Gene Expression From Random Libraries of Yeast Promoters

Abstract

Genomewide techniques to assay gene expression and transcription factor binding are in widespread use, but are far from providing predictive rules for the function of regulatory DNA. To investigate more intensively the grammar rules for active regulatory sequence, we made libraries from random ligations of a very restricted set of sequences. Working with the yeast Saccharomyces cerevisiae, we developed a novel screen based on the sensitivity of ascospores lacking dityrosine to treatment with lytic enzymes. We tested two separate libraries built by random ligation of a single type of activator site either for a wellcharacterized sporulation factor, Ndt80, or for a new sporulation-specific regulatory site that we identified and several neutral spacer elements. This selective system achieved up to 1:10 4 enrichment of the artificial sequences that were active during sporulation, allowing a high-throughput analysis of large libraries of synthetic promoters. This is not practical with methods involving direct screening for expression, such as those based on fluorescent reporters. There were very few false positives, since active promoters always passed the screen when retested. The survival rate of our libraries containing roughly equal numbers of spacers and activators was a few percent that of libraries made from activators alone. The sequences of 100 examples of active and inactive promoters could not be distinguished by simple binary rules; instead, the best model for the data was a linear regression fit of a quantitative measure of gene activity to multiple features of the regulatory sequence.

Incorporating nucleosomes into thermodynamic models of transcription regulation

“…One promising direction for testing these hypotheses is studying the activation dynamics of synthetic promoter variants containing different combinations of the promoter elements discussed in this study (boundary elements and transcription factor sites). Such largescale libraries aimed at exploring the effects of transcription factor binding on expression have already started to emerge (Ligr et al 2006;Gertz et al 2009). However, with the wealth of new hypotheses gained by this work, more elaborate variant libraries aimed also at studying the effects of nucleosomes on transcription can be designed.…”

Splicing of designer exons reveals unexpected complexity in pre-mRNA splicing

“…We contend the opposite, that a prerequisite to understanding how individual elements combine to yield an emergent property requires an understanding of how the individual elements act, first alone, and then in simple combinations. The way in which transcriptional regulatory signals combine to produce what is often a binary decision is in many ways analogous to the splicing decision problem, and can also be explored using synthetic promoters (Alper et al 2005;Ligr et al 2006). Additional examples of this approach lie in the de novo design of proteins (Kuhlman et al 2003) and cell membranes (Tanaka and Sackmann 2005).…”

Compensation for differences in gene copy number among yeast ribosomal proteins is encoded within their promoters

“…Variants of this approach were successfully applied to reveal ordered activation of genes in various pathways in bacteria (Kalir et al 2001;Zaslaver et al 2004) and to generate libraries of synthetic promoters in bacteria (Cox et al 2007) and yeast (Ligr et al 2006;Murphy et al 2007;Gertz et al 2009), which provided much insight into the rules that underlie combinatorial cis-regulation. However, since they require genetic engineering of one strain for each tested promoter, these approaches are harder to implement.…”