The Two Unknowns of Nucleosomes: How They Are Formed and How They Are Removed
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In eukaryotes, the genomic DNA is highly packaged inside the nucleus of a cell by forming beads-on-a string-like structure called nucleosomes. The propensity of a ~150-bp duplex DNA to form a nucleosome, termed the "nucleosome affinity", varies over a few orders of magnitude depending on the DNA sequence. Although nucleosome affinity is thought to be determined by DNA bending stiffness, the exact relationship between the two is not clear. Besides their role in DNA packaging, nucleosomes can repress gene expression by preventing transcription factors from accessing their DNA binding sites. Some nucleosomes, however, do not directly occlude transcription factor binding sites, and therefore their role in gene expression remains unknown. In the first part of this talk, I will explain how we measure looping kinetics and permanent curvature of various sequences of DNA using single-molecule FRET (fluorescence resonance energy transfer) and gel electrophoresis. I will then estimate nucleosome affinity of different sequences based on these measurements. In the second part, I will explain how we quantify gene expression pattern using fluorescence microscopy of yeast cells and demonstrate two examples where a nucleosome exhibits opposite effects on gene expression. Based on these results, I will present a model for nucleosome removal prior to transcription initiation.