Transcription regulation dynamics

Abstract

A large body of previous research indicates the importance of dynamics in transcription regulation, yet our understanding of this aspect is quite poor. The overall aim of the work described in this thesis is to study transcription regulation dynamics. This started out with what seems an apparently simple question, namely how is the different dynamic response of housekeeping and regulatable genes achieved? Crucially for this study, we made use of sets of genes that depend on the exact same activator for transcription. The analyses reveal the mechanisms behind the difference in dynamics between housekeeping and regulatable genes to a great level of molecular detail. The results show how the dynamics of TF binding can have a different effect on transcription based on the molecular properties associated with different classes of promoters. Besides explaining this important difference between these two general classes of genes for the first time, the study also highlights the importance of studying TF-DNA binding dynamics for a proper molecular mechanistic understanding of cellular processes. Because of the lack of methods to properly measure TF on- and off-rates for many individual sites in parallel in vivo, a method was developed for genome-wide off-rate measurements. This method (DIVORSEQ: determining in vivo off-rates by sequencing), first required extensive optimization of one of its component parts, that is the ChIP DNA binding measurement protocol, in order to yield results quantitatively comparable between different samples. Optimization experiments for almost all individual steps of ChIP led to an optimized protocol, which is presented in detail. By applying DIVORSEQ using this optimized ChIP protocol, off-rates were successfully determined genome-wide for the TF Abf1. To the best of our knowledge this is the first study to directly determine in vivo off-rates for a single TF for hundreds of sites in parallel. Abf1 is a general regulatory factor in yeast, akin to chromatin pioneering TFs in larger eukaryotes, and with several previously ascribed functions, for example in organizing chromatin, regulating transcription and termination. The acquired TF-off rates cover a wide range of different values: average TF residence times at different loci range from 4.5 to 37 minutes. These are analyzed in the context of the different roles of Abf1. Besides showing that DIVORSEQ provides very meaningful measurements of TF binding stability, the analyses show how different binding dynamics of the same TF can contribute to different aspects of its function. The work described in this thesis demonstrates the importance of understanding TF-DNA binding dynamics in order to understand how gene expression is regulated. In addition, a powerful new tool for quantifying these dynamics is presented