title: Phase Separation of the Methyl-CpG-Binding Domain (MBD) Protein Family Underlies Heterochromatin Organization
Creators: name: Zhang, Hui
Publisher: name: UNSPECIFIED
Year: 2022

description: In the last decade, phase separation has been proposed as a mechanism for the formation of a variety of subcellular compartments such as chromatin compartmentalization in eukaryotic cell nuclei. Yet, the requirements for chromatin compartmentalization have not been well characterized and several overlapping factors have been implied. This study aimed to gain insight into the function and underlying mechanisms of the MBD family proteins (MBDs) on chromatin organization. Firstly, I characterized the phase separation properties, functions, and dynamics of the MBDs in vitro and in vivo. I found that the MBDs have different phase separation properties and functions in heterochromatin organization. In vitro, MBD1∆CxxC3 formed only irregular aggregates, while MBD2, MBD3, MBD4, and MeCP2 formed liquid-like spherical droplets in certain conditions. In vivo, MBD1∆CxxC3 and MBD3 did not influence heterochromatin compartment size, while MBD1, MBD2, MBD4, and MeCP2 promoted the heterochromatin clustering in a dose-dependent manner. Besides, MBD1, MBD1∆CxxC3, and MBD4 were shown to decrease the roundness of heterochromatin compartments, while MBD2, MBD3, and MeCP2 did not. I also detected the molecule exchange dynamics of MBDs in between heterochromatin compartments and surrounding environments and found that all MBDs are highly dynamic. In conclusion, the LLPS properties were: MeCP2 &gt; MBD2 &gt; MBD3 &gt; MBD4 ≥ MBD1∆CxxC3. The functions in heterochromatin compartment size were: MeCP2 &gt; MBD4 &gt; MBD2 &gt; MBD1∆CxxC3 ≈ MBD3. The functions in heterochromatin compartment roundness were: (MBD3 &gt;) MeCP2 &gt; MBD2 &gt; MBD4 &gt; MBD1∆CxxC3. The protein mobility was: MeCP2 &lt; MBD2 &lt; MBD1∆CxxC3 &lt; MBD4. The immobile fractions were: MeCP2 ≈ MBD2 &gt; MBD4 ≈ MBD1∆CxxC3. Thus, the heterochromatin organization could be regulated by the MBDs mediated phase separation. MBD2 and MeCP2 show the highest liquid-liquid phase separation (LLPS) properties in vitro and more significantly influence the heterochromatin compartment structure. Secondly, I elucidated the functional difference of MBD2 isoforms in vivo and in vitro. MBD2 has three isoforms, MBD2a, MBD2b, and MBD2c. MBD2a is the longest isoform and contains N-terminus, MBD-TRD, and the C-terminus, while MBD2b and MBD2c contain the conserved MBD-TRD, but lack either the N-Terminus (MBD2b) or the C-Terminus (MBD2c). All MBD2 isoforms showed the ability of phase separation with distinct properties. MBD2b exhibited overall similar but weaker phase separation properties than the full-length MBD2a. MBD2b was less enriched in heterochromatin compartments and promoted the heterochromatin compartment clustering to a lesser extent than MBD2a. MBD2b showed a faster molecule exchange between heterochromatin compartments and surrounding environments. Compared to MBD2a, MBD2c showed similar molecule dynamics and enrichment in the heterochromatin compartments but did not influence the heterochromatin compartment size. In vitro, the purified MBD2c formed only irregular aggregates. Besides, the N-terminus formed only irregular aggregates in all conditions tested and showed the ability to recruit DNA. The MBD-TRD alone did not form any condensates but formed irregular aggregates in the presence of DNA. The C-terminus could form either irregular aggregates or spherical droplets depending on buffer conditions that are not influenced by DNA. Thus, polymer-polymer phase separation (PPPS) also plays a role in MBD2 function, especially for MBD2c. Consistently, high levels of MBD2-MBD-TRD and MBD2c did not influence the total occupancy of heterochromatin while the high levels of MBD2b promoted the spreading of the heterochromatin region. In conclusion, the N-terminus is responsible for protein-DNA interaction, the MBD-TRD is responsible for PPPS and heterochromatin localization, and the C-terminus is responsible for the LLPS. The three isoforms of MBD2 exhibit distinct functional properties due to a combination of the three regions. Thirdly, I demonstrated that MeCP2 alone could form liquid-like spherical droplets via self-interaction-induced LLPS. The fold enrichment of MeCP2 in the droplets in vitro and heterochromatin compartments in vivo was similar. MeCP2 LLPS could be promoted by various factors that somehow mimic the in vivo physiological conditions. DNA promoted the LLPS of MeCP2 in both DNA length and concentration-dependent manner by providing additional sites for multivalent weak protein-DNA interactions. Crowders promoted the MeCP2 LLPS by increasing the local protein concentration and enhancing the weak interactions via the “excluded volume” effect. The weak MeCP2-MeCP2 and MeCP2-DNA interactions were characterized by the emergence of a properly confined diffusion population in the droplets and heterochromatin compartments. Cytosine methylation restricted the size of MeCP2 condensates in vivo and in vitro with more confined overall mobility. The specific mC-DNA interaction contributed to the emergence of the static population with low to static diffusion in MeCP2 condensates in vivo and in vitro. The phase separation properties of MeCP2 were altered by the RTT-related nonsense mutations. In vivo, the MeCP2 promoted the heterochromatin compartment clustering, which was attenuated by RTT-related nonsense mutations. The phase separation properties and function of RTT-related nonsense mutations in heterochromatin compartment clustering are negatively correlated with the severity in RTT patients. RTT-related nonsense mutations influence the heterochromatin organization via impaired LLPS properties. These results provide new insights into the function and underlying mechanism of the MBD protein family in heterochromatin organization and thus contribute to our general understanding of phase separation in nuclei architecture.