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

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

doi:10.1038/s41598-017-12010-5

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.

Highlights

Dynamic binding of transcription factors (TFs) to their DNA recognition sites is crucial to mediate induction or repression of specific genes[1,2]
To optimize L-crosslinking as a method for studying DNA-protein interactions in living cells, we analyzed the crosslinking yield achieved in human cells using an advanced, flexible and userfriendly UV light source, in this case a dedicated, compact fs UV laser
The high repetition rate (RR) and high pulse and/or average energy and wavelength tunability of this laser system (Supplementary Fig. S1c) makes it an attractive tool for generating ultrashort UV pulses able to induce highly-efficient DNA-protein crosslinking in living cells

Summary

Introduction

Dynamic binding of transcription factors (TFs) to their DNA recognition sites is crucial to mediate induction or repression of specific genes[1,2]. The main method used to map DNA-protein interactions in vivo on an (epi)genome-wide scale[5,6] is chromatin immunoprecipitation (ChIP) coupled with sequencing (ChIP-seq)[7] This conventional technique binds proteins to DNA by crosslinking, generally using formaldehyde[8]. We report on the potential applications of a novel custom tunable fs UV laser system and its integrated version with a microfluidic system designed to induce L-crosslinking in living human cells in a shorter space of time and with fewer cells compared to other methods. This UV laser device overcomes current limitations and induces highly efficient DNA-protein crosslinking. This would provide a greater insight into the functional states of individual cells, which often host rare events undetectable in bulk measurements, with major implications for biological research and medicine

Methods

Results

Conclusion