Visualization of H3K9me3 in Embryoid Bodies Using Genetically Encoded Fluorescent Sensor MPP8-Green

Abstract

Objective: Histone modifications play a crucial role in shaping the epigenetic landscape of chromatin, influencing its structure and function. These histone modifications change throughout the cell cycle and during processes such as morphogenesis, differentiation, stress adaptation, and aging. Among these, histone H3 Lys9 trimethylation (H3K9me3) plays a critical role in gene silencing and cellular identity. Despite the importance of histone modifications in regulating gene expression, the dynamic visualization of these modifications in live cells remains a significant challenge. Here, we aim to develop a method to track H3K9me3 changes of induced pluripotent stem cells (iPSCs) during spontaneous differentiation into embryoid bodies (EBs) with the genetically encoded fluorescent sensor. Methods: We created a stable iPSC line, KUIFMSi004-A-1, expressing the MPP8-Green fluorescent sensor, which binds H3K9me3. This sensor combines two copies of the natural M-phase phosphoprotein 8 (MPP8) reader domain with the green fluorescent protein mNeonGreen. The iPSC line was induced to form EBs, and the distribution of H3K9me3 was monitored using live-cell fluorescence microscopy. Results and Discussion: The expression of the MPP8-Green sensor allowed real-time visualization of H3K9me3 modifications during spontaneous differentiation of iPSCs into EBs. We observed two distinct groups of cells with different patterns of H3K9me3 distribution: one with characteristic chromatin “dots” and another with diffuse sensor distribution. The sensor enabled tracking of epigenetic landscape changes during differentiation, providing insights into the dynamics of H3K9me3 during early embryoid body formation. Conclusions: Our study demonstrates the potential of using the MPP8-Green sensor to monitor the dynamic changes of H3K9me3 during iPSC differentiation. This method offers a novel approach for studying the temporal and spatial regulation of histone modifications in live cells, advancing our understanding of epigenetic regulation during development.