Permutational analysis of Saccharomyces cerevisiae regulatory elements

Abstract

Gene expression in Saccharomyces cerevisiae is regulated at multiple levels. Genomic and epigenomic mapping of transcription factors and chromatin factors has led to the delineation of various modular regulatory elements – Enhancers (UAS), core promoters, 5’ untranslated regions (5’ UTR) and transcription terminators/3’ untranslated regions (3’ UTR). However, only a few of these elements have been tested in combinations with other elements and the functional interactions between the different modular regulatory elements remains under explored. We describe a simple and rapid approach to build a combinatorial library of regulatory elements and have used this library to study 26 different enhancers, core promoters, 5’UTRs and transcription terminators/3’ UTRs to estimate the contribution of individual regulatory parts in gene expression. Our combinatorial analysis shows that while enhancers initiate gene expression, core promoters modulate the levels of enhancer mediated expression and can positively or negatively affect expression from even the strongest enhancers. Principal component analysis (PCA) indicate that enhancer and promoter function can be explained by a single principal component while UTR function involves multiple functional components. The PCA also highlights outliers and suggest differences in mechanisms of regulation by individual elements. Our data also identify numerous regulatory cassettes composed of different individual regulatory elements that exhibit equivalent gene expression levels. These data thus provide a catalog of elements that could in future be used in the design of synthetic regulatory circuits.

Limitations of composability of cis-regulatory elements in messenger RNA

“…However, our observations of 1.5 fold change in the relative effect of terminators depending on coding sequence and promoter choice highlight the quantitative limitations of the assumption of composability. Our results echo previous findings of idiosyncratic and context-dependent interactions between CREs (18). Previous work by Kosuri et al (17) suggests that screening a larger library of CREs would reveal a subset with stronger quantitative interactions.…”

“…However, 5% of the constructs had, on average, a 13-fold deviation. A recent study in budding yeast also found strong pairwise interactions between different promoter regions (18). Experiments with promoter-terminator swaps in budding yeast revealed 2-fold changes due to promoter-terminator interactions (3), even though these CREs are separated by hundreds of nucleotides of coding sequence.…”

Learning the Regulatory Code of Gene Expression

“…Despite the importance of the whole gene regulatory structure in gene expression, very few combinations of regulatory elements have been tested and their functional interactions remain poorly explored. To estimate the contribution of individual regulatory parts in gene expression, a combinatorial library of regulatory elements including different enhancers, core promoters, 5′ UTRs and transcription terminators was constructed in S. cerevisiae ( Dhillon et al, 2020 ). A strong interaction was found between enhancers and promoters, showing that, while enhancers initiate gene expression, core promoters modulate the levels of enhancer-mediated expression and can positively or negatively affect expression from even the strongest enhancers.…”

Advances in promoter engineering: Novel applications and predefined transcriptional control

“…Replicates of the same UAS region or UASs with similar properties can also yield suites of promoters with graded expression. [ 7,79–81 ] Promoter strength can often be enhanced by increasing the number enhancing elements with the UAS region, as has been shown in both S. cerevisiae [ 82 ] and Yarrowia lipolytica . [ 83 ] In S. cerevisiae , UASs from the CLB2, CIT1, and TEF1 promoters were combined in tandem with various core promoters, creating a promoter 2.5x stronger than the TDH3 promoter, one of the strongest promoters in S. cerevisiae .…”