DNA is the blueprint of life that provides instructions for producing various functional proteins within a cell. But DNA in living cells exists in a protein environment/packaging system called "chromatin" that controls when functional proteins are produced, or when the instructions from DNA to produce proteins are silenced. The way chromatin controls the production of proteins can be influenced by age of the organism, environment that the organism is living in, and whether the organism is diseased. Much is still unknown about how chromatin regulates the production of proteins, but a better understanding could lead to advances in our knowledge of how organisms adapt to an environment or how better to treat certain diseases. This project will leverage novel molecular biology techniques to better understand key components of the complex human chromatin organization by progressively rewriting (or rebuilding) the system within budding yeast, a much simpler laboratory model. This will enable the dissection of the complicated regulatory circuits that control the production of functional proteins from DNA in humans, other animals, and other multicellular organisms. Two postdoctoral researchers and one graduate student will be trained in state-of-the-art molecular biology methods and analyses. Results from the research will be disseminated broadly by a yeast art program to be exhibited on the New York City subway, by partnering with the New York City "biobus" program, and by presentations at a new "epigenome engineering" meeting.
The epigenome equips many eukaryotes with the ability to generate stable and distinct cell types from a single identical genome. This multipurpose ability stems from interconnected chromatin organizing pathways acting at multiple length scales across chromosomes to regulate gene expression, maintain cell identity, and adapt to environmental changes. What rules lead to one cell's chromatin organization versus another? Advances in genome-wide methodologies have revealed the patterns underlying chromatin architectures, but inferring causal effects remains difficult. Mechanistic biochemical approaches that require complex protein purifications and use minimal chromatin substrates have limited ability in recapitulating living systems. Multigene knockout studies can lead to cellular dysfunction and pleiotropy, making it difficult to decouple overlapping phenotypic effects. In this research project, human epigenetic pathways will be reconstituted within the eukaryote budding yeast (Saccharomyces cerevisiae) at four levels to provide a bottom-up approach for unraveling principles of chromatin organization and inherited gene expression. The four levels are: (1) Histones: expanding the ability to generate diverse human histone variant architectures. (2) Heterochromatin: human pathways such as Polycomb Group Proteins will be imported into budding yeast to generate the repressive histone Post-Translational Modifications (PTMs) H3K27me, H3K9me, H2AK119ub and CpG methylation. (3) Euchromatin: the COMPASS complex in yeast will be replaced by the human pathway that generates the activating histone PTM H3K4me. (4) Topologically Associating Domain (TAD) structures: human-like TAD structures will be engineered using human cohesin and CTCF complexes.
This award reflects NSF's statutory mission and has been deemed worthy of support through evaluation using the Foundation's intellectual merit and broader impacts review criteria.
