(A-33) Heterochromatin Protein 1 (HP1) dimer mechanics: Potential implications for genome regulation

Introduction:: Mechanical force is transmitted to the nucleus via cytoskeleton and the LINC complex. Active and passive response of the nucleus and chromatin to mechanical force influences gene transcription by changing the accessibility of mechanosensitive transcription factors or directly altering chromatin and DNA interactions. Heterochromatin Protein 1 (HP1), a chromosomal protein, plays an important role in nuclear and chromatin mechanical maintenance. HP1 is accumulated into heterochromatin and compacts chromatin by bridging the chromatin fibers, contributing to the mechanical rigidity of the nucleus. Additionally, HP1 has been shown to further recruit heterochromatin-related proteins and facilitate liquid-liquid phase separation in vitro, which may also be a key contributor to nuclear stiffness. The chromatin crosslinking and phase separation by HP1 are influenced by the HP1 dimerization via its chromoshadow domain (CSD). The CSD-CSD dimerization of HP1 is mediated by α-helix regions of each CSD (see Figure 1). The strength of the HP1 dimerization varies phase separation states of HP1 and chromatin condensation levels in silico. The mechanical properties of CSD-CSD α-helix-mediated interactions in the HP1 dimer can, therefore, help understand molecular and chromatin assembles and mechanical properties of the chromatin and nucleus. However, how HP1 dimerization strength changes under a mechanical environment and how HP1 interactions achieved via CSD-CSD respond to mechanical force remain unclear. To reveal the detailed molecular mechanistic roles of HP1, we employed steered molecular dynamics simulations to analyze the mechanical behavior and strength of CSD-CSD α-helix-mediated interactions under the force-applied condition.

Materials and Methods:: GROMACS 2019.6 software package was used for the atomic MD simulation and corresponding analyses. The crystal structure of the CSD-CSD of the HP1 dimer is available on the protein database (PDB ID: 3I3C). CHARMM36 was applied to the system as the force field. The CHARMM-modified TIP3P water model was employed to solvate all the systems. The sodium and chloride counter ions were added to the system, and all the simulations were performed at the physiological condition, the 150 mM concentration of NaCl. After energy minimization (50000 steps), NVT and NPT equilibration (5 ns for each), and relaxation (200 ns), pulling force at a constant velocity, 0.5 Å/ns, was applied to one of the CSD for the force-applied condition for 20 ns. CSD-CSD binding via α-helix disrupted within the 20 ns simulation time. The no-force condition was defined as the further 20 ns relaxation after the first 200 ns relaxation. Hydrogen bonds were counted when the distance between possible acceptor and donor pairs was within 0.35 nm and the angle between the hydrogen, oxygen, and nitrogen was below 30°. The short-range electrostatic interactions were calculated by the modified Particle Mesh Ewald (PME). The salt bridge interaction ratios were calculated by the number of time steps, where the oppositely charged atoms were within 4.0 Å, divided by the whole time steps.

Results, Conclusions, and Discussions:: The molecular dynamic simulation trajectory data of the CSD-CSD dimer subjected to mechanical force before breaking its bonds were compared to those under the no-force condition. The root-mean-square-fluctuation (RMSF) of the interactive α-helix of the CSD-CSD in the force-applied condition showed higher fluctuations in the amide (Asn and Gln) group and charged residues such as Lys, Arg, and Glu. The average number of hydrogen bonds in the α-helix was similar between no-force and force-applied conditions. On the other hand, the average electrostatic energies of the α-helix interaction in the force-applied condition were significantly higher than in the no-force condition having around the 20 kJ/mol difference. From these results, we hypothesized that Lys and Glu residues, which showed high fluctuations in the RMSF and opposite charges, made salt bridge interactions. Interestingly, the salt bridge interaction ratios in the force-applied condition were significantly greater than in the no-force condition. These results indicate that Lys-Glu salt bridge interactions induce the strong binding of the HP1 dimer responding to mechanical force. A recent computational study examined phase separation developments of chromatin and HP1 dimers by changing HP1 dimer interaction energy from 2kBT to 4kBT, 6kBT, and 10kBT (about 5.15, 10.3, 15.5, and 25.8 kJ/mol at the body temperature 310 K) and observed four phases; mixed no-separation, liquid-like droplets, droplets with absorbed polymer, and coated polymer regimes. Our results of electrostatic interaction changes due to the Lys-Glu salt bridge caused by the force application in CSD-CSD were in the range, which can alter the phase separation states, thus, may suggest HP1 forms mechano-sensitive phase separation. To the best of our knowledge, this is the first insight into the detailed molecular behaviors of HP1 against mechanical force, shedding light on the potential mechano-sensitive HP1 phase separation. The molecular mechanics of the CSD-CSD dimer of HP1 shown in this study may define the mechanical foundation of HP1 dimerization, liquid-liquid phase separation, chromatin condensation and interactions, and ultimately mechano-genome regulation.

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