Intellectual Merit: How our DNA is packaged into cells and is accessed for processes such as gene expression remains a fundamental question in modern biology. A major contributor to DNA packaging and its accessibility are a class of proteins called histones. These proteins wrap DNA into repeating units called nucleosomes, which are the fundamental building blocks of chromatin. Remarkably, histones are decorated with an array of distinct chemical modifications or 'tags' that control their function and the underlining DNA they package. While much has been learned about the modification types and where on the histones these modifications reside, a great deal less is known about how histone modifications contribute to chromatin biology. One modification in particular, methylation of lysine 79 on histone H3 (H3K79), is mediated by the histone methyltransferase Dot1 and contributes to gene silencing, cell cycle control and DNA damage response in yeast. The mammalian counterpart of yeast Dot1, DOT1L, is essential for regulation of embryonic development, cardiac function and promotion of leukemia. Despite an established role of Dot1 and H3K79 methylation in cellular growth and development, the mechanisms whereby Dot1 contributes to chromatin organization and gene expression are poorly understood. Thus, the long-term goal of this project is to uncover the fundamental mechanisms by which Dot1 functions in cellular biology. This goal will be accomplished by addressing the following key questions: 1) How is Dot1 recruited to chromatin to methylate H3K79? 2) Once on chromatin, do other histones and their modifications contribute to Dot1-mediated H3K79 methylation? 3) How does H3K79 methylation actually signal in chromatin to regulate chromatin organization and gene transcription? Our hypothesis is that Dot1 and H3K79 methylation contribute to gene expression through multiple, regulated steps that include i) recruitment and binding of Dot1 to chromatin, ii) functional interplay of histone tails that stimulate methylation followed by iii) the recruitment and/or exclusion of effector proteins that "read" H3K79 methylation. Because Dot1 and H3K79 methylation are conserved across species, we will address the foregoing questions by taking advantage of the highly tractable model organism Saccharomyces cerevisiae, using a combination of biochemical and genetic experiments. These studies will provide fundamental insights into how histone methylation contributes to chromatin organization and gene expression.
Broader Impacts: This project will provide opportunities for undergraduate students, graduate students, and post-doctoral trainees to experience scientific discovery, learn modern technologies and develop critical thinking, planning, and communication skills. The proposed experiments involve a variety of classical genetics, molecular biology, biochemical, and proteomic approaches making them excellent vehicles for training new scientists. Projects involving yeast are particularly amenable to undergraduate participation because yeast cells are easy to grow and manipulate. There will also be numerous opportunities for participation by underrepresented minorities through my involvement with the UNC Post-Baccalaureate Research Education Program (PREP), for which I am a preceptor. As our studies will produce high-impact findings, we will disseminate our discoveries to the scientific community and the general public through open access journal publications in addition to local and national on-line news outlets. Along with my plans of partnering with the Kenan Fellows for Curriculum & Leadership Development Program, which pairs K-12 teachers with university mentors to develop new teaching modules at local schools, this proposal will have a significant and concrete impact on scientific education at multiple levels.
