Periodic Reporting for period 4 - chromo-SUMMIT (Decoding dynamic chromatin signaling by single-molecule multiplex detection)
Berichtszeitraum: 2021-11-01 bis 2022-10-31
Chromatin is decorated with combinations of post-translational modifications (PTMs) on the histone proteins. This language of chemical modifications serve to regulate the structure, dynamics and function of chromatin and thus of all processes involving the genome, including gene expression, DNA replication and repair. Histone PTMs, which are written or erased by specific enzyme systems, either directly alter chromatin structure and dynamics, or they serve as binding sites for a host of structural proteins or enzymatic systems. Together, all these molecular components form complex signaling networks, which produce distinct biological outputs.
Chemical methods to synthesize defined chromatin fibers, containing specific histone PTM patterns, combined with physico-chemical methods to directly observe the consequence of chromatin modifications are promising tools to disentangle these complex regulatory networks. Indeed, this is the goal of the current project, which focuses on chromatin signaling during DNA damage repair, and is organized into three objectives.
I) Establish chemical methods for the installation of PTMs involved in DNA repair and develop methods to multiplex single-molecule methods to simultaneously study biochemical reactions on multiple differently modified chromatin fibers.
II) Directly observe ubiquitin signaling in the DNA damage response, focusing on a number of E3 ubiquitin ligases that play a key role in DNA damage signaling.
III) Develop methods to directly study the effect of histone PTMs involved in the DNA damage response on chromatin structure and dynamics during DNA repair.
Together, these studies yield molecular information on a complex signaling pathway in chromatin and will be of broad use for chemical and biomedical research with applications beyond the chromatin field.
Chemical methods to synthesize defined chromatin fibers, containing specific histone PTM patterns, combined with physico-chemical methods to directly observe the consequence of chromatin modifications are promising tools to disentangle these complex regulatory networks. Indeed, this is the goal of the current project, which focuses on chromatin signaling during DNA damage repair, and is organized into three objectives.
I) Establish chemical methods for the installation of PTMs involved in DNA repair and develop methods to multiplex single-molecule methods to simultaneously study biochemical reactions on multiple differently modified chromatin fibers.
II) Directly observe ubiquitin signaling in the DNA damage response, focusing on a number of E3 ubiquitin ligases that play a key role in DNA damage signaling.
III) Develop methods to directly study the effect of histone PTMs involved in the DNA damage response on chromatin structure and dynamics during DNA repair.
Together, these studies yield molecular information on a complex signaling pathway in chromatin and will be of broad use for chemical and biomedical research with applications beyond the chromatin field.
We developed a method to multiplex single-molecule studies of modified chromatin substrates, termed XSCAN (Makasheva et al., JACS 2021). This method now enables us to detect chromatin interaction dynamics of DNA damage factors to multiple differently modified chromatin fibers at the same time in one single-molecule measurement, which allows us to directly determine how such protein read-out the histone PTM code in real time. We could apply this methodology to reveal how the genome editor Cas9 can invade nucleosomes, and the methods is now applied in various project to decode chromatin regulation.
We have investigated chromatin ubiquitylation by two E3 ligases, involved in DNA repair and gene repression, RNF168 and variant PRC1 (vPRC1). Utilizing chemical methods to synthesize ubiquitylated H1, we could show that the activity of RNF168 is stimulated by distinct polyubiquitinated H1 molecules within chromatin. We further developed methods to observe vPRC1 interaction dynamics with chromatin, and even directly detected chromatin ubiquitination reactions on the single-molecule scale. These studies provide novel insights how chromatin ubiquitin ligases are regulated.
We have developed methodologies to directly observe chromatin structure and dynamics, producing the first landscape for chromatin dynamics. This involved the combination of different single-molecule methods (in collaboration with Prof. C. Seidel, U. Düsseldorf) with a novel data analysis pipeline and theoretical modeling (Kilic et al., Nat. Commun. 2018).
We developed methods to synthesize histone proteins containing DNA damage related histone PTMs, and revealed that one of those modifications – ubiquitylation on lysine 15 in histone H2A – unravels chromatin fibers to facilitate repair processes (Kilic, Boichenko et al., Chem. Sci. 2018).
Another H2A modification, ubiquitination on K119 (H2Aub1), is critical for gene silencing during development. In collaboration with Prof. J. Müller, MPI Biochemistry, we observed however that excess of H2Aub1 disrupted chromatin silencing. Single-molecule imaging revealed that chromatin containing high levels of H2Aub1 is no longer able to compact and thus genes are derepressed. These experiments reveal that the exact balance of H2A ubiquitination is critical for development (Bonnet et al. Genes Dev 2022).
DNA binding proteins exploit chromatin dynamics to invade chromatin structure. We have observed how pioneer transcription factors can invade and open chromatin structure (Mivelaz et al., Mol Cell 2020), and how histone variants, such as the centromeric histone CENP-A, alter chromatin structure to allow DNA access for DNA binding factors (Nagpal & Fierz, bioRxiv, 2022).
Together, these studies show that chromatin structure is highly dynamic and directly regulated by PTMs and histone variants, enabling chromatin invasion of regulatory factors.
The overall results of this project have disseminated in various conferences and scientific meetings, and have been published in peer-reviewed manuscripts, including:
1. Kilic, S., Boichenko, I., Lechner C. and Fierz B., A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage, Chemical Science, 2018, 9, 3704-3709
2. Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor, Makasheva K., Bryan, L.C. Anders, C., Panikularm, S., Jinek, M., Fierz, B., J. Am. Chem. Soc., 2021, 143, 40, 16313–16319
3. Kilic, S., Felekyan S., Doroshenko O., Boichenko I., Dimura M., Vardanyan H, Bryan LC., Arya G, Seidel, CAM* & Fierz B.*, Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α, Nature Commun., 2018, 9(1):235.
4. Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor, Mivelaz, M., Cao, A.-M. Kubik, S., Zencir, S., Hovius, R., Boichenko, I., Stachowicz, A.M. Kurat, C.F. Shore, D., Fierz B., Mol. Cell, 2020, p488-500.e9
5. Bonnet J, Boichenko I, Kalb R, Le Jeune M, Maltseva S, Pieropan M, Finkl K, Fierz B*, Müller J*. PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction. Genes Dev. 2022 Nov 10. doi: 10.1101/gad.350014.122.
We have investigated chromatin ubiquitylation by two E3 ligases, involved in DNA repair and gene repression, RNF168 and variant PRC1 (vPRC1). Utilizing chemical methods to synthesize ubiquitylated H1, we could show that the activity of RNF168 is stimulated by distinct polyubiquitinated H1 molecules within chromatin. We further developed methods to observe vPRC1 interaction dynamics with chromatin, and even directly detected chromatin ubiquitination reactions on the single-molecule scale. These studies provide novel insights how chromatin ubiquitin ligases are regulated.
We have developed methodologies to directly observe chromatin structure and dynamics, producing the first landscape for chromatin dynamics. This involved the combination of different single-molecule methods (in collaboration with Prof. C. Seidel, U. Düsseldorf) with a novel data analysis pipeline and theoretical modeling (Kilic et al., Nat. Commun. 2018).
We developed methods to synthesize histone proteins containing DNA damage related histone PTMs, and revealed that one of those modifications – ubiquitylation on lysine 15 in histone H2A – unravels chromatin fibers to facilitate repair processes (Kilic, Boichenko et al., Chem. Sci. 2018).
Another H2A modification, ubiquitination on K119 (H2Aub1), is critical for gene silencing during development. In collaboration with Prof. J. Müller, MPI Biochemistry, we observed however that excess of H2Aub1 disrupted chromatin silencing. Single-molecule imaging revealed that chromatin containing high levels of H2Aub1 is no longer able to compact and thus genes are derepressed. These experiments reveal that the exact balance of H2A ubiquitination is critical for development (Bonnet et al. Genes Dev 2022).
DNA binding proteins exploit chromatin dynamics to invade chromatin structure. We have observed how pioneer transcription factors can invade and open chromatin structure (Mivelaz et al., Mol Cell 2020), and how histone variants, such as the centromeric histone CENP-A, alter chromatin structure to allow DNA access for DNA binding factors (Nagpal & Fierz, bioRxiv, 2022).
Together, these studies show that chromatin structure is highly dynamic and directly regulated by PTMs and histone variants, enabling chromatin invasion of regulatory factors.
The overall results of this project have disseminated in various conferences and scientific meetings, and have been published in peer-reviewed manuscripts, including:
1. Kilic, S., Boichenko, I., Lechner C. and Fierz B., A bi-terminal protein ligation strategy to probe chromatin structure during DNA damage, Chemical Science, 2018, 9, 3704-3709
2. Multiplexed Single-Molecule Experiments Reveal Nucleosome Invasion Dynamics of the Cas9 Genome Editor, Makasheva K., Bryan, L.C. Anders, C., Panikularm, S., Jinek, M., Fierz, B., J. Am. Chem. Soc., 2021, 143, 40, 16313–16319
3. Kilic, S., Felekyan S., Doroshenko O., Boichenko I., Dimura M., Vardanyan H, Bryan LC., Arya G, Seidel, CAM* & Fierz B.*, Single-molecule FRET reveals multiscale chromatin dynamics modulated by HP1α, Nature Commun., 2018, 9(1):235.
4. Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor, Mivelaz, M., Cao, A.-M. Kubik, S., Zencir, S., Hovius, R., Boichenko, I., Stachowicz, A.M. Kurat, C.F. Shore, D., Fierz B., Mol. Cell, 2020, p488-500.e9
5. Bonnet J, Boichenko I, Kalb R, Le Jeune M, Maltseva S, Pieropan M, Finkl K, Fierz B*, Müller J*. PR-DUB preserves Polycomb repression by preventing excessive accumulation of H2Aub1, an antagonist of chromatin compaction. Genes Dev. 2022 Nov 10. doi: 10.1101/gad.350014.122.
We developed XSCAN, a new method to multiplex single-molecule experiments, which goes beyond the state of the art. This method enable us to directly compare competitive binding of chromatin factors to multiple chromatin states. This method is now widely applied in the lab for multiple systems
We are established new methods to poly-ubiquitylated chromatin proteins in a highly defined fashion, enabling to dissect the regulation of RNF168.
New single-molecule approaches allowed us to visualize chromatin ubiquitylation on the single-molecule level for vPRC1, enabling to dissect the regulatory mechanism of this chromatin factor.
Combining several single-molecule FRET methods, we could show for the first time that chromatin fibers are highly dynamic, demonstrating dynamics from microseconds to seconds, and that DNA damage related chemical modifications alter chromatin structure and dynamics. This enabled us to investigate how chromatin is regulated by histone ubiquitylation and histone variants, enabling efficient DNA repair, regulating gene silencing and mitosis.
We are established new methods to poly-ubiquitylated chromatin proteins in a highly defined fashion, enabling to dissect the regulation of RNF168.
New single-molecule approaches allowed us to visualize chromatin ubiquitylation on the single-molecule level for vPRC1, enabling to dissect the regulatory mechanism of this chromatin factor.
Combining several single-molecule FRET methods, we could show for the first time that chromatin fibers are highly dynamic, demonstrating dynamics from microseconds to seconds, and that DNA damage related chemical modifications alter chromatin structure and dynamics. This enabled us to investigate how chromatin is regulated by histone ubiquitylation and histone variants, enabling efficient DNA repair, regulating gene silencing and mitosis.