atlas

To tackle the genome complexity it is of utmost importance to obtain a genome-wide view of factor binding to DNA and its dynamic interplay with chromatin and other chromatin associated factors. Only such information, together with the data provided by the multiple genome sequencing projects, will allow deciphering genome-encoded information into signal transduction programs which regulate cell identity, homeostasis, proliferation, and death. The understanding of such genome-encoded information will act as a resource to study pathology. To this aim the most valuable tool is the ChIP technology. The conventional chemical crosslink, does by far not reach the precision provided by the current tiling array or seq technology. ATLAS will develop a high precision, global LChIP technology.

Transfer between scientific areas.

ATLAS will validate its global LChIP technology by interfacing top level scientists and major existing European research efforts. We are excited that partners from three EU consortia will validate the new technology, thus providing rapid scientific output and introduction into the scientific community. Obviously, once developed the LChIP technology will be made available to the scientific community.

Innovation and risks.

Ultrafast lasers, oscillators, and amplifiers have undergone a dramatic evolution over the last 30 years. Laser pulses have become shorter and laser systems more reliable and user-friendly. The recent advances in the generation and amplification of ultrafast laser pulses have opened up new possibilities in laser–matter interaction, spectroscopy, biophotonics, and nanophotonics. At the moment, the most relevant commercial options offer the possibility to have either an ultrashort laser oscillator (low energy per pulse, very high repetition rate) or amplified laser systems (high energy per pulse, repetition rate up to 5-10 kHz). ATLAS will benefit from both high repetition rate and high energy per pulse at the same time. ATLAS will combine these characteristics into a novel laser source/setup whose development will require a continuous information exchange between the LC and the scientific partner within a trial and error process. Light Conversion (#2, LC) offers the unique advantage of being a laser designer who chose to follow the route of developing laser systems combining the two above essential requirements. Therefore, ATLAS will develop a custom version of the new diode pumped femtosecond Ytterbium laser system based on design principles of commercial system PHAROS produced by LC (#2). We wish to point out that, based on the present technology, a realistic trade-off between high laser pulse repetition rate and high energy per pulse needs to be and will be found to reach a cross-linking efficiency higher than ordinarily achieved by means of chemical methods.

Strategy.

To overcome the major limitations for XChIP use (see 1.1), ATLAS has gathered an international multidisciplinary consortium of high caliber mathematicians, chemists, physicists, molecular biologists (both array specialists and signal transduction specialists) and molecular oncologists with the aim to complement/replace XChIP by laser- crosslinking and establish global LChIP technologies. In addition the LChIP approach opens a new experimental dimension that cannot be addressed with current technologies such as binding dynamics.

ATLAS will develop a laser system adapted to DNA-protein crosslinking. We have already provided proof-of-principle that an early-stage UV laser can be used for LChIP in vivo at efficiencies (determined in a direct LChIP-XChIP comparison in both relative and absolute ranges of DNA-protein crosslink), which are ten times higher than those of formaldehyde crosslink1 (Fig. 1, 2 and ATLAS unpublished results). In a second phase L-ChIP will be set up and validated in two modes of operation. On the one hand, LChIP will be applied to individual promoters to study dynamics, on the other hand global LChIP will be set up and used to address genome function-related questions. A particular strength of the ATLAS project is its tight link to other European consortia; top specialists will use LChIP technologies to address distinct aspects of genome complexity. Moreover, ATLAS will combine fluorescence-activated cell sorting with (global) LChIP technologies for genome wide studies in subset of cells in biological populations. ATLAS will construct the prototype of a microfluidic system for fluorescence-activated cell sorting connected with a dual pulse femtosecond laser for in situ crosslinking up to the single cell level. With these settings it will be for the first time possible to crosslink cells selectively from heterogeneous populations according to pre-defined parameters.

chemical principles and mechanism(s) of photo-crosslinking Construction of femtosecond double - pulse, two
color tunable laser Optimization of EXPERIMENTAL conditions
Development & validation of LChIP and global-lChIP technologies IN BIOLOGICAL SYSTEMS
Development & optimisation of cell sorting L-Chip technologies: APPLICATION IN BIOLOGICAL SYSTEMS