INVESTIGATING THE ROLE AND REGULATION OF H3K36 METHYLATION IN NEUROECTODERMAL LINEAGE COMMITMENT OF MOUSE EMBRYONIC STEM CELLS

Date

2023-8-28

Author

Sezginmert, Dersu

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Embryonic development is a complex process orchestrated by spatiotemporally regulated factors. Embryonic stem cells (ESCs) serve as invaluable in vitro models for studying these developmental dynamics. Epigenetic mechanisms, crucial in this regulation, guide pluripotent ESCs towards specific lineages. A deeper understanding of these mechanisms is essential for addressing developmental disorders. A previously conducted mass spectrometry analysis revealed an elevation in H3K36me2 levels during neuroectoderm differentiation, while such an increase was absent in endoderm differentiation. Our preliminary data hints a distinctive role for H3K36 methylation in neuroectoderm differentiation. After these findings were validated by Western blot, expression changes in H3K36 methylation regulatory system, including methyltransferases, demethylases, and readers, were analyzed. RT-qPCR analyses of neuroectoderm, complemented with existing RNA-seq data for endoderm, indicated a central role for H3K36 methylation in pluripotency exit and/or differentiation. Notably, Nsd2 and Zmynd11 expression levels were upregulated during differentiation into both lineages, whereas Kdm4c and Msh6 were found to be expressed higher in mESCs compared to differentiated states. Conversely, lineage-specific expression patterns were noted for Nsd1 and Setd5. Finally, through a CRISPR knock out screening done to identify the critical epigenetic factors for neuroectoderm differentiation, we revealed that Nsd1, Setd5 and Kdm2b are critical for neuroectoderm differentiation. These findings highlight the importance of H3K36me2 regulation for lineage choice and exit from pluripotency, with regulatory proteins showing stage- or lineage-specific expression changes. Further work is needed to uncover the precise mechanisms by which this modification and its associated proteins influence neuroectoderm differentiation.

Subject Keywords

Epigenetics, H3K36 Methylation, Mouse Embryonic Stem Cells, Neuroectoderm Differentiaiton

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https://hdl.handle.net/11511/105301

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Graduate School of Natural and Applied Sciences, Thesis