This action funds an NSF Postdoctoral Research Fellowship in Biology for FY 2011, Intersections of Biology and Mathematical and Physical Sciences. The fellowship supports a research and training plan in a host laboratory for the Fellow at the intersection of biology with physics. The title of the research and training plan for this fellowship to Maria Kamenetska is "Unwinding DNA: measuring mechanical stiffness of a single DNA molecule to understand histone control over gene transcription." The host institution for this fellowship is Yale University with sponsoring scientists Drs. Lynne Regan and Simon Mochrie.
Histone proteins package DNA into chromatin. This research explores how histones control the unwinding of DNA to open it for the steps that lead to protein synthesis. Changes in histones are believed to affect the pliability of chromatin and thus the accessibility of DNA for transcription. Single molecule nano-manipulation and force-spectroscopy techniques from physics are being used to quantitatively assess the affect of histone modification on DNA. Chromatin re-constituted with exclusively wild-type or modified histones is being prepared for studies that use optical tweezers and AFM-based force-spectroscopy techniques to stretch single chromatin tethers and measure their mechanical and structural properties. These experiments enable an unambiguous comparison of pliability of individual DNA molecules with different histone compositions and thus reveal the role of histones in controlling gene expression.
Training objectives include learning molecular biological techniques and acquiring relevant biochemical skills to prepare homogenous preparations of chromatin fibers. Broader impacts include educational outreach to biology students to learn single molecule nano-manipulation techniques and generally promoting the dissemination of physical approaches in biological science at the host institution. Also, outreach includes developing and teaching a physics-based module to supplement the biology curriculum for Breakthrough New Heaven, a summer program for disadvantaged middle-school students.
The focus of this project has been to understand the dynamics of a DNA-protein structure called chromatin which controlls gene expression inside our cells. Understanding factors influencing gene expression may help us understand the biology of cancer as well as the special features of stem cells. In particular, I have focused on studying the nucleosome, which is the basic building block of chromatin and consists of roughly 150 base pairs of DNA wrapped around a complex of eight histone proteins. My approach to studying chromatin centers on combining biophysical methods, such as protein expression and purification, with single molecule techniques borrowed from the physical sciences. For this project, I have expressed and purified wild-type and variant (mutant) histone proteins, reconstituted nucleosomes using these histones and used single molecule force spectroscopy to measure the affect of the variant histones on chromatin dynamics. In the course of the project, I helped develope a novel force spectroscopy-based technique for measuring single molecule transitions far from equilibrium. This technique greatly increases data acquisition on a single biological molecule and reduces total measurement time. In addition, I have measured kinetics of wild type nucleosome unwinding and rewinding in different salt conditions. That work resulted in the discovering of an alternate unwinding pathway for the nucleosome. Currently, I am in the process of measuring the unwinding and rewinding rates of nucleosomes containing variant histones. Overall, the work conducted under this grant will help better understand how cells use histone variants to regulate gene expression.
