spot), to form an elongated, beaded structure from up to 10 microns long. Arrays later recondense by 3 to 8 hrs of hormone treatment, consistent with earlier studies that demonstrate a transient activation of the MTMV promoter in these cells. The degree of decondensation is proportional to the amount of transcript produced by the array, as detected by RNA FISH. Decondensation is blocked by two different drugs that inhibit polymerase II, DRB, and a-amanitin. These observations demonstrate a role for polymerase in producing and maintaining decondensed higher order chromatin. These same properties of higher order chromatin structure are also found in transcriptionally active domains of several hundred kilobases that we have identified and studied in human chromosomes 6 and 22. Thus, at the light microscope level, higher order chromatin is characterized by beaded structures that elongate upon transcriptional activation, and are strictly dependent upon transcription to maintain the decondensed state.
NCI Core Fluorescence and Imaging Facility
The NCI Core Fluorescence and Imaging Facility provides the NCI intramural research community with access to several different state-of-the-art 3D microscopes, and computers to visualize and process image data. Facility personnel are available for consultation about the design of imaging experiments and/or the analysis of image data. The facility also periodically sponsors seminars and tutorials on microscopy techniques in cooperation with others on the NIH campus.
This page was last updated on 6/10/2013.