Robert Kingston Laboratory
The Kingston lab is interested in understanding the fundamental mechanisms of how eukaryotic enzymes can modify chromatin. One focused approach is to isolate chromatin-modifying proteins and to test such complexes in functional assays, with the thesis that assay outcomes will inform the in vivo function of these protein machines.
Another important goal is to understand how long-range chromatin interactions are established in the cell nucleus, and this effort is leading to new strategies toward defining such interactions in vivo.
In the eukaryotic cell, DNA is dynamically regulated by higher order packaging of the DNA into chromatin. The fundamental unit of chromatin is the nucleosome - DNA wrapped around a core of histone proteins. These nucleosomes can be compacted or expanded to greatly affect its accessibility to the transcriptional activity of RNA polymerase, ultimately leading to changes in gene expression. How is this dynamic regulation achieved?
One prime mechanism for chromatin regulation is modification of the chromatin by protein enzymes, some of which utilize ATP as an energy source. A general goal of this lab is to identify such chromatin remodeling enzymes and dissect their function with biochemical assays. We have deconstructed the functional core of the Polycomb Group complexes in animals, and have fundamentally dissected the domain activities of other ATP-dependent chromatin remodeling complexes. The lab has recently begun to examine the effects of long-range interactions in chromatin templates that may be mediated by chromatin remodeling complexes, and it is an important goal in this lab to begin to probe long-range chromatin interactions by developing new in-vitro diagnostic tools and functional biochemical assays.
Polycomb Group Complexes and other ATP-dependent Chromatin Remodeling Enzymes
To maintain the differentiated tissues in any organism, cell-type specific genes must remain "on" for the lifetime of the organism in one cell type, and "off" for the lifetime of the organism in other cells. Thus, the same gene can be kept in an "on" state across numerous cell divisions in one cell, but be kept in an "off" state across numerous cell divisions in a nearby cell. This type of regulation, frequently referred to as epigenetic regulation, occurs at least in part by maintaining different states of the chromatin over a gene when it is on as compared to when it is off. When a gene is maintained in an off state, the chromatin packaging that gene is maintained in a state that inhibits transcription factor function, and thus blocks transcription of the gene. When a gene is maintained in an on state, the chromatin is maintained in a configuration that is permissive for transcription.
The nucleosome is the fundamental building block of chromatin, and genetic studies imply that altering nucleosome structure and plays a central role in epigenetic regulation. Research projects in the Kingston laboratory are designed to characterize, using biochemical approaches, protein complexes that are involved in epigenetic regulation. One set of projects focuses on the mechanism of ATP-dependent remodeling complexes, with the goal of understanding how these complexes use the energy of ATP hydrolysis to alter nucleosome structure and how these alterations can contribute to overall chromosome dynamics and to transcriptional regulation. A second set of projects focuses on the Polycomb-group (PcG) of genes. Complexes of that are encoded by PcG genes are required to maintain chromatin in a repressed state, and current projects use purified forms of these complexes to characterize how the complexes regulate chromatin structure and transcription of chromatinized templates. A key area of future analysis will be to understand how these and other complexes contribute to the regulation of higher order chromatin structure.
Examining long-range chromatin interactions in live cells
Regulated changes in chromatin structure result in changes in nucleosome position/conformation and changes in the binding of sequence-specific regulatory factors. These changes in architecture cause changes in the ability of nucleases and chemical agents to cleave packaged genomic DNA. Study of these changes in cleavage over the past twenty five years has led to important advances in understanding regulatory processes and to the identification of regulatory elements. One significant conceptual advantage of using cleavage mapping to probe for regulatory events is that it is not necessary to have prior knowledge of gene-specific factors that act on the region of interest; the cleavage maps can define important aspects of regulation and can define sequences that might be key sites for binding of gene-specific regulators.
Our goal is to devise technology that will allow chromatin structure to be examined over very large (100 kb or greater) regions of the genome. The focus is on mapping cleavage sites for chemicals and enzymes whose activity is known to display sensitivity to changes in chromatin structure. Development of this technology will not only provide an important, largely unbiased, mechanism for searching for novel regulatory elements, but will also provide a tool to increase our understanding of long-range changes in chromatin structure.