Quantifying Transcription Dynamics of CUP1 gene of S. cerevisiae at the Single‐Molecule Level

Abstract

Transcription factors (TFs) show rapid and reversible binding to gene promoters to activate transcription in live cells. Transcription often occurs in sporadic bursts, however it is yet poorly understood how the dynamics binding of TFs is coordinated with gene bursting. Gene promoters are generally occupied by the nucleosomes and it is known that the presence of a nucleosome at the gene promoter reduces the accessibility for TF binding. Additionally, nucleosomes are known to evict the TFs from the gene promoters. Hence, chromatin remodeling plays an instrumental role in modulating TF binding. Due to advancements in the Single‐Molecule Imaging approaches, quantification of the transcription parameters such as initiation rate, transcript production time, burst frequency, burst duration and burst size has become achievable. We developed and employed several Single‐Molecule approaches (Single‐Molecule Tracking (SMT), live imaging of CUP1 transcription site (TS) by PP7 stem‐loop approach and smFISH modeling of CUP1 transcription) to quantify the transcription dynamics of the CUP1 gene of S. cerevisiae. In cell population, we observe slow sequential cycles of CUP1 transcription in conditions of the prolonged heavy metal stress. By applying gene bursting model, we extract the dynamics of the transcription parameters within the slow cycle from live TS and smFISH data and correlate these parameters with the dynamics of TF and POLII. The chromatin remodeler RSC speeds up the search process of the TF Ace1p for its Response Elements (RE) at the CUP1 promoter. RSC chromatin remodeler also affects the transcription of CUP1. We investigate the effects of RSC remodeler on the slow cycle of CUP1. Modulation of burst frequency yields more effective and productive response than the modulation of burst amplitude. We are interested in cyclical expression of the genes in stress response.Support or Funding InformationThis work was supported by Intramural Research Program of the National Institutes of Health (NCI, CCR).