Biochemical and biophysical studies of the regulation of chromatin folding and self-association by the N-terminal histone tails
Date of Issue2016-11-29
School of Biological Sciences
Nanyang Technological University
As the carrier of genomic DNA in eukaryotic cells, chromatin is one of the key factors involved in gene regulation. Many studies have revealed the general behavior of chromatin, whose basic feature is the assembly of nucleosome into array fibers. It has been identified that many factors, like cation environment, histone tails, post-translational modifications, linker histones and linker DNA, have great importance for chromatin higher order structure. Chromatin compaction is necessary for packaging of DNA in the nucleus and occurs by both intra-array folding and inter-array fiber-fiber association. However, little is known about the mechanism of inter- and intra-array interaction at the molecular level. A close contact between the surfaces of the nucleosomes, called nucleosome stacking, is a common structural feature of the condensed state of chromatin, been observed in almost all condensed chromatin. As one of the most significant post-translational modifications, acetylation of the N-terminal tail of histone H4 aa lysine 16 (H4K16) was proved to be involved in many cellular processes like transcription and DNA double strand break (DSB) repair. Several in vitro studies with reconstituted nucleosome arrays indicated that this modification disrupted nucleosome stacking leading to partial unfolding. In this thesis, we adopted a novel approach to investigate the importance of the H4 tail mediated nucleosome interaction on array folding and self-association (fiber-fiber interaction). We reconstituted nucleosome arrays using 12-601-177 DNA and various K16 mutated or modified Xenopus histone H4, including H4K16C, H4K16N, H4K16Q, H4K16R, H4K16me3, H4K16Ac and H4K16Q+CH2 (one thioether bond longer than H4K16Q), to investigate the mechanism of the H4K16 mediated chromatin folding and self-association using sedimentation velocity analytical ultracentrifugation (AUC) and precipitation assay measurements. Novel chemical approaches were employed to produce homogeneous and site-specifically modified H4K16 proteins. H4K16Ac was prepared by using a novel semi-synthetic approach we previously developed. H4K16 mutants and modifications were generated to reveal the possible factors that would affect the H4K16 functions. The importance of the H4K16 positive charge on the NCP-NCP interaction was investigated. Effect of side chain was also studied by comparing the modifications with conserved positive charge but bulkier side chain or varied length of side chain on H4K16. Our result showed the significance of positive charge of H4K16 in affecting nucleosome- nucleosome stacking and chromatin compaction. With the loss of positive charge, arrays became less compact compared to wild type array under high salt concentration. The length of side chain also displayed an important effect for maintaining the H4 tail mediated NCP-NCP interaction. Under the maximal folding condition, arrays with shorter side chain at H4K16 were found to be less compact. H4K16N, with a charge-removed and shorter side chain, failed to interact with surrounding residues of adjacent NCP and showed a similar disruptive effect on array folding as H4K16Ac. The detrimental effect on chromatin folding caused by H4K16Ac was proposed to be the consequence of cumulative effect of charge removal and bulky size of side chain. The interaction of the histone H4 N-terminal tail positive charge region (R17-R23) and the H2A acidic patch region is highly important interaction for NCP stacking. We investigated how charge changes of residues in these two regions affected array folding and self-association. Mutation constructs with positive charge removal on the H4 positive charge region include double mutations (R19L and K20L, RK-LL) and triple mutations (R17L, R19L and K20L, RRK-LLL). The mutant construct with charge neutralization of the residues in the H2A acidic patch was H2A-STT (D90S, E91T and E92T). It was found that the disruptive effect on stacking and folding caused by positive charge neutralization on the H4 N-terminal tail positive charge region (RK-LL and RRK-LLL) was similar to the effect caused by negative charge neutralization on the H2A acidic patch (H2A-STT). No cumulative effect on array unfolding was observed in array with charge neutralization on both these two regions (STT+RRK-LLL). In our study of nucleosome array self-association, the efficiency of salt-induced self-association behavior was generally in correlation with the change of charges of arrays. However, with no change of net charge, array reconstituted with H4 mutations (RRK-LLL) and H2A mutations (STT) required more Mg2+ to self-associate than wild type array, which was possibly due to the irrecoverable H2A-H4 interaction. Our result demonstrated the role of H2A-H4 interaction on chromatin self-association. Besides that, we also investigated how nucleosome arrays behaved under mixed-ion circumstances: (1) K+ and Mg2+, (2) Na+ and Mg2+. It was found that the addition of K+ or Na+ promoted the efficiency of Mg2+ induced self-association, while K+ and Na+ played opposite roles in Mg2+ induced chromatin folding.