UC Berkeley

Differentiation programs such as meiosis depend on extensive gene regulation to mediate cellular morphogenesis. The molecular events associated with gene regulation in budding yeast meiosis are particularly well-documented at both the transcriptional and the translational levels. Through the integrated study of these two steps in gene regulation, a novel mode of gene repression was identified whereby an mRNA does not produce the protein coded within it, but rather plays a purely regulatory role (Chapter 2, Chapter 3).

We first discovered this by investigating the regulation of the kinetochore gene NDC80 (Chapter 2). The program of meiosis requires transient removal, and thus inactivation, of the outer kinetochore, the complex that connects microtubules to chromosomes. We found that in budding yeast, this occurs by reducing the abundance of a limiting subunit, Ndc80. Central to this mechanism is the developmentally controlled transcription of an alternate NDC80 mRNA isoform (Long Undecoded Transcript Isoform, LUTI), which itself cannot produce protein due to regulatory upstream ORFs (uORFS) in its extended 5'-leader. Instead, transcription of the LUTI represses the canonical NDC80 mRNA expression in cis through the deposition of Set1-dependent histone H3K4 dimethylation, Set2-dependent H3K36 trimethylation, and increased nucleosome occupancy to establish a repressive chromatin state in the downstream canonical NDC80 promoter and thereby inhibit Ndc80 protein synthesis.

We further investigated whether other genes may be regulated in a similar fashion (Chapter 3). We identified 74 genes that have 5'-extended leaders in meiotic prophase, all but two of which have at least one ATG uORF, and the vast majority of which are translationally repressed. While the translational down-regulation of these extended transcripts is near universal, the transcriptional repression of the canonical isoform is more variable. The features important for transcriptional repression at the NDC80 locus are associated with down regulation of the canonical transcript in the newly identified LUTIs. More specifically, H3K36 trimethylation and a decrease in strong positioning of the +1 nucleosome correlate with a greater decrease of the canonical transcript upon LUTI induction. Higher LUTI abundance also correlates with greater repression.

Together we have delved deeply into the regulation of how a novel regulatory mRNA affects the expression of a single gene, and we provide evidence that the same mechanics of gene repression occur at additional loci throughout the genome during meiotic prophase.