Subsequent projects

Prof. Dr. Joachim Griesenbeck
University of Regensburg
Fakultät für Biologie -
Institut für Biochemie, Genetik und Mikrobiologie


Prof. Dr. Hinrich Boeger
University of California, Santa Cruz
Molecular Cell & Developmental Biology

Structural analysis of single gene molecules by electron and atomic force microscopy

In previous collaborations, we have investigated the mechanism of transcriptional activation of individualgenes in the eukaryotic model organism S. cerevisiae (hereafter called yeast) (Boeger et al., 2008; Brown et al., 2013; Hamperl et al., 2014a, 2014b). To this end we have developed an experimental method for the isolation of molecules of defined genes from yeast chromosomes. This has allowed us to investigate both the protein composition of individual genes in different functional states by mass spectrometry, and to analyse the nucleosome configurations of single gene molecules by electron microscopy. Both approaches have greatly advanced knowledge and understanding of the structural and compositional dynamics of defined chromatin domains; and novel insights into the mechanistic relationship between chromatin structure and transcription were thus obtained.

We wish to continue our collaboration with the goal to further advance electron microscopic (EM) techniques in both laboratories, and to explore the possibility of atomic force microscopy (AFM) for the structural analysis of single gene molecules. AFM microscopy will be performed in collaboration with Holger Schmidt (Electrical Engineering, UCSC); and we would like to include Dr. John van Noort (University of Leiden), an expert on AFM and magnetic force microscopy, who is interested in the acquisition of our chromatin ring isolation method for his research on the elastic properties of chromatin fibers. The hope is that AFM will provide an easier and more efficient experimental tool for the structural analysis of single gene molecules than EM.

References:

Boeger, H., Griesenbeck, J., and Kornberg, R.D. (2008). Nucleosome retention and the stochastic nature of promoter chromatin remodeling for transcription. Cell 133, 716–726.
Brown, C.R., Mao, C., Falkovskaia, E., Jurica, M.S., and Boeger, H. (2013). Linking stochastic fluctuations in chromatin structure and gene expression. PLoS Biol. 11, e1001621.
Hamperl, S., Brown, C.R., Garea, A.V., Perez-Fernandez, J., Bruckmann, A., Huber, K., Wittner, M., Babl, V., Stoeckl, U., Deutzmann, R., et al. (2014a). Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae. Nucleic Acids Res. 42, e2.
Hamperl, S., Brown, C.R., Perez-Fernandez, J., Huber, K., Wittner, M., Babl, V., Stöckl, U., Boeger, H., Tschochner, H., Milkereit, P., et al. (2014b). Purification of specific chromatin domains from single-copy gene loci in Saccharomyces cerevisiae. Methods Mol. Biol. Clifton NJ 1094, 329–341.

 

Primary project: Molecular mechanism of gene activation: Chromatin transition at the yeast PHO5 promoter

 

Final Report

Subsequent funding by BaCaTeC enabled the continuation of an established collaboration between the research groups of Hinrich Boeger at the UC Santa Cruz and Joachim Griesenbeck at the University of Regensburg.  Over the years, the above collaboration had led to significant insights in the regulation of eukaryotic gene transcription [1]-[11].  While the earlier studies relied mostly on biochemical and cell biological analyses, subsequent funding by BaCaTeC was intended to extend the methodological approaches to the structural level.  The recent breakthrough in single molecule structure analyses of complex biological assemblies promises that it will be possible to understand important structure-function relationships in the process of gene regulation.  For this reason, one goal of the subsequent funding was to initiate a collaboration with the group of John van Noort (University Leiden).

BaCaTeC funding enabled a productive meeting of Hinrich Boeger, Joachim Griesenbeck, and John van Noort in December 2016 in Regensburg.  The meeting set the basis for a project to test if selected gene loci, which were isolated under native conditions from S. cerevisiae, are amenable to single-molecule force spectroscopy.  These analyses yielded first insights in the biophysical properties of defined, native genetic material.  The results of this study were recently published in a renowned scientific journal [12].  In 2016, the three groups applied for funding within the Human Frontier Science Program - unfortunately, without success.  The work published in collaboration with the group at the University of Leiden should now enable to apply again for third party funding.

References

1. Boeger, H., Griesenbeck, J., Strattan, J. S. & Kornberg, R. D. Nucleosomes unfold completely at a transcriptionally active promoter. Mol. Cell 11, 1587–1598 (2003).
2. Griesenbeck, J., Boeger, H., Strattan, J. S. & Kornberg, R. D. Affinity purification of specific chromatin segments from chromosomal loci in yeast. Mol. Cell. Biol. 23, 9275–9282 (2003).
3. Boeger, H., Griesenbeck, J., Strattan, J. S. & Kornberg, R. D. Removal of promoter nucleosomes by disassembly rather than sliding in vivo. Mol. Cell 14, 667–673 (2004).
4. Griesenbeck, J., Boeger, H., Strattan, J. S. & Kornberg, R. D. Purification of defined chromosomal domains. Meth. Enzymol. 375, 170–178 (2004).
5. Boeger, H., Griesenbeck, J. & Kornberg, R. D. Nucleosome retention and the stochastic nature of promoter chromatin remodeling for transcription. Cell 133, 716–726 (2008).
6. Lorch, Y., Griesenbeck, J., Boeger, H., Maier-Davis, B. & Kornberg, R. D. Selective removal of promoter nucleosomes by the RSC chromatin-remodeling complex. Nat. Struct. Mol. Biol. 18, 881–885 (2011).
7. Mao, C., Brown, C. R., Griesenbeck, J. & Boeger, H. Occlusion of regulatory sequences by promoter nucleosomes in vivo. PLoS ONE 6, e17521 (2011).
8. Brown, C. R., Mao, C., Falkovskaia, E., Jurica, M. S. & Boeger, H. Linking stochastic fluctuations in chromatin structure and gene expression. PLoS Biol. 11, e1001621 (2013).
9. Hamperl, S. et al. Compositional and structural analysis of selected chromosomal domains from Saccharomyces cerevisiae. Nucleic Acids Res. 42, e2 (2014).
10. Hamperl, S. et al. Purification of specific chromatin domains from single-copy gene loci in Saccharomyces cerevisiae. Methods Mol. Biol. 1094, 329–341 (2014).
11. Brown, C. R. et al. Chromatin structure analysis of single gene molecules by psoralen cross-linking and electron microscopy. Methods Mol. Biol. 1228, 93–121 (2015).
12. Hermans, N. et al. Toehold-enhanced LNA probes for selective pull down and single-molecule analysis of native chromatin. Sci Rep 7, 16721 (2017).

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