Molecular and Structural Determinants of Chromatin Folding
Open Access
- Author:
- Correll, Sarah Jane
- Graduate Program:
- Genetics
- Degree:
- Doctor of Philosophy
- Document Type:
- Dissertation
- Date of Defense:
- June 02, 2011
- Committee Members:
- Sergei A Grigoryev, Dissertation Advisor/Co-Advisor
Kristin Ann Eckert, Committee Member
Sarah Bronson, Committee Member
Laura Carrel, Committee Member
Sergei A Grigoryev, Committee Chair/Co-Chair
Jiyue Zhu, Committee Member - Keywords:
- linker histone
nucleosome
chromatin structure
analytical ultracentrifugation - Abstract:
- Most of the genetic information needed to direct cellular processes is encoded in the chromosomal DNA of the eukaryotic cell, which needs to be highly organized to fit inside the cell nucleus while remaining accessible to cellular factors. This is accomplished through the association of DNA with proteins to form chromatin fibers. At the primary level, DNA is wrapped around an octamer of core histone proteins, forming the nucleosome. Additional levels of compaction (higher-order structures) result from interactions between core histone tails, linker histone proteins, cations, and architectural proteins. The goal of this thesis was to develop an experimental approach to determine the individual contributions of various chromatin components in the overall formation and regulation of higher order structures. This approach utilized a high affinity nucleosome binding DNA sequence to construct reconstituted chromatin arrays that position nucleosomes with single nucleotide precision. Using these reconstituted chromatin arrays, chromatin higher order structures were tested for interactions both within (folding) and between (self-association) arrays, through biochemical assays such as sedimentation velocity experiments using analytical ultracentrifugation and reversible self-association assays. By using the reconstituted array system to alter translational and rotational nucleosome settings, we observed a negative correlation between linker DNA length and chromatin folding. This relationship was not strictly linear, indicating that for short linker DNA lengths (≤177 bp) typical of lower eukaryotes, rotational settings were important for compact chromatin structure. However, alterations to the rotational setting of chromatin arrays with linker DNA ~200 bp (typical of higher eukaryotes) did not reveal any alterations to chromatin higher order structure, leading to the possibility that long linker DNA can absorb variations in rotational settings. Additional studies evaluated chromatin higher order structure in the presence of high mobility group (HMG) architectural proteins. For HMGA1, which has been implicated in chromatin condensation in vivo, we found that it was not sufficient to alter chromatin structure in vitro. In contrast, for HMGN5, which has been shown to unfold constitutive heterochromatin in vivo, we observed that it was sufficient to unfold chromatin fibers in vitro and that both its nucleosome binding domain and acidic C-terminal domain were necessary for impairing linker histone dependent chromatin folding. We also examined chromatin folding in a set of experiments where core histone tails were subjected to enzymatic conversion of arginine to citrulline by the PAD4 enzyme. These experiments revealed that citrullination of several residues of core histones was sufficient to impair linker histone dependent chromatin folding. In addition, we discovered that linker histone H5 was citrullinated by PAD4 but this citrullination was not required for chromatin unfolding. We argue that understanding the role of individual components of chromatin to the overall structure is important in dissecting the complex interactions relating chromatin structure to genetic and epigenetic regulation. Specific factors and interactions revealed in our studies may be targeted by DNA- and protein-modifying factors to alter chromatin structure and restore defects in gene expression associated with impaired cell differentiation and cancer.