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How eukaryotic cells regulate the expression of their genetic information remains a fundamental question in cell biology. Specific proteins organize the DNA (the carrier of genetic information) of eukaryotes into chromatin. The chemistry and structure of chromatin undergo dynamic changes depending on the transcriptional state of genes. Although investigations on the role of chromatin in transcriptional regulation have become a main focus of recent research, the underlying mechanisms are still far from being understood.
A detailed molecular analysis eventually requires isolation of pure, natural chromatin closely reflecting the in vivo situation. We have established a novel technique to purify single-copy genes from chromosomal loci in the yeast Saccharomyces cerevisiae to near homogeneity, conserving the chromatin structure of distinct transcriptional states [1-4]. Future research pursues two lines of investigations:
(i) Determination of structure and composition of natural chromatin in different functional states by electron microscopy (EM) and mass spectrometry
(ii) Description of the interplay between chromatin remodeling and transcription using isolated natural chromosomal domains and purified protein factors in vitro.
References:
1. Boeger, H., Griesenbeck, J., Strattan, J. S., and Kornberg, R. D. (2003). Nucleosomes unfold completely at a transcriptionally active promoter. Mol Cell 11, 1587-1598.
2. Boeger, H., Griesenbeck, J., Strattan, J. S., and Kornberg, R. D. (2004). Removal of Promoter Nucleosomes by Disassembly Rather Than Sliding In Vivo. Mol Cell, in press.
3. Griesenbeck, J., Boeger, H., Strattan, J. S., and Kornberg, R. D. (2003). Affinity purification of specific chromatin segments from chromosomal Loci in yeast. Mol Cell Biol 23, 9275-9282.
4. Griesenbeck, J., Boeger, H., Strattan, J. S., and Kornberg, R. D. (2003). Purification of Defined Chromosomal Domains. Methods Enzymol 375, 170-178.
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Final report:
To increase our understanding about gene regulation, we have extended our studies on transcriptional activation of the PHO5 locus in the yeast S. cerevisiae. The PHO5 gene is coding for an acidic Phosphatase, whose expression is tightly regulated. We had previously discovered that changes occurring in the regulatory PHO5 promoter region in course of transcriptional activation are accompanied with loss of nucleosomes (the core particles of chromatin) [1]. We could also determine that nucleosomes vanish from the promoter DNA, because they are disassembled [2]. During the past 15 months we have further focused on the molecular mechanism of the disassembly reaction.
Funding by BaCaTeC provided the financial support for three visits of Dr. Griesenbeck at the University of Stanford (February and June 2005, June 2006) and two stays of Dr. Boeger at the University of Regensburg (June 2005 and 2006). During these periods it was possible to accumulate experimental data, which led to new insights about the mechanism and the functional significance of chromatin remodeling [3]. We developed a mathematical model for the chromatin transition whose implications will be tested in future studies.
In Spring 2006 Dr. Boeger assumed the position of Assistant Professor of Molecular, Cell and Developmental Biology at the University of California at Santa Cruz, where he is currently establishing his own research group. We intend to continue this fruitful cooperation between the laboratories of Professor Kornberg at Stanford, Professor Boeger in Santa Cruz, and Dr. Griesenbeck in Regensburg. The support by BaCaTeC has significantly contributed to the success of our scientific efforts.
[1] Boeger, H., Griesenbeck, J., Strattan, J. S., and Kornberg, R. D. (2003). Nucleosomes unfold completely at a transcriptionally active promoter. Mol Cell 11, 1587-1598.
[2] Boeger, H., Griesenbeck, J., Strattan, J. S., and Kornberg, R. D. (2004). Removal of promoter nucleosomes by disassembly rather than sliding in vivo. Mol Cell 14, 667-673.
[3] Boeger, H., Griesenbeck, J., Kornberg, R. D. (2006). Conservation of a single nucleosome, and the dynamics of transcriptional activation (submitted).
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