ATLAS

Development of Laser-Based Technologies and Prototype Instruments for Genome-Wide Chromatin ImmunoPrecipitation Analyses

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 Obiettivo del progetto (Objective)

'Deciphering the complexity of chromatin-encoded information is the prerequisite for understanding the regulatory circuits governing development and (patho)physiology. Transcription factors and epigenetic modulators translate chromatin-embedded information in a dynamic and cell/gene context specific manner to orchestrate homeostasis, growth and differentiation. To date, the most powerful and commonly used approach is immunoprecipitation of chemically cross-linked chromatin (XChIP) coupled with single gene or global analysis using DNA tiling arrays (ChIP-chip) or parallel single molecule sequencing (ChIP-seq). At present, serious limitations of the XChIP technology preclude factor-DNA interaction studies at dynamic ranges below minutes. Moreover, conventional XChIP cannot be used to study samples of <106 cells or cell populations within complex biological samples. Based on established proof-of-principle experiments, the multidisciplinary ATLAS consortium will develop novel types of femtosecond (fs) UV tunable lasers to induce highly efficient DNA-protein crosslinking for ChIP analyses with unprecedented precision and reproducibility, and extending the present dynamic time range by order of magnitudes. ATLAS will further validate LaserChIP (LChIP) technologies and integrate them e.g. with manipulation of irradiated (frozen) tissues slices and microfluidic cell sorting systems. Combining LChIP with proximity ligation approaches will facilitate the analyses of cell-selective (epi)genetic programs on small pre-defined cell populations down to the single cell level. Integration of innovative SMEs with physicists, oncologists, biologists, chemists and mathematicians secures efficient introduction and application of LChIP in basic and translational research. This consortium has the technical and commercial expertise as well as the capacities to successfully develop and commercialize laser-based ChIP technologies.'

Introduzione (Teaser)

EU-funded scientists have developed an ultra-fast laser system enabling the analysis of transient molecular binding interactions. They used it to 'witness' activity between individual molecules that mediate gene expression.

Descrizione progetto (Article)

Chromatin consists of deoxyribonucleic acid (DNA) tightly wound around proteins called histones. The winding and binding limits the access of transcription factors (TFs, molecules that modulate DNA transcription) to the DNA. An 'opening' of the complex is therefore required for gene expression and proper cell functioning.

The modulation of chromatin structure is itself a highly dynamic process with complex regulation and whose dysfunction can result in aberrant gene expression. Understanding the mechanisms as well as the spatio-temporal patterns of binding in a single cell or in small groups of cells is critical to a full understanding of gene expression. The recently developed chromatin immunoprecipitation (ChIP) assay has been quite valuable for identification of histone modifications and of binding patterns between TFs and chromatin.

However, immunoprecipitation of chemically cross-linked chromatin (XChIP) does not allow factor-DNA interaction studies on a temporal scale less than a few minutes, thus prohibiting the evaluation of transient chromatin binding. In addition, it does not facilitate the study of small populations of less than a hundred or so cells. With EU-funding of the ATLAS project, scientists developed laser-based ChIP (LChIP) that overcomes these disadvantages and brings with it numerous other benefits.

Ultraviolet (UV) irradiation with an ultra-short pulse laser source creates a stable crosslink of zero length in a very short time due to the inherent photo-reactivity of excited nucleotides in the DNA. There is less perturbation of the biological system than with chemical cross-linkers such as formaldehyde and one can now study transient interactions occurring on very fast timescales. Further, the optimisation of DNA amplification methods enables the use of very small numbers of cells.

Ground-breaking laser ChIP technology delivered by ATLAS now enables the study of transient binding interactions between chromatin and other molecules not detectable with conventional methods and critical to gene expression. The analysis of cell-selective genetic and epigenetic programmes on small subpopulations of cells down to the single-cell level should push the frontiers of scientific discovery. ATLAS is expected to stimulate enhanced understanding of physiological and pathological cellular processes leading to the development of novel genetic therapies. Please also see: http://vimeo.com/15927539, http://vimeo.com/15928069