Distinctive Klf4 mutants determine preference for DNA methylation status

Abstract

Reprogramming of mammalian genome methylation is critically important but poorly understood. Klf4, a transcription factor directing reprogramming, contains a DNA binding domain with three consecutive C2H2 zinc fingers. Klf4 recognizes CpG or TpG within a specific sequence. Mouse Klf4 DNA binding domain has roughly equal affinity for methylated CpG or TpG, and slightly lower affinity for unmodified CpG. The structural basis for this key preference is unclear, though the side chain of Glu446 is known to contact the methyl group of 5-methylcytosine (5mC) or thymine (5-methyluracil). We examined the role of Glu446 by mutagenesis. Substituting Glu446 with aspartate (E446D) resulted in preference for unmodified cytosine, due to decreased affinity for 5mC. In contrast, substituting Glu446 with proline (E446P) increased affinity for 5mC by two orders of magnitude. Structural analysis revealed hydrophobic interaction between the proline's aliphatic cyclic structure and the 5-methyl group of the pyrimidine (5mC or T). As in wild-type Klf4 (E446), the proline at position 446 does not interact directly with either the 5mC N4 nitrogen or the thymine O4 oxygen. In contrast, the unmethylated cytosine's exocyclic N4 amino group (NH2) and its ring carbon C5 atom hydrogen bond directly with the aspartate carboxylate of the E446D variant. Both of these interactions would provide a preference for cytosine over thymine, and the latter one could explain the E446D preference for unmethylated cytosine. Finally, we evaluated the ability of these Klf4 mutants to regulate transcription of methylated and unmethylated promoters in a luciferase reporter assay.

Structural Basis for the Versatile and Methylation-Dependent Binding of CTCF to DNA

“…In the structure of ZF3-7 in complex with DNA containing a 5mC at position 12, the methyl group is in a van der Waals cage surrounded by the side chain carbon Cδ atom of E362, the aromatic side chain of Y343, and the guanidine group of R339, which recognizes the 3′ Gua at position 13 (Figure 3I). The distinct effects of methylation at C 2 and C 12 on binding affinity are due to the difference in the amino acid (D452 or E362) used in the interaction, with Asp preferring to bind unmodified cytosine and Glu preferring methylated cytosine (Choo and Klug, 1997; Hashimoto et al, 2016; Liu et al, 2013) (Figure S3). This Glu preference for 5mC could also apply to methylated C 14 and E336 (Figure 2P).…”

Overlapping functions of Krüppel-like factor family members: targeting multiple transcription factors to maintain the naïve pluripotency of mouse embryonic stem cells

“…S6). Interestingly, these four residues cluster out of the third zincfinger domains when Klf4 binds to the target sequence with the first and second zinc fingers (Hashimoto et al, 2016), suggesting their function in the interaction with other protein(s) rather than the target DNA sequence, or with an additional target DNA sequence besides the one bound by the three zinc-finger domains of Klf4 (Schuetz et al, 2011). The specific function of the zinc-finger domain in Klf4 was shared by that of Drosophila luna, which shows the highest amino acid homology with conserved exon-intron organization in the Drosophila genome, suggesting that the pluripotency-associated function of the zinc-finger domain is a co-optional use of the evolutionarily conserved function as in the case of the evolution of Sox2 (Niwa et al, 2016).…”

Structural basis of human PR/SET domain 9 (PRDM9) allele C–specific recognition of its cognate DNA sequence

“…Thus, the RD dipeptide can accommodate changes from C:G (with G-Arg interaction) to G:C (with C-Asp interaction). We note that Asp can bind unmodified cytosine (21,26,31). The same adaptability could apply to ZF13, which contains the Arg-Asn (RN) dipeptide at positions Ϫ8 and Ϫ7 (shaded green in Fig.…”