WP 1: Physical measurements of TF-DNA binding

Objectives

The overall objective of this work package is to obtain a quantitative description of binding events of prototypical transcription factors (TFs) to their binding sites (TFBSs) in promoters of inflammatory genes, on naked DNA, reconstituted chromatin and living cells .

Specific aims:

• Quantification of affinity constants (KD, Kon, Koff) for selected inflammatory TFs (NF−κB, IRF, AP1) to canonical binding sites and their variants in vitro.
• Identification of functional heterodimers and relationship between sequence specificity and binding affinity at the single molecule level.
• Characterization of the occupancy for each TF on naked DNA. Characterization of TFBSs occupancy in an “in vitro reconstituted chromatin context” for each TF.
• Role of histone H1, chromatin remodelling machines and histone chaperones on the time resolved interactions of each TF on in vitro reconstituted chromatin templates.
• Quantification of dynamic constants for protein-chromatin interactions in vivo on active endogenous promoters under standard and perturbed conditions.
• Analysis of protein-protein interactions in vivo on actively transcribed promoters, containing canonical and variant TFBSs.
• Modelling of TFs binding on naked DNA and chromatin (described in WP 4)