Author: Nathaniel L. Burge

Publisher:

Published: 2022

Total Pages: 0

ISBN-13:

The eukaryotic genome is highly compacted by a complex of histone proteins and DNA to form chromatin. On the smallest level, the 4 core histones form a histone octamer complex wrapped ∼1.65 times with DNA to form a structure termed a nucleosome. A single strand of genomic DNA contains many nucleosomes, one roughly every 200 bp of DNA. An additional histone, histone H1, binds the outside of a nucleosome and compacts nucleosomes and chromatin into higher order structures. Compaction of chromatin by H1 and other factors play a critical function in regulating multiple fundamental processes in a cell including DNA repair, DNA replication, and transcription. We investigated the mechanisms H1 employs to regulate transcription factor binding using minimalistic in vitro experiments with nucleosomes and arrays of nucleosomes. One mechanism H1 utilizes to reduce transcription factor (TF) binding to a nucleosome is shifting the equilibrium of spontaneous nucleosome partial unwrapping in the nucleosome entry/exit region to the wrapped state while also increasing the extent of the wrapping. This further blocks a TF from accessing its binding site as the site is sterically blocked when the nucleosome is wrapped. We found a small 16 amino acid region of the C terminal domain (CTD) of H1 is responsible for altering the wrapping of a nucleosome and the reduction of TF binding to DNA. In addition, when a nucleosome partially unwraps and a TF binds, H1 remains bound to the nucleosome, but the CTD dissociates from the linker DNA where the TF binds. We also investigated the effect of relevant H1 post translational modifications (PTMs) on altering nucleosome wrapping and subsequent TF binding but found these PTMs have at most very modest effects on these processes, indicating these PTMs may act through other mechanisms. Our results support a model where the beginning of the H1 CTD is critical for altering nucleosome wrapping and compaction and warrants further investigation into differences between H1 isoforms on nucleosome wrapping, possible asymmetric effects on linker DNA arm binding and dynamics, and alternative mechanisms H1 PTMs employ to alter transcription. Intrinsic or extrinsic factors that alter H1 binding to chromatin may also play a role in regulating transcription. Some histone chaperone proteins can bind H1 and alter its binding to chromatin, positioning them as potential regulators of transcription. We tested the effect of the histone chaperones sNASP and Nap1 on altering H1 binding, wrapping of nucleosomes, and TF binding to nucleosomes. sNASP and Nap1 altered H1 binding to nucleosomes, but did so with a roughly 500 fold difference between them despite having similar binding affinities for free H1. The chaperones also reduced the effect H1 has on nucleosome wrapping. Since H1 induced nucleosome wrapping is altered by these chaperones, we would expect TF binding to be altered in their presence, however we found the chaperones altered TF binding to nucleosomes and DNA themselves complicating our measurements. The results point to future investigations into the effect of these histone chaperones on TF binding themselves through possible nonspecific binding due to their charged properties and whether these chaperones can bind H1 that is bound to a nucleosome. We further explored the effect Nap1 has on the binding kinetics of H1 to nucleosomes and nucleosome arrays. Interestingly, we measured two binding rates of H1 to both nucleosomes and arrays. In the presence of Nap1, both binding rates of H1 to nucleosomes and arrays were greatly reduced. In addition, we monitored exchange of H1 between nucleosomes and arrays. H1 bound to nucleosomes exchanged after 10 minutes at most while H1 bound to arrays exchanged slowly over hours. Nap1 greatly increased the exchange rate of H1 with arrays while having little effect on nucleosomes. Our experiments, particularly with nucleosomes, should be repeated to ensure measured differences between nucleosomes and arrays are repeatable. Future experiments may focus on different salt concentrations and H1 isoforms to distinguish between the two measured rates. Our results suggest that Nap1 may be responsible for altering H1 binding dynamics to chromatin, positioning it as a potential regulator of transcription via this mechanism.