atlas

The specific objectives of the ATLAS project are:

- Development and validation of a custom femtosecond laser prototype for optimized UV-laser photo-induced crosslinking with high efficiency. In the first 12 months, a new dedicated ultrashort laser system will be developed that will be able to deliver high-energy, UV, fs-pulses (in the energy range of several microjoule per pulse) at a repetition rate variable within six orders of magnitude, between 1Hz and 1MHz. The wavelength of such pulses will be variable in the range 250-280 nm, in order to match the first UV absorption band range of DNA bases. The combined features of high energy/pulse and high repetition rate are not commercially available at the moment for any known ultrashort laser source.

- To define the laser parameters for optimal laser induced cross-linking LChIP two sequential steps will be needed. In the first we will determine the structure and energy levels (MOs) of the DNA bases which are excited by the laser pulse. The second step will consist of building up the excitation model for the DNA base-aminoacid interaction in living cells, based on the parameters determined in the first step. The macroscopic parameters characterizing the DNA-laser interaction will be accounted for in the final stage of development. The model will be built, tested and then run for a range of parameters as they occur in practice. The final benefit will consist in indicating the values or the restricted range of values for an optimal interaction between the laser and the molecular system.

- Development and implementation of laser system upgrades with dedicated Optical Parametric Amplifier (OPA) that allows performing two-color, double pulse irradiation of the sample, thereby significantly minimizing cell damage; optimizing DNA-protein crosslink and enables to explore the possibility to photo-crosslink proteins with high efficiency by laser-technology. The developed OPA will have unique features in terms of high energy/pulse together with the high pulse repetition rate and will have a wide tunability, covering the 200 nm – 2600 nm spectral window. This will also render possible to exploit specific wavelength windows in case specific crosslink mechanism would require it.

- Determination of the chemical nature of laser-induced DNA-protein cross-linking, using the synergy between theoretical calculations and study of increasingly complex chemical models. Factors effecting the excitation of the DNA and the protein, the formation of intermediate reactive species, and the creation of the new DNA-protein bonds will be addressed in a sequential manner, from the isolated building blocks of each macromolecule to reacting adjacent monomers and the long-range effects that can be found in larger systems with tertiary structure, in particular when proximity (and affinity) is enforced. This multi-level detailed description of the photochemical behavior of DNA in a protein environment will provide the knowledge for rational design and optimization of the crosslinking experimental conditions. The identification of the intermediate species involved in the DNA-protein bond-making process and their corresponding electronic states will also lead to the possibility of fine-tuning the laser wavelength in the visible range to selectively link the desired fragments.

- Integration of LChIP with microfluidic-based cell sorting platforms. The laser system dedicated to pump/probe sample irradiation will be integrated into the fluorescence-activated microfluidic cell sorter (#2, 3, 7). This setup will allow analysis of selective subsets using much lower number of cells than required for conventional approaches. Moreover, this approach will allow the dynamic crosslinking of cells in a flux and number dependent manner.

- Application of LChIP with i) microscopic manipulations of frozen tissue slices; ii) microfluidic station for experiments in cellular subpopulations; iii) global study of epi-modifications and TFs binding (MYC, ER, SPs) in time resolved manner upon selected treatments (f.e. Epi-drugs) # CO1;

- Application of the LChIP and global LChIP for time resolved studies of the dynamics of factor binding at model promoters and for global analyses using microarrays (LChIP-CHIP) and massive parallel single molecule sequencing approaches (LChIP-Seq). These studies will unravel mechanisms of transcription activation and deciphering transcriptional pathways and networks (#4, 5).

- Use of LChIP-CHIP to identify chromosome (and subsequently genome) - wide putative direct DNA binding sites for p160 co-activators which possess putative DNA binding domains by LChIP-CHIP, and comparison of direct and indirect chromatin binding patterns for transcription factors that bind directly and indirectly (nuclear receptors) or only indirectly (i.e., CBP) to chromatin (#4).

- Optimization of amplification methods of very small amounts of DNA obtained by LChIP (#6), allowing the use of low numbers of cells as starting material. Development and characterization of antibodies to obtain LChILL and LChIP grade antibodies; emphasis will be on the production of antibodies against epitope peptides that will be masked by formaldehyde. These antibodies will have a dramatically higher IP efficacy allowing the use of epitope-tagging approaches independent of the presence of lysines. Optimized protocols will be established for LChIP-seq and LChIP-chip #6. Application of the laser fixation for the analyses of very small number of cells using proximity ligation approaches (LChILL) (#5 and #6).

- Kit(s) for LChIP (and for DNA amplification for LChIP) will be developed by #6. The LChIP kit will contain controls such as LChIP grade antibodies and physically crosslinked cells as well as optimized buffers, PCR primers and adapted protocol.

- The new developed machine joining the Laser and the microfluidic station will be optimized including dedicated software (#10); ATLAS will contribute to the creation and automation of the system for commercial exploitation.

All partners will be involved in tackling basic questions of transcription regulation possible with the global LChIP technology. The extraordinary large dynamic range of this technology will allow separating stochastic from productive complex formation on chromatin. Aspects like factor sliding (one-dimensional diffusion) along and temporal recruitment to chromatin will be addressed.