The fast and the direct Unravelling gene regulatory networks with acute depletion strategies

DNA is the blueprint of life and every cell is specialized to use the DNA in its unique manner. This promotes cell identity and function. Around 2% of DNA contains genes, which encode for proteins. The activation and inactivation of genes are usually defined as gene regulation. Regulation of gene activity is generally promoted by the activity of transcription and the regulation of the molecular machinery that activates transcription. Most of the DNA does not contain genes, rather it contains DNA sequences with a regulatory function, defined as Regulatory Elements (RE). RE serve to activate genes at the appropriate level, time and space. Furthermore, the DNA is packaged into chromatin, a three-dimensional assembly of DNA and proteins that fits into the cell nucleus. Chromatin structure contributes to gene activation. Understanding the mechanisms of gene regulation is fundamental to deciphering cell behavior and function. In this thesis, we determine the dynamicity of various factors directly regulating gene activation. We used acute depletion strategies to study the direct role of key players involved in gene regulation, to determine the mechanisms of chromatin organization and to identify functional regulatory regions. Using a time-resolved determination of the early molecular events that control gene activity, we uncover novel insights into the factors involved in gene regulation, determining a novel function for chromatin dynamics, the main molecular factors important for gene regulation and develop a framework to identify RE relevant for gene activation.