Super-resolution compatible DNA labeling technique reveals chromatin mobility and organization changes during differentiation

Abstract

Chromatin dynamics play a crucial role in cellular differentiation, yet tools for studying global chromatin mobility in living cells remain limited. Here, we developed a novel probe for the metabolic labeling of chromatin and tracking its mobility during neural differentiation. The labeling system utilizes a newly developed silicon rhodamine-conjugated deoxycytidine triphosphate (dCSiRTP). We show that this dCTP is efficiently delivered into living human induced pluripotent stem cells (iPSCs) and neural stem cells (NSCs) via a synthetic transporter (SNTT1). Using correlative confocal microscopy and stimulated emission depletion (STED) super-resolution microscopy, we quantified the sizes of labeled chromatin domains. Time lapse super-resolution microscopy combined with single particle tracking revealed that chromatin mobility decreases during the transition from iPSCs (pluripotent state) to NSCs and neurons (differentiated state). This reduction in mobility correlates with the differentiation state, suggesting a role for chromatin dynamics in cellular plasticity. Concomitant mechanistic insights obtained from MNase digestion assays, chromatin compaction and histone modification analyses revealed a decrease in chromatin accessibility during neuronal differentiation, indicating that chromatin adopts a more constrained and compacted structure. These findings provide new insights into chromatin regulation during neurogenesis.