Investigating the structure of chromatin in vitro: the roles of H4 K16 acetylation, linker histone H5, and the DNA template

• Main Author: Calestagne-Morelli, Alison
• Other Authors: Ausio, Juan
• Language: English; en
• Published: 2008
• Subjects: chromatin; acetylation; histone; gene activation; UVic Subject Index::Sciences and Engineering::Biology::Microbiology; UVic Subject Index::Sciences and Engineering::Chemistry::Biochemistry
• Online Access: http://hdl.handle.net/1828/946

It has been frequently postulated that genome or domain-wide histone post-translational modifications induce structural changes to chromatin. Until recently, however, experimental evidence for this hypothesis was lacking. H4 K16 acetylation is the first, and only, of all of the possible post-translational modifications to be directly linked to changes in chromatin conformation. I was interested in clarifying the mechanism by which H4 K16Ac exerts its modulatory effect. To characterize the role of this modification I reconstituted mononucleosomes with H4 K16Ac isolated from chicken erythrocytes by weak cation-exchange HPLC. Analytical ultracentrifuge (AUC) and MgCI2 solubility data suggested that H4 K16Ac structurally relaxes the association of nucleosomal DNA with the histone octamer by weakening intra-nucleosomal DNA-histone electrostatic interaction. Similar to early studies on H4 K16 acetylation, evidence suggests that the phosphorylation of linker histones promotes chromatin decondensation and increases DNA accessibility. In an effort to initiate a study characterizing the structural effects of chromatin fibers containing phosphorylated linker histories, I have optimized a method of purification for nonphosphorylated and monophosphorylated linker histone H5 from erythrocytes of anemic chickens. Preliminary AUC data of oligonucleosomes containing the nonphosphorylated control linker histones (manipulated to the same degree as the experimental monophosphorylated linker histones) showed a salt-dependent sedimentation trend that was consistent with expected values at low ionic concentrations. The purification method described is valuable as it results in a high yield of pure post¬translationally modified H5 product suitable for oligonucleosome reconstitutions. In the third component of this thesis, I describe the creation of a DNA template, Pbsn-208(10), which contains a Rattus norvegicus probasin promoter nucleosome positioning sequence flanked on both sides by 5 tandem repeats of the Lytechinus variegatus nucleosome positioning sequence. Unlike other DNA templates used in the study of chromatin compaction in vitro, the Pbsn-208(10) allows not only the reconstitution of homogenous chromatin fibers but also the differentiation of the middle of the fiber from its flanking ends. Thus, the effects of PTMs on histone-DNA interactions and nucleosome conformation in the center of a chromatin fiber can now be easily investigated. In addition, the Pbsn-208(10) template can help to identify the precise location of H1/H5 within the nucleosome.