ZBTB7A regulates primed-to-naïve transition of pluripotent stem cells via recognition of the PNT-associated sequence by Zinc Fingers 1-2 and recognition of γ-globin -200 gene element by Zinc Fingers 1-4.

ZBTB7A, a transcription factor containing a tandem array of four Cys2-His2 zinc fingers (ZFs), is vital for multiple physiological events through directional binding to different genomic loci. Our previously determined crystal structure of ZBTB7A in complex with a GCCCCTTCCCC sequence revealed that all the four ZFs (ZF1-4) are involved in binding to γ-globin -200 gene element to repress fetal hemoglobin expression. Recently, it has been reported that ZBTB7A drives primed-to-naïve transition (PNT) of pluripotent stem cells through binding to a 12-bp consensus sequence ([AAGGACCCAGAT], referred to as PNT associated sequence). Here, we report a crystal structure of ZBTB7A ZF1-3 in complex with the PNT associated sequence. The structure shows that ZF1 and ZF2 primarily contribute to recognizing the GACCC core sequence mimicking the half part (GCCCC) of γ-globin -200 gene element via specific hydrogen bonding and van der Waals contacts. The mutations of key residues in ZF1-2 remarkably reduce their binding affinities for the PNT associated sequence in vitro, and cannot restore EpiSCs to the naïve pluripotent state in vivo. Collectively, our studies demonstrate that ZBTB7A mainly employs its ZF1-2 to recognize the PNT associated sequence but recognizes γ-globin -200 gene element via ZF1-4, providing insights into the molecular mechanism for the diversity of ZBTB7A's genomic localization.