This award is funded under the American Recovery and Reinvestment Act of 2009 (Public Law 111-5).
Intellectual Merit: The transformative accomplishment of this research will be the development of single molecule strategies to detect, quantify, and map multiple targets in a single nucleosome simultaneously at any locus within a living cell. These single molecule tools will enable researchers in both basic and applied sciences to uncover dynamic aspects of chromatin modifications directing gene expression that cannot be revealed by current approaches. The ultimate goal is to develop transformative technologies to advance chromatin biology and gene regulation studies in any cell type into the single living cell realm. Near-term objectives supporting our ultimate goal are: 1) To identify any desired chromosomal locus by optimizing nuclear delivery and locus-specific targeting in single living cells; and 2) To quantify multiple histone modifications and histone variants within individual or neighboring nucleosomes simultaneously at targeted loci. This interdisciplinary proposal brings together a chromatin biologist and a biophysicist to devise revolutionary single cell platforms to evaluate multiple active or repressive histone modifications simultaneously in individual nucleosomes. This high-risk approach will set the precedence for understanding nuclear delivery, distribution, and targeting of multiple chromatin probes to ensure efficient signal to noise ratio for the quantification of epigenetic targets. Once developed, these powerful tools will be applicable to multiple areas of research to profoundly impact an array of disciplines and fields supported by the NSF's Molecular and Cellular Biosciences programs. Deliverables include: (i) an optimized strategy for nuclear probe delivery and targeting, (ii) identification and quantification of multiple modifications within a single nucleosome and the extent to which individual modifications are required for others to occur, (iii) elucidation of histone modifications present within single nucleosomes at any defined locus in a living cell.
Broader Impacts: The power of this approach comes from its sensitivity to detect single molecules in single cells; its modular design that promotes customization; and its applicability to a wide range of basic and applied research questions related to gene regulation, DNA replication and repair, and chromosome dynamics in cells. Educational impact will include the cross-disciplinary training of a postdoctoral researcher and a graduate student in the fields of Biochemistry and Biophysics, opening new avenues for career development. The project also has the capacity to support multidisciplinary research and curriculum development. Training of undergraduates will be achieved through the involvement of sophomore/junior level students from the Discovery Undergraduate Research Internship Program, the Biochemistry undergraduate research program, the Summer Research Opportunity Program or the Summer Undergraduate Research Fellowship program at Purdue.
