Eukaryotic DNA is organized into a structural polymer called chromatin which ultimately controls important DNA processing functions such as transcription, DNA repair and DNA replication. The fundamental unit of chromatin is the nucleosome which is made up of about 146 base pairs wrapped around a histone core. The histone core contains 2 copies each of the histones H2A, H2B, H3 and H4. Long strings of nucleosomes compact into higher order structures which are not well known, but play a pivotal role in DNA accessibility. There are many factors that affect higher order structure and compaction of chromatin including inter and intra nucleosome interactions, incorporation of linker histones (H1), and post translational modifications. This dissertation includes a detailed study of some of these mechanisms.
The first study looks at the H3 N-terminal tail which is long, unstructured and heavily modified in vivo . Using Electron Paramagnetic Resonance and site directed spin labeling; we were able to observe the dynamics of the H3 tail within compacted 17-mer nucleosome arrays. We find that these tails maintain their mobility as the arrays compact and self-associate despite previous studies that suggested these tails make inter- and intra-nucleosome contacts during compaction. We conclude that these contacts are transient and permit the tails to maintain mobility and accessibility.
The second study looks at how H1 affects transcription. Using Fluorescence Resonance Energy Transfer and Protein Induce Fluorescence Enhancement, we observed the binding of H1 to the nucleosome, while also being able to detect the binding of a transcription factor (TF) within the entry-exit region of the nucleosome. We find that H1 represses TF binding by a factor of three for unmodified nucleosomes. These results indicate that H1 does not block butonly suppresses DNA accessability and that H1 exchange is not the only process by which DNA can be accessed. We also observed H1 interacting with the linker DNA, and we found that the H1 remains bound to the linker DNA even as the DNA unwraps from the nucleosome to allow the TF to bind. This result is contrary to the widely held belief that H1 dissociates completely from DNA in transcriptionally active regions.
We also studied how histone fold PTMs impact H1 repression of TF binding. We find that the H3(K56ac), located in the entry-exit region of the nucleosome, extinguishes H1 repression of TF binding while H3(K122ac), located at the dyad, does not reduce H1 repression of TF binding. It appears that H3(K56ac) defines regions of chromatin that can not be regulated by H1.
The third study was a mechanical engineering study that uses the well-studied transcription factor, LexA, to create a functional protein-containing DNA Origami hinge. We attached a synthetic oligonucleotide to the LexA dimerization domain which was designed to anneal to one of the hinge staples. A LexA recognition sequence was folded into the other side of the hinge. In this context, when LexA binds to its recognition sequence it acts as a latch. While some hinges were observed with LexA bound, further studies are being conducted to improve this system.