TerryYamaguchi_CS037981_32ChIP_101624

Sp5 is a zinc-finger transcription factor that postively regulates canonical Wnt signaling and binds to key Wnt-signaling components such as TCF7. TCF7, also known as T-cell factor 1, is a transcription factor that binds to DNA via its high mobility group (HMG) domain. Importantly, TCF7 forms protein-protein complex with other cofactors, such as TLE, to regulate canonical Wnt signaling. TLE is a transcriptional-linked enhancer/repressor protein that binds TCF7 to repress canonical Wnt signaling. The goal of the project was to investigate the binding pattern of Sp5 in in vitro derived NMPs. Additionally, we investigated whether the binding patterns of TCF7 and TLE factors changed in the presence and absence of Sp5 and Sp8 in in vitro derived NMPs. Overall design: ES cells were differentiated in serum free N2B27 media and treated with bFGF (12ng/ml) from Day0 to Day2 followed by 3uM CHIR and 12ng/ml bFGF from Day2 to Day3. For dox-inducible Sp5 overexpression cells, 1ug/mL dox was added from Day2 to Day3. Cells were harvested and fixed in 1% formaldehyde solution for 15min, quenched in 0.125M glycine and flash-frozen. Nuclear extracts were incubated with anti-Flag, anti-TCF7, and anti-TLE antibodies (F3165 Sigma-Aldrich, C63D9 Cell Signaling,TLE(C-19):sc-13373 Santa Cruz, respectively) for chromatin immunoprecipitation followed by sequencing.