Fig. 3: Rapid degradation of SOX2 via a knock-in degron confirms a role of SOX2 in both local and global histone acetylation.

a Diagram illustrating the construction of SOX2-dTAG gene with an in-frame FLAG-FKBP12^F36V degron knocked in at the SOX2 N-terminus. b, c WB analysis showing the levels of SOX2, SOX2-dTAG, histone acetylation, and EP300 in parental and SOX2-dTAG K450 cells without and with dTAG treatment for 3 h, 12 h, and 24 h. Quantification of histone acetylation levels were performed using Image J program. In b antibodies against SOX2, ACTIN were probed on the same gel. The samples originate from the same experiment, but FLAG, H3ac and H4ac were processed on separate gels in parallel. In c antibodies against SOX2, H3 were probed on the same gel. The samples originate from the same experiment, but FLAG, EP300, H3K27ac and H4ac were processed on separate gels in parallel. d Heatmap comparison of histone H4 acetylation ChIP-seq signals in SOX2-dTAG K450 cells treated without or with dTAG for 3 h and 24 h, ranked by acH4 intensities in SOX2-dTAG K450 cells without dTAG treatment. e, f UCSC genome browser snapshots of a representative KRT cluster containing KRT5 and KRT6A (e) and GOLT1B (f) with normalized signals of H4ac from SOX2-dTAG K450 cells treated without or with dTAG for 3 h and 24 h. g Profiles of H4ac ChIP-seq signals from SOX2-dTAG K450 cells treated without or with dTAG for 3 h and 24 h. h Profiles of H4ac ChIP-seq signals on SOX2 containing peaks from SOX2-dTAG K450 cells treated without or with dTAG for 3 h and 24 h. i Profiles of H4ac ChIP-seq signals on SOX2 absent peaks from SOX2-dTAG K450 cells treated without or with dTAG for 3 h and 24 h. j ChIP-qPCR analysis of the SOX2 binding on KRT6A and KRT5 genes in SOX2-dTAG K450 cells treated without or with dTAG for 3 h and 24 h. k ChIP-qPCR analysis of the EP300 binding on KRT6A and KRT5 genes in SOX2-dTAG K450 cells treated without or with dTAG for 3 h and 24 h. ChIP-qPCR data (j, k) are represented as mean +/- SD from 4 biological replicates, n = 4. P values were determined by two-sided unpaired t test. *, P < 0.05, ***, P < 0.001. In b, c experiments were repeated independently at least 2 times with similar results. Representative data from one experiment is shown. In d-i one ChIP-seq replicate was performed, and data are shown from two analytical replicates. Data in j, k are from three biological replicates.