Objectives
The goal of our research is to develop and use a set of complementary approaches in nanobiotechnology and related areas to study chromatin, a biomolecule critical in maintaining gene structure and function. We are using these approaches to study chromatin structure, function, and dynamics, with a focus on nucleosome-binding proteins (e.g., PARP-1 and H1) and chromatin remodeling complexes (e.g., SWI/SNF).
Methods
Our studies bring together approaches from nanobiotechnology, as well as from biophysics, biochemistry, and genomics. The integration of these approaches allows us to examine chromatin and associated factors in an unprecedented way both under highly defined conditions with single molecules and in the complex native environment of living cells, all at nano-scale resolution. Single molecule optical traps are devices capable of manipulating and detecting sub-nanometer displacements for sub-micron dielectric particles, making them very useful for the manipulation and study of single molecules of DNA or protein attached to those dielectric particles. Atomic force microscopy (AFM) provides three-dimensional views of biological molecules with nanometer resolution. AFM can also be extended to the determination of the molecular identities of specific components within complexes or mixtures. Multi-photon microscopy (MPM) provides real-time views of gene regulation in living tissue that are of unprecedented clarity and resolution. The deep-penetrating and low background MPM is ideal for imaging tissues and cells. In vitro reconstitutions allow for the assembly of macromolecular complexes (e.g., molecular motors, nucleosomes) with defined compositions for use in single molecule mechanistic analyses. Genomic microarrays are solid surfaces arrayed in a nano-scale pattern with a DNA probe set synthesized in situ representing defined regions of the genome. High density microarrays can be used to map the binding of factors and the position of nucleosomes across the genome.
Summary
Chromatin is a dynamic polymer of nucleosomes (DNA wrapped around a protein core of histone proteins) whose biochemical and biophysical properties play important and specific roles in determining the structure and function of chromatin in vivo. Our group is using the complementary approaches described above to study the nano-structure and dynamics of chromatin. Specificially, we are determining (1) the mobility of canonical and variant histones within chromatin, (2) the effect of nucleosome-binding proteins on the structure of chromatin, and (3) the mechanisms by which chromatin remodeling complexes alter nucleosome structure and stability. As highlighted in the next section, we have made progress in a number of key areas related to the aims of the project over the past year (Year 2 of the project). These studies have (1) explored the mechanisms of nucleosome remodeling by the SWI/SNF remodeling complex using Holliday junctions in an angular trap as a “nano-sensor” for remodeling activity, (2) examined histone dynamics in vivo using MPM, and (3) determined the genomic localization and effects of the nucleosome-binding protein PARP-1 on chromatin structure using genomic microarrays, in vitro reconstitutions, and AFM. In addition, our work over the past year has produced reagents and refined techniques that will be used for the studies planned in Year 3 (e.g., recombinant SWI/SNF complex for in vitro reconstitutions, new GFP expression constructs and fly lines for MPM). Our integrative approach and productive interactions among the team members is providing a molecularly detailed and biologically relevant view of the dynamics of chromatin.
Accomplishments
- The Wang lab has used nanofabricated quartz cylinders in an angular optical trap to create a Holliday junction in DNA that may be used as a “nano-sensor” for motor proteins. Working closely with the Kraus lab, the Wang lab has obtained intriguing preliminary data on translocation of the nucleosome remodeling enzyme SWI/SNF against the junction.
- The Lis and Webb labs have used MPM to monitor histone dynamics in living cells. Specifically, they have created a fly line expressing paGFP tagged H2B in order to directly track how these changes occur. By generating flies coexpressing mCherry tagged LacI with transgenic inserts of LacO sequences flanking the Hsp70 gene loci, they can now identify and specifically activate molecules at these loci before gene activation and track changes in structure over time.
- The Kraus and Lis labs have used biochemical, genomic, and gene-specific assays to determine the localization and chromatin remodeling effects of the nucleosome binding protein PARP-1. These studies have shown that PARP-1 localizes to promoter regions and, for many promoters, excludes the binding of the linker histone H1 (another nucleosome binding protein).
