ZBTB7A regulates primed-to-naive transition of pluripotent stem cells via recognition of the PNT-associated sequence by zinc fingers 1-2 and recognition of gamma-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 four ZFs (ZF1-4) are involved in binding to ?-globin -200 gene element to repress fetal haemoglobin expression. Recently, it has been reported that ZBTB7A drives primed-to-naive 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 ?-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 epiblast stem cells to the naive pluripotent state in vivo. Collectively, our studies demonstrate that ZBTB7A mainly employs its ZF1-2 to recognize the PNT-associated sequence but recognizes ?-globin -200 gene element via ZF1-4, providing insights into the molecular mechanism for the diversity of ZBTB7A's genomic localization.