Comparison Of The Binding Ability Of M1 And M2 Bind To CTCF And Identification Of Critical Base Pairs Required Motif M1 And M2

CCCTC-binding factor(CTCF)is a ubiquitously expressed and highly conserved 11 zinc-finger protein.CTCF binds throughout the genome via combinatorial subsets of its 11 zinc fingers and plays multiple functions in the genome.Early studies have distinguished two different CTCF binding motifs M1 and M2.However,it is unclear in binding affinity of CTCF to both M1 and M2.In this study,we generated GST-CTCF-ZFs construct,induced and purified fusion protein.We compared the different binding ability of CTCF-ZFs to both M1 and M2 using EMSA and ChIP-qPCR assays.We further designed a series of mutations of Mland M2 to determine the positions in CTCF binding site which are required for both M1 and M2 to bind CTCF.The results showed that:1.The binding CTCF-ZFs to M1 is much stronger than M2 both in vitro and in vivo:M1 could bind to 0.2 ?g in vitro purified GST-CTCF-ZFs and lead to a supershift band,but supershift band gradually became stronger with the increased amount of GST-CTCF-ZFs from 0.6 ?g to 2.5 ?g;M1 demonstrated a Kd of 1.0 × 10-11 M contrasting with Kd values of 2.9 × 10-10 M for CTCF-M2 interactions;competition analysis suggested that the binding of CTCF-ZFs to M2 is easily abolished by M1,but not vice versa.2."TGG"(three base pairs in M1)might be required for M1 binding to CTCF:replacement of the"TGG" with "GTT" in M1 resulted in a drastic loss of the DNA binding ability of CTCF-ZFs,mutated one nucleotide or a few nucleotides within or around the "TGG" did not abolish the binding shift of CTCF to Ml.3.Every single base pair of M2 might required for M1 binding to CTCF:mutation of 1 bp in three selected positions resulted in loss of binding between CTCF and M2.