UC San Diego

Gene regulation is an intrinsic feature of the cell cycle, where Heterochromatin Protein 1alpha (HP1alpha) is a key component. It has been proposed that HP1alpha helps form and maintain heterochromatin regions via its biomolecular condensate properties. The partitioning of HP1alpha between cellular condensates and the nucleoplasm is highly dynamic similar to the behavior of HP1alpha during liquid-liquid phase separation (LLPS). HP1alpha’s ability to undergo phase separation is due to its multivalent interaction capacity enabled by the structural features of HP1alpha. For Aim 1 of the thesis research, the LLPS of HP1alpha was investigated in the presence of 205 bp DNA and four peptides from Shugoshin-1 (Sgo1), Chromatin Assembly factor-1 (CAF-1), Lamin B Receptor (LBR), and histone protein H3 (H3). For Aim 2 of the thesis research, the LLPS of HP1alpha was investigated in the presence of longer protein binding partner (PBP) constructs from Sgo1, LBR, and p150 subunit of CAF-1 (CAF1-p150). In general, the positively charged peptides and PBP constructs enhanced the LLPS of HP1alpha, and the negatively charged peptides and PBP constructs disrupted the LLPS of HP1alpha. In addition, binding partners (DNA, LBR, and Sgo1) could compensate for missing properties of HP1alpha, such as the phosphorylation of the NTE and the hinge region to promote the LLPS of HP1alpha. Under LLPS conditions, it was demonstrated that the peptides and PBP constructs can specifically (via the PVXVL motif interaction for HP1alpha) and non-specifically (via non-specific electrostatic interactions) regulate the partitioning behavior of different components between the liquid droplet phase and the bulk aqueous environment. Overall, the findings contributed to the understanding of the multivalent interaction network during the LLPS of HP1alpha and further demonstrated the ability of binding partners to tune the LLPS properties of HP1alpha. This study reveals potential specific and non-specific regulating mechanisms for the partitioning of HP1alpha and its binding partners between two distinct phases that is potentially an important mechanism for gene regulation.