A novel approach for transcription factor target identification: Studies of the Saccharomyces cerevisiae cell cycle and the human beta-globin locus

Transcription factors are key regulatory proteins that mediate the processes of cellular differentiation and proliferation by influencing the expression of tens to hundreds of different genes. Identifying the set of genes regulated by a transcription factor helps to define the function of that transcriptional regulator, but also lends some understanding toward how specific developmental processes are manifested on a molecular level. This dissertation describes a new approach to gene target identification termed chIp-chip, which couples chromatin i&barbelow;mmunop&barbelow;recipitation and DNA chin technology to comprehensively identify the direct DNA-binding targets of transcription factors across the genome.; Two eukaryotic systems have been explored in this dissertation: the yeast Saccharomyces cerevisiae cell cycle and the human beta-globin locus. The importance of transcription in propelling the yeast cell cycle is reviewed in Chapter 1. It describes the key transcription factors that participate in regulating the waves of transcriptional activity in the cell cycle and discusses how the chIp-chip approach and other genomic methods have expanded the knowledge of this subject in the past five years. Chapter 2 focuses on two of the yeast cell-cycle transcription factors introduced in Chapter 1 that are critical for initiating the yeast cell cycle: SBF (S&barbelow;wi4-Swi6 cell-cycle box b&barbelow;inding f&barbelow;actor) and MBF (M&barbelow;luI cell-cycle box b&barbelow;inding f&barbelow;actor). The results of chIp-chip analysis with these two factors are presented. The next two chapters are devoted to the characterization of genes that were identified as targets of SBF. The evidence presented in Chapter 3 implicates two previously uncharacterized genes, TOS1 (T&barbelow;arget o&barbelow;f S&barbelow;BF) and TOS2 in polarized cell growth. Chapter 4 describes the results of chIp-chip analysis of nine transcription factors that are downstream of SBF. In the final chapter (Chapter 5), the chIp-chip approach has been applied in a mammalian system and describes the binding of the erythroid-specific transcription factor GATA-1 throughout the human beta-globin locus. The work encompassed by this dissertation has hopefully lead to a better understanding of the transcriptional control of the yeast cell cycle and human globin gene expression and has provided a tool for further study of transcriptional regulation in all systems.