A Syntheic Biology Study on Transcriptional Regulation in Yeast

Transcription initiation in yeast encompasses the binding of activators and/or repressors to upstream regulatory sequences, recruitment of chromatin regulators, general transcription factors (TFs) and the RNA polymerase II machinery to the promoter region. Advances in synthetic biology, DNA synthetic technologies, and high-throughput methodologies in recent years have led to new approaches to study gene regulation, mainly at the transcription initiation step. These approaches include the design of libraries (e.g. promoters, regulatory regions) and high-throughput methodologies (e.g. sorting, deep sequencing) in order to screen many variants at a fraction of the time.

We study the effect generated by various chimeric proteins (bacterial DNA-binding domain fused to yeast chromatin modifiers), on gene regulation and chromatin state. We do so by designing a systematically-fashioned yeast minimal promoter library, containing ~70k synthetic regulatory elements. The library is based on truncated promoters, which includes a synthetic bacterial regulatory sequence (BRS). In addition, a small library of synthetic TFs made of a bacterial DNA-binding domain (DBD) fused to a yeast chromatin modifying domain was generated. The synthetic TF chimeras are targeted to the BRS region, which contains specific recognition sites for the bacterial DBDs. The BRSs vary by the binding sites type, binding sites number, distance relative to the truncated promoter and the relative positioning of the binding sites (in-phase/out of phase). This system design will enable us to study the regulatory effect each synthetic fusion on the chromatin state and gene expression output in our system.