Regulation of cell size and the size dependence of gene expression

Volume is a fundamental morphological feature of cells, influencing a wide variety of cellular processes. Because it is coupled to so many processes, cells employ regulatory mechanisms to ensure cells exhibit a limited range of sizes. Recent work in budding yeast has shown that a key cell cycle regulator, the G1/S transcriptional inhibitor Whi5, is synthesized independent of cell size. The dilution of Whi5 in larger cells links cell size to G1/S cell cycle progression. However, it has also been shown that growth over the full cell cycle does not depend on cell size at birth, termed an "adder". It has been proposed that this observation suggests that cell size is controlled over the course of the full cell cycle, leading to an apparent contradiction. Here we show that cell size control occurs independently in different parts of the cell cycle and does not reflect a molecular mechanism measuring growth during the full cell cycle. Consistent with previous results, we find that cell size sets the rate of entry into the cell cycle during the pre-Start period. We also identify the key parameters predicting the rate of entry into cytokinesis at the end of the post-Start period. We use these parameters to build a phenomenological cell cycle model that recapitulates observations of growth and size distributions for cells without explicit coupling between cell cycle phases. Our model predicts that changes to the rate of progression through either phase of the cell cycle should disrupt the adder behavior and we show that mutants in genes controlling G1/S size control breaks the adder. The rate of passage through Start depends on volume, which is thought to depend on the size-independent expression of Whi5. This type of gene expression scaling is unusual because although cells of a given type may span a range of sizes, most proteins and RNA are maintained at constant, size independent, concentrations, rather than amounts. This ensures that biochemical reactions proceed independently of cell size. The identification of WHI5, whose gene product differs from this pattern, raises two fundamental questions: (1) Are there additional genes whose synthesis is decoupled from cell volume? (2) If most gene expression is proportional to cell size, what molecular mechanism promotes cell-size-independent gene expression? To address these questions, we analyzed flow cytometry data collected using the yeast GFP-fusion library. We identified approximately 200 genes whose expression is not proportional to cell volume. Gene ontology analysis revealed that non-scaling genes are enriched for genes with roles in DNA-templated processes and membrane transport. This suggests that cells employ differential protein synthesis to coordinate protein requirements with the scaling properties of cellular structures. Membranes are expected to scale as size2/3 and DNA content is independent of size. To understand the mechanisms that underlie size-independent gene expression, we used transcriptional reporters of non-scaling genes, including WHI5, and determined that cell-size-independent regulation of some genes is due to non-scaling transcription rates. Targeted analysis of the WHI5 promoter showed that the region between 1000 bases and 550 bases upstream of the translation start site are required for cell-size-independent gene expression. This suggests there is a molecular element within this region required for non-scaling gene expression. Finally, we identify a partitioning mechanism ensuring proteins are partitioned in dividing cells in amounts that are independent of asymmetric sizes of the mother and daughter cells. Tight chromatin association ensures that proteins are segregated in equal amounts despite asymmetric division. Consistent with this model, while Whi5 is normally partitioned in equal amounts, a Whi5 protein that lacks the domain required for association with transcription factors is partitioned in proportion to the mother-daughter cell size ratio. Taken together, our work demonstrates a functional role for differential size-dependency of protein synthesis and gives insights into the underlying molecular mechanism(s).

Keywords: Biology.
Advisor: Skotheim Jan
Cyert Martha S.
Stearns Tim 
Straight Aaron