Cell proliferation and differentiation depend on rigorously controlled gene expression. In eukaryotic cells, chromatin plays an essential role in gene regulation and thus is subject to intense investigation in recent years. However, the vast majority of these experiments were bulk assays averaging thousands to millions of cells. These averaging processes tend to mask cell-to-cell variability and dynamics. As a result, chromatin is usually described in a homogeneous and static fashion, and little is known about how they contribute to the gene expression noise and dynamics. Elucidating these properties will significantly improve our mechanistic understanding of many cellular processes related to gene regulation.
My research in the past few years focused on a specific nucleosome positioning pattern called “nucleosome-depleted-region (NDR)”. It was recently found that >90% of yeast promoters, as well as many promoters in fly and human cells, contain NDRs. Despite the abundance and conservation of promoter NDRs, their functional significance and formation mechanism are not well understood. I pursued these questions in budding yeast, but what we learned is likely to have general implications for all eukaryotic species. I used a variety of methods, including biochemical and biophysical methods, genetics and bioinformatics. Among them, a very critical technique is called time-lapse fluorescence microscopy, where we continuously observe live cells over many generations so that they grow from a single cell into a micro-colony. The time-lapse assay allows us to simultaneously measure the average level and cell-to-cell variability of gene expression, activation / repression dynamics, and the correlation between gene expression pattern across cell generations.
CLN2 promoter is cell-cycle regulated by activators SBF and Rme1. These activator binding sites are localized inside a ~300 bp NDR. Using the CLN2 promoter as a model, we identified multiple locally-bound factors that are responsible for the NDR generation. These nucleosome-depleting factors are functionally-separated from activators: they remove nucleosomes without much direct effect on gene expression; while the activators enhance gene expression without any effect on nucleosome positioning.
By perturbing the nucleosome coverage on CLN2 promoter and probing the corresponding transcriptional activity in single-cell time-lapse microscopy, we discovered that promoters containing NDRs lead to reliable, once-per-cycle gene activation. In striking contrast, promoters lacking NDRs induce bimodal, “on / off” activation in individual cell cycles, which displays short-term memory, or epigenetic inheritance, from the mother cycle. These results provided significant insight for the mechanism of transcription noise suppression, the dynamics of transcriptional activation in eukaryotic cells, as well as the sequence composition and spatial organization of functional promoter.
The long term goal of my lab is to identify sequence and chromatin features that affect level, noise and dynamics of gene expression, to understand how these chromatin features are established and characterize their cell-to-cell variability and dynamic change, and finally, to explore how these molecular processes affect cell phenotype. In terms of technique, my lab will employ and improve recently developed single cell time-lapse assay which allows the simultaneous measurement of gene expression level, noise, dynamics and propagation. We will also devise new biophysical methods to probe chromatin structures at single molecule level.