Structural biology and mechanism of large macromolecular complexes that interact with and alter chromatin states
University of Colorado at Boulder, Department of Chemistry and Biochemistry, JSCBB (East Campus)
To determine the molecular mechanisms by which nucleosomes are assembled and disassembled
To determine the structure and function of Poly (ADP-ribose) polymerase 1 during DNA damage repair
To understand how inhibitors or Poly (ADP-ribose) polymerase 1 (currently in clinical trials) function
To understand kinetochore assembly through interaction of protein with centromere-specific nucleosomes
Some current areas of interest are:
Interaction of Chromatin with Nuclear Proteins
Many nuclear proteins bind nucleosomes in vivo, with profound effects on chromatin architecture, leading to varied biological outcomes. We use a combination of approaches to investigate the thermodynamics and architecture of such complexes, and to monitor the structural changes in chromatin and interacting proteins. Several of the factors under investigation are of clinical importance, either as targets for anticancer drugs (e.g. Poly-(ADP-ribose) polymerase (PARP-1, 2, and 3), or because mutations in the corresponding gene are correlated with disease states (e.g. MeCP2). We are also interested in how the kinetochore is assembled through the specific recognition of centromere-specific nucleosomes with the ‘alphabet’ soup of kinetochore proteins.
Histone Chaperones and Nucleosome Assembly Factors
Histone chaperones are structurally diverse proteins that bind histones and assist in the various steps of nucleosome assembly and disassembly. We study the structure of several histone chaperone complexes with histones and nucleosome assembly intermediates, and have developed assays to study their mechanism in vitro and in vivo. We find that different histone chaperones promote different steps of nucleosome assembly and disassembly. Therefore, we have started to investigate synergies between histone chaperones, and between chaperones and ATP-dependent chromatin remodeling factors. Mechanisms by which various histone chaperones are targeted to their respective sites of action are also investigated.
Transcription in a Chromatin Context
Nucleosomes present formidable barriers for the transcription machinery. We are using a recombinant in vitro system to study how RNA polymerase II navigates through nucleosomes, and determine the role of various nuclear factors in displacing nucleosomes in front of the polymerase, and in reassembling them in its wake (Fig. 3). This system also allows us to dissect the role of epigenetic modifications on RNA polymerase progression.
Probes to target Nucleosomes in the Cell in a DNA sequence specific mannner
We are working on approaches to specifically recognize a DNA sequence in the cell only when it is in the context of a nucleosome. Because the vast majority of human DNA is organized in nucleosomes, such reagents might be useful complements to other gene-editing tools that act on free DNA .