(357b) Monitoring Constitutive Heterochromatin Reorganization during Dopaminergic-like Neuron Differentiation

Establishment of heterochromatin and remodeling of DNA and histone modifications are essential for lineage commitment and neuronal development. Neuronal differentiation also results in heterogeneous population including neurons with different maturity and supporting cells such as glia and astrocyte. However, whether and how epigenetic modifications drives heterogeneous heterochromatin establishment and further induces changes in gene expression profile during differentiation remains elusive. In this study, we utilized SH-SY5Y, a neural precursor like cell as our model system and established a stable SH-SY5Y cell line expressing both CpG^me and H3K9me3 probe. SH-SY5Y was differentiated into dopaminergic-like neurons induced by retinoic acid. We monitored global changes of CpG^me and H3K9me3 simultaneously during differentiation, and observed shrinkage of nucleus and increase of puncta number and size of both CpG^me and H3K9me3. CpG^me and H3K9me3 rich foci showed partial colocalization under diffraction limit confocal microscopy as expected. We then used fluorescence lifetime imaging microscopy (FLIM) to probe interaction between CpG^me and H3K9me3 FRET probe with higher spatial resolution. Our single cell tracing experiment showed decrease of fluorescence lifetime of CpG^me probe during differentiation, suggesting formation of more compact heterochromatin. In summary, we developed novel FRET probes to track heterochromatin organization with 10 nm resolution and observed increased degree of interaction between constative chromatin marker CpG^me and H3K9me3 during neuronal differentiation.