Position-Dependent Dynamic Competition between Chd1 and the Pioneer Factor Rap1 on Nucleosomes

Single-molecule analysis of yeast promoter activation by pioneer Transcription Factors and Chromatin Remodelers

The regulatory role of chromatin is especially pronounced at upstream activating sequences (UAS) where transcription factors bind and regulate nearby genes. In an inactive state, the presence of nucleosomes inhibits TF binding. When the gene is activated, this region is found depleted of nucleosomes (it is called nucleosome depleted region (NDR)) and are occupied by TFs. Pioneer transcription factors (pTF) can transiently bind to their binding sites on nucleosomes in UAS and trigger NDR formation. This requires the action of chromatin remodeling complexes (CRs), which collaborate with TFs 1. My project concerns the investigation of how pTF together with CR establish active promoter architecture.

Previous experiments in our lab showed that the yeast pTF Rap1 collaborates with the CR RSC to displace promotor nucleosomes, paving the way for long-lived bound states on newly exposed DNA 2. Based on these findings we are now establishing an in vitro MNase-seq screen to dissect how pTFs can modulate CR activity. In the initial studies I will focus on Rap1 and the CRs RSC and Chd1. In parallel I am dissecting the mechanism of the collaboration between pTFs and CRs and kinetics of the reaction using single-molecule FRET. In the future I will follow up with other pairs of CRs and pTFs using single-molecule fluorescence experiments and MNase-seq experiments on synthetic nucleosomes and also validate these interactions in vivo.

[1] Barisic, D., Stadler, M.B., Iurlaro, M. & Schubeler, D. Mammalian ISWI and SWI/SNF selectively mediate binding of distinct transcription factors. Nature 569, 136-140 (2019). [2] Mivelaz et al., Chromatin Fiber Invasion and Nucleosome Displacement by the Rap1 Transcription Factor, Molecular Cell (2019).