Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses

Angela Nebbioso; Rosaria Benedetti; Mariarosaria Conte; Vincenzo Carafa; Floriana De Bellis; Jani Shaik; Filomena Matarese; Bartolomeo Della Ventura; Felice Gesuele; Raffaele Velotta; Joost H.A. Martens; Hendrik G. Stunnenberg; Carlo Altucci; Lucia Altucci

doi:10.1038/s41598-017-12010-5

Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses

Angela Nebbioso, Rosaria Benedetti, Mariarosaria Conte, Vincenzo Carafa, Floriana De Bellis, Jani Shaik, Filomena Matarese, Bartolomeo Della Ventura, Felice Gesuele, Raffaele Velotta, Joost H.A. Martens, Hendrik G. Stunnenberg, Carlo Altucci, Lucia Altucci

Research output: Contribution to journal › Article › peer-review

12 Citations (Scopus)

Abstract

Interactions between DNA and proteins are mainly studied through chemical procedures involving bi-functional reagents, mostly formaldehyde. Chromatin immunoprecipitation is used to identify the binding between transcription factors (TFs) and chromatin, and to evaluate the occurrence and impact of histone/DNA modifications. The current bottleneck in probing DNA-protein interactions using these approaches is caused by the fact that chemical crosslinkers do not discriminate direct and indirect bindings or short-lived chromatin occupancy. Here, we describe a novel application of UV laser-induced (L-) crosslinking and demonstrate that a combination of chemical and L-crosslinking is able to distinguish between direct and indirect DNA-protein interactions in a small number of living cells. The spatial and temporal dynamics of TF bindings to chromatin and their role in gene expression regulation may thus be assessed. The combination of chemical and L-crosslinking offers an exciting and unprecedented tool for biomedical applications.

Original language	English
Article number	11725
Journal	Scientific Reports
Volume	7
Issue number	1
DOIs	https://doi.org/10.1038/s41598-017-12010-5
Publication status	Published - 1 Dec 2017
Externally published	Yes

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Nebbioso, A., Benedetti, R., Conte, M., Carafa, V., De Bellis, F., Shaik, J., Matarese, F., Della Ventura, B., Gesuele, F., Velotta, R., Martens, J. H. A., Stunnenberg, H. G., Altucci, C., & Altucci, L. (2017). Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses. Scientific Reports, 7(1), Article 11725. https://doi.org/10.1038/s41598-017-12010-5

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abstract = "Interactions between DNA and proteins are mainly studied through chemical procedures involving bi-functional reagents, mostly formaldehyde. Chromatin immunoprecipitation is used to identify the binding between transcription factors (TFs) and chromatin, and to evaluate the occurrence and impact of histone/DNA modifications. The current bottleneck in probing DNA-protein interactions using these approaches is caused by the fact that chemical crosslinkers do not discriminate direct and indirect bindings or short-lived chromatin occupancy. Here, we describe a novel application of UV laser-induced (L-) crosslinking and demonstrate that a combination of chemical and L-crosslinking is able to distinguish between direct and indirect DNA-protein interactions in a small number of living cells. The spatial and temporal dynamics of TF bindings to chromatin and their role in gene expression regulation may thus be assessed. The combination of chemical and L-crosslinking offers an exciting and unprecedented tool for biomedical applications.",

author = "Angela Nebbioso and Rosaria Benedetti and Mariarosaria Conte and Vincenzo Carafa and {De Bellis}, Floriana and Jani Shaik and Filomena Matarese and {Della Ventura}, Bartolomeo and Felice Gesuele and Raffaele Velotta and Martens, {Joost H.A.} and Stunnenberg, {Hendrik G.} and Carlo Altucci and Lucia Altucci",

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AU - Nebbioso, Angela

AU - Benedetti, Rosaria

AU - Conte, Mariarosaria

AU - Carafa, Vincenzo

AU - De Bellis, Floriana

AU - Shaik, Jani

AU - Matarese, Filomena

AU - Della Ventura, Bartolomeo

AU - Gesuele, Felice

AU - Velotta, Raffaele

AU - Martens, Joost H.A.

AU - Stunnenberg, Hendrik G.

AU - Altucci, Carlo

AU - Altucci, Lucia

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AB - Interactions between DNA and proteins are mainly studied through chemical procedures involving bi-functional reagents, mostly formaldehyde. Chromatin immunoprecipitation is used to identify the binding between transcription factors (TFs) and chromatin, and to evaluate the occurrence and impact of histone/DNA modifications. The current bottleneck in probing DNA-protein interactions using these approaches is caused by the fact that chemical crosslinkers do not discriminate direct and indirect bindings or short-lived chromatin occupancy. Here, we describe a novel application of UV laser-induced (L-) crosslinking and demonstrate that a combination of chemical and L-crosslinking is able to distinguish between direct and indirect DNA-protein interactions in a small number of living cells. The spatial and temporal dynamics of TF bindings to chromatin and their role in gene expression regulation may thus be assessed. The combination of chemical and L-crosslinking offers an exciting and unprecedented tool for biomedical applications.

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Time-resolved analysis of DNA-protein interactions in living cells by UV laser pulses

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