Abstract 1497 Structural studies of EPOP-EloB-EloC interaction

Embryonic stem cells (ESCs) are a unique cell line that retains pluripotency.Once the ESCs are triggered by certain chemical processes, they start to undergo differentiation, which ultimately leads to the development of three embryonic germ lines in a developing embryo.Mitogen-activated protein kinase (MAPK) cascades encompass different signaling pathways and play a critical role in embryonic development.MAPK signaling has been shown to play a role in promoting the differentiation of ESCs and regulating the pluripotency of stem cells.However, at the transcriptional level, transcription factor Vezf1 has been shown to affect the MAPK signaling pathway.Our results showed that in Vezf1 knockout murine embryonic stem cells (mESCs), MAPK signaling is downregulated thus maintaining the pluripotency state of ESCs.Wide genomic analysis of knockout Vezf1 mESCs revealed that the transcription factor CTCF may be a pivotal factor in the regulation of MAPK signaling alongside some genes such as MAP7 and IRS-1 due to its opportunistic promoter binding.To test if CTCF plays a role in the downregulation of MAPK signaling and pluripotency, Vezf1 knockout mESCs were transiently transfected with a CTCF shRNA using lipofectamine, and different assays like western blotting and rt-q-PCR were performed to test the pluripotency state and MAPK signaling.Results showed that upon CTCF knocked down mESCs, MAPK signaling was upregulated, specifically IRS-1 as it is tied to MAPK signaling.Given that CTCF is absent, further analysis can be done on other MAPK signaling genes to test their expression levels and role in the development process.