In Vivo Single-molecule Tracking of CHD Family Chromatin Remodeler Hrp3 Defines the Chromatin Context-dependent Binding Dynamics in S. Pombe

ATP-dependent chromatin remodelers of the CHD family regulate genome organisation and transcription, yet their dynamic behaviour in native chromatin remains unclear. Here, we performed in vivo single-molecule tracking of the CHD remodeler Hrp3 in live Schizosaccharomyces pombe cells to quantify its chromatin interaction dynamics. By generating domain-deletion mutants (Δchromo, ATPaseK406A (ATPase-dead), Δcoupling region, DNA binding domains (ΔSANT, ΔSLIDE), and ΔDUF), we systematically dissected the contribution of each domain to DNA binding in vivo. While some observations align with previous in vitro studies, key differences highlight the critical influence of the cellular chromatin environment on remodeler function. We find that Hrp3 exhibits specific binding only with constitutive heterochromatin, but not with euchromatin or facultative heterochromatin. We tested the effect of altered histone acetylation and methylation on the chromatin binding dynamics of Hrp3. Our data demonstrated the chromatin context-dependent binding of Hrp3: reduced acetylation allows binding of Hrp3 with euchromatin, increased acetylation reduces binding of Hrp3 with heterochromatin, and reduced methylation increases the binding of Hrp3 with heterochromatin. Interestingly, we found specific binding of Hrp3 with mitotic chromosomes, suggesting its role in maintaining heterochromatin silencing during mitosis and probably contributing to epigenetic memory. Collectively, our results demonstrate that Hrp3 chromatin binding is highly sensitive to epigenetic modifications and chromatin compaction and cannot be fully predicted from in vitro studies alone. Given the conservation of CHD remodelers and their links to human diseases, this study provides important insights into how epigenetic therapies, such as HDAC inhibitors, may modulate chromatin remodeler dynamics and influence gene expression.