The commitment to differentiate into new cell types is a crucial step in the development of animals, plants and many other eukaryotes. This project will study how this important process is regulated using yeast as the model system, and will uncover new mechanisms for controlling cell fate decisions. The project will also expand the pool of talented students who will pursue careers in science by exposing gifted students from a magnet high school to the process of scientific discovery; providing college students between their junior and senior year with hands-on research opportunities; and training graduate students in hypothesis-driven molecular genetic research.
Similar to differentiation programs in multicellular organisms, sporulation in yeast is governed by a transcriptional cascade. The transcriptional cascade of sporulation consists of early, middle and late sets of genes. Commitment to sporulation is not acquired until middle genes are expressed. Ndt80 is the transcription factor that activates middle promoters, and it is transcribed just before middle genes are expressed. The NDT80 promoter can be viewed as a platform where a switch-like output is generated that commits cells to gametogenesis. Studies performed in this laboratory indicate that the NDT80 promoter is assembled into a state resembling silenced heterochromatin specifically during meiotic development. This heterochromatic state (referred to as "gatekeeper chromatin") requires a DNA-binding repressor protein named Sum1 that recruits a histone deacetylase named Hst1 (a close relative of Sir2). At the end of meiotic G2 (just prior to the commitment point) Sum1 is phosphorylated on multiple residues by a cyclin-dependent kinase (CDK), and a meiosis-specific CDK-like kinase named Ime2. A central hypothesis that will be tested in this project is that the phosphorylation of Sum1 downregulates the Hst1 sirtuin to trigger the transition of heterochromatic gatekeeper chromatin to a state in which Sum1 is exchangeable with DNA. The resulting fractional occupancy of the Sum1 repressor permits low-level NDT80 expression. Commitment is acquired when newly translated Ndt80 protein competitively displaces Sum1 from DNA to initiate a positive autoregulatory loop (Sum1 and Ndt80 competitively bind to a single DNA element in the NDT80 promoter). In this project, this "gatekeeper chromatin model" for commitment will be tested. The specific goals of the project are 1) To establish an assay to monitor the exchangeability of the Sum1 protein in chromatin and 2) to combine sum1 phosphosite mutants and analog-sensitive kinases to create a system that synchronously undergoes the off/on gatekeeper chromatin transition at NDT80 in response to specific phosphomodifications, and to use this system to define biochemical steps controlling this transition.
