My first research project focuses on the relationship between chromatin structure and function. A new gel electrophoresis method, referred to as Quantitative Agarose Gel Electrophoresis (QAGE) has recently been developed. It allows physical measurements of the size, surface charge density and shape of defined chromatin templates assembled in vitro (Fletcher et al., 1994). In collaboration with Dr. Hansen (UTHSCSA) and Drs. Hager (NIH) and Fletcher (University of Miami). The the technology has been applied to in vivo-assembled chromatin.

Agarose multigel electrophoresis (see Fig. 1) has been used to characterize the structural features of genomic mouse mammary tumor virus (MMTV) promoters. The mouse 3134 cells utilized for these studies contain ~200 stably integrated tandem repeats of a 2400 base-pairs MMTV promoter fragment. Genomic promoters were liberated by restriction digestion of isolated nuclei, recovered in a low salt nuclear extract, and electrophoresed in multigels consisting of nine individual agarose running gels (see Fig. 2). Specific bands were detected and characterized by Southern and Western blotting. We find that transcriptionally inactive promoters contained bound H1 and TBP, and were present to varying extents in both untreated and dexamethasone (DEX)-treated cells. The basally active promoter present in untreated cells was bound to RNA Pol II, TBP, and Oct1, and contained acetylated H3. The DEX-activated promoter possessed similar composition as the basal promoter, and also contained bound the chromatin remodeling complex sub-unit Brg1. Strikingly, all functional forms of the MMTV promoter condense into higher order secondary and tertiary chromatin structures in the presence of Mg²⁺. These results suggest that local nucleosome-nucleosome interactions, and their spatial effects on chromatin fiber organization, are required for proper transcription initiation from genomic MMTV promoters (Georgel et al., Genes & Dev. 2003)(see Fig.3).   

The relevance of our studies of in vivo-assembled chromatin using the QAGE method extends far beyond what was determined during the initial investigation of the MMTV promoter in mouse 3134 cells. Perhaps most importantly, the potential applications of QAGE are general, and not limited to studies of one specific genomic locus or to the process of transcriptional activation. In the context of transcription, one of the next logical steps is be to study a single copy genomic locus, since the 200 tandemly repeated copies of the MMTV promoter utilized for our initial experiments represent a partially artificial system.

My current ongoing project is designed to study the changes in chromatin structure potentially linked to activation of the Drosophila Heat Shock Protein 70 promoter. The HSP70 chromatin structure will be analyzed from in vivo-recovered chromatin as well as in vitro-assembled nucleosomal arrays using drosophila embryo extract (also referred to as Fly Embryo extract or FEE), initially characterized by Becker and Wu (1992). A large number of promoters have been investigated using FEE and related extract-ATP-dependent chromatin assembly systems. Although the nucleosome repeat length and overall patterns of nucleosome distributions obtained with the FEE closely resemble that of the native in vivo configuration, the use of the crude extract for nucleosome assembly has not permitted the subsequent characterization of the higher order structure of chromatin. The use of QAGE has the potential to help characterizing the structural changes that occur upon sequential addition of transcription factors or chromatin-associated proteins such as remodeling complexes as well as histone modifying enzymes.  

- Fletcher, T. M.; Krishnan, U.; Serwer, P.; Hansen, J. C. Biochemistry 1994, 33, 2226-2233.
- Georgel, P. T.; Fletcher, T. M.; Hager, G. L.; Hansen, J. C. Genes & Dev 2003. 17, 1617-1629.
- Becker, P. B.; Wu, C. Mol Cell Biol 1992, 12, 2241-2249.
- Georgel, P.T. Biochem. Cell Biol. 2002, 80(3), 295-300.