Transcription factors bind to regulatory regions to help activate or repress genes. Compaction of the genome into nucleosomes helps reduce the physical space that must be sampled to find in vivo targets, yet forces transcription factors and histones to compete for access to DNA. The equilibrium of this competition can be altered via post-translational modifications of the histone core and regulation of nucleosome position. I examined the differential contributions of DNA sequence, transcription factors, and chromatin to the regulation of gene expression in the model organism, Saccharomyces cerevisiae. While transcription factors bind DNA in a sequence specific fashion, the contribution of DNA in determining in vivo usage of consensus sequences is specific to the biological role of the transcription factor itself. Chromatin shows limited changes in different growth conditions, but localized changes surrounding transcription factor binding sites are evident. Chromatin stability appears to be regulated by the underlying DNA sequence of the genome and modified by active mechanisms such as post-translational modifications. Together, these processes establish stable chromatin in the body of genes and unstable chromatin at promoters. Depletion of nucleosomes is sufficient to alter transcription of 50% of the yeast genome, suggesting that chromatin plays a major role in regulating gene expression via regulating DNA accessibility to transcription factors as well as less direct effects, such as blocking assembly of the transcriptional machinery.