Synthesis and characterization of linker histone acetylation

Abstract

Chromatin is a complex formed by DNA and proteins whose main function is to compact long DNA molecules into more compact, denser structures. The basic unit of chromatin is the nucleosome. The linker histone is one of the nucleosome binding partners that are important components of chromatin in eukaryotic cells. The major function of the linker histone is to bind with the nucleosome as well as DNA to remodel the chromatin structure so that regulates genetic metabolism. Over the last few decades, several attempts have been made to reveal that the linker histone interacts with the nucleosome particles and is condensed into a higher-order chromatin structure till the Cryo-EM structure is released. Characterizing post- translational modifications (PTMs) is one of the most common ways to examine a protein. However, the attempt to characterize the linker histone PTMs by the structural biology method is quite difficult. Therefore, we focused on investigating the linker histone PTMs by biochemical/biophysical approaches. In this thesis, we performed systematic research on the acetylation of the linker histone H1.4, one of the most important human somatic linker histone variants. Compared with other variants, H1.4 shows a higher affinity to nucleosomes and a higher ability to pack the chromatin structure which represents to be a good research candidate. And lysine acetylation is thought to be the gene transcription activator whose working mechanism is worthwhile to be investigated. The chemical biology approaches were applied to synthesize the H1.4 acetylation analogs and the in-vitro characterizations were conducted by fluorescence-based and ITC-based assays. We found that the globular/C-terminal domain acetylation plays an important role in regulating H1 dynamics in binding with the nucleosome. More importantly, the results of the in-cell fluorescence recovery after photobleaching and immunofluorescence assays agree well with the in-vitro experimental results.