Fluorescence Imaging Group
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Contact Information Research Overview Group Members Fluorescence Imaging Facility Affiliations Open Positions |
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Jim McNally, Principal Investigator |
Contact Information
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James G. McNally, Ph.D. LRBGE-National Cancer Institute The National Institutes of Health 41 Library Drive Bethesda, MD 20892 |
Email: mcnallyj@mail.nih.gov Phone: (301) 402 - 0209 Fax: (301) 496 - 4951 Office: B516 Bldg. 41 |
Research Overview:
There are two research areas within the group: transcription factor dynamics and higher order chromatin structure.
Transcription factor dynamics
Some transcription factors are only transiently bound to their promoters even as transcription is occurring. This "fast cycling" occurs on a time scale of seconds. Since many components of the transcription complex also exhibit fast cycling, it is not known whether a stable transcriptional complex ever forms. We are addressing these questions with biophysical techniques to measure the residence times of various promoter factors under normal conditions and in the presence of various mutants that might be involved in regulating residence times. A current focus is to establish the reliability of these various biophysical techniques by comparing the estimates produced by fluorescence recovery after photobleaching, fluorescence correlation spectroscopy and single particle tracking.
A related project is to investigate a slower form of transcription factor dynamics at promoters. This "slow cycling" occurs on a time scale of tens of minutes and can reflect changes in promoter accessibility that modulate the fraction of promoters that can be bound by transcription factors across the cell population. This we know can arise from changes in nucleosome occupancy at promoters, but little is known about the underlying clock that drives these changes. This is the subject of our current interest.
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| Model for concurrent fast and slow cycling. Changes in promoter accessibility generate the slow cycle of promoter occupancy detected by ChIP which measures the total number of factors bound (green ovals). However, each factor cycles on and off the promoter on a fast time scale generating the fast cycle detected by FRAP. See Karpova et al. (2008) for details. |
Selected publications:
| • | Karpova, T.S., Kim, M.J., Spriet, C., Nalley, K., Stasevich, T.J., Kherrouche, Z., Heliot, L. and McNally, J.G. (2008). Concurrent fast and slow cycling of a transcriptional activator at an endogenous promoter. Science 319:466-469. |
| • | Mueller, F., Wach, P. and McNally, J.G. (2008) Evidence for a common mode of transcription factor interaction with chromatin as revealed by improved quantitative FRAP. Biophys. J. 94:3323-3339. |
| • | Stavreva, D.A., Müller, W.G., Hager, G.L., Smith, C.L. and McNally, J.G. (2004) Rapid GR exchange at a promoter: coupling to transcription and regulation by chaperones and proteasomes. Mol. Cell Biol. 24:2682-2697. |
| • | Sprague, B.L. and McNally, J.G. (2005) FRAP analysis of binding: proper and fitting. Trends Cell Biol. 15:84-91. |
| • | McNally*, J.G., Mueller*, W.G., Walker, D., Wolford, R. and Hager, G.L. The glucocorticoid receptor: rapid exchange with regulatory sites in living cells. Science 287:1262-1265. (2000). * co-first authors |
Higher order chromatin structure
There is much to be learned about how chromatin is folded inside the nucleus, and about how it is unfolded for transcription to occur. Using light microscopy, we have shown that transcription induces 400 kb regions of chromatin to unfold into a series of adjacent clusters that can be explained by a simple self-avoiding random walk of a 10 nm fiber. We have also demonstrated that transcription occurs at the edges of these chromatin clusters, and that highly decondensed chromatin is extruded from the clusters. These observations have provided new support for the pol II factory model and a stationary polymerase. We are now using x-ray microscopy to obtain images of whole nuclei with correlative fluorescence microscopy to identify transcriptionally active sites. This work, which is supported by external funds from the Human Frontier Sciences Program, has the potential to yield 3D images at 30 nm resolution of intact, cryo-preserved nuclei that have not been subjected to chemical fixation, staining or sectioning.
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| Model for transcription site formation based on the data in Müller et al. (2007). Transcribed chromatin is threaded through a stationary polymerase producing loops of decondensed DNA that surround the sites of transcription. |
Selected publications:
| • | Müller, W.G., Rieder, D., Karpova, T.S., John, S., Trajanoski, Z. and McNally, J.G. (2007) Organization of chromatin and histone modifications at a transcription site. J. Cell Biol. 177:957-67. |
| • | Müller, W.G., Rieder, D., Kreth, G., Cremer, C., Trajanoski, Z. and McNally, J.G. (2004) Generic features of tertiary chromatin structure as detected in natural chromosomes. Mol. Cell Biol. 24:9359-9370. |
| • | Müller, W.G., Walker, D. Hager, G.L., McNally, J.G. (2001) Large-scale chromatin decondensation and recondensation regulated by transcription from a natural promoter. J. Cell Biol. 154: 33-48. |
Group Members
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Jim McNally, Principal Investigator | |
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Tatiana Karpova, Staff Scientist and Manager of NCI Core Fluorescence Imaging Facility | |
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Min Kim, Research Assistant | |
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Davide Mazza, Post-doctoral Research Fellow | |
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Florian Mueller, Post-doctoral Research Fellow | |
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Hillary Mueller, Research Assistant | |
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Tim Stasevich, Post-doctoral Research Fellow |
Affiliations
Open Positions
We welcome inquires about working with our group. Interested parties should contact Jim McNally at mcnallyj@mail.nih.gov.