Regulation of the Heterochromatin Protein 1 by Phosphorylation of Histone H3 and the HP1 Hinge Domain

Chromatin, a polymer formed from DNA, histones, and associated proteins, is the physiological form of genetic information in all eukaryotic cells. Posttranslational modification of histones, such as acetylation, methylation, and phosphorylation, regulates various DNA-dependent processes, ranging from transcription to replication, DNA repair, and apoptosis. A key mechanism by which histone modifications exert these effects is by recruitment of specific binding partners (effector proteins), that in turn direct downstream functions. Insight into the underlying mechanisms are of great importance for a full understanding of chromatin structure and function. One of these effector proteins, Heterochromatin Protein 1 (HP1), plays important roles in heterochromatin formation. It is recruited to chromatin by interaction with methylated lysine 9 of histone H3 (H3K9me). However, it has remained enigmatic how HP1 reversibly dissociates from chromatin during mitosis, while the histone mark that recruits the protein, H3K9me, persists. In the first part of my thesis, my collaborators and I show through a combination of in vitro and in vivo experiments that this release depends on a novel mechanism, “methyl-phos switchingâ€, in which two nearby histone marks collaborate to accomplish the dynamic regulation of effector protein binding. Phosphorylation of histone H3 at serine 10, immediately adjacent to HP1’s binding site at H3K9me, at the onset of mitosis interferes with HP1 binding to H3K9me, resulting in the release of the effector protein. In the second part of my thesis, I investigate to what extent posttranslational modification of HP1 itself is involved in the regulation of the effector protein. I identify ten novel phosphorylation sites for the three human HP1 isoforms (α, β, γ), most of which map to the HP1 “hinge region†and are specifically phosphorylated in mitosis. For one highly conserved site, HP1α serine 92 phosphorylation, I identify Aurora B as the responsible kinase in vivo. In vitro data suggest that ...