Denys-Drash syndrome associated WT1 glutamine 369 mutants have altered sequence-preferences and altered responses to epigenetic modifications

Abstract

Mutations in human zinc-finger transcription factor WT1 result in abnormal development of the kidneys and genitalia and an array of pediatric problems including nephropathy, blastoma, gonadal dysgenesis and genital discordance. Several overlapping phenotypes are associated with WT1 mutations, including Wilms tumors, Denys-Drash syndrome (DDS), Frasier syndrome (FS) and WAGR syndrome (Wilms tumor, aniridia, genitourinary malformations, and mental retardation). These conditions vary in severity from individual to individual; they can be fatal in early childhood, or relatively benign into adulthood. DDS mutations cluster predominantly in zinc fingers (ZF) 2 and 3 at the C-terminus of WT1, which together with ZF4 determine the sequence-specificity of DNA binding. We examined three DDS associated mutations in ZF2 of human WT1 where the normal glutamine at position 369 is replaced by arginine (Q369R), lysine (Q369K) or histidine (Q369H). These mutations alter the sequence-specificity of ZF2, we find, changing its affinity for certain bases and certain epigenetic forms of cytosine. X-ray crystallography of the DNA binding domains of normal WT1, Q369R and Q369H in complex with preferred sequences revealed the molecular interactions responsible for these affinity changes. DDS is inherited in an autosomal dominant fashion, implying a gain of function by mutant WT1 proteins. This gain, we speculate, might derive from the ability of the mutant proteins to sequester WT1 into unproductive oligomers, or to erroneously bind to variant target sequences.

Figure 1.

Normal WT1 prefers adenine and the three Q369 mutants prefer guanine in the 3′ triplet. (A) The consensus DNA binding sequence of WT1, adopted from (28). The pie-diagrams show the distribution of the base in the middle position of the 3′ triplet (GXG) from the ChIP-chip (28) and ChIP-seq (40). (B–E) Binding affinities of normal WT1 and three Q369 mutants (in the –KTS isoform) with oligos containing various base pairs in the middle position of the 3′ recognition triplet (position ‘X’ in panel a). (H–K) Binding affinities of normal WT1 and three Q369 mutants in the +KTS isoform with various oligos. (F) Binding affinities of the equivalent three-finger Egr1, which contacts a Glu (E) in the corresponding position of WT1 Q369 (29). (G) The Q369P of WT1 prefers T, followed by 5mC, to all other bases by a factor of 10 or more.

Figure 2.

Structural analysis of purine (adenine or guanine) binding in the 3′ triplet. (A–E) Structure of normal WT1 in complex with the A:T base pair. (A) Schematic representation of the ZF2 (from N-to-C termini) and the recognition sequence (from 3′-to-5′ of the top strand). The complementary strand (bottom) has a 5′ overhanging thymine used for the crystallization study. (B) R372, Q369, and R366 of ZF2 (in blue) interact, respectively, with Gua7, Ade8, and Gua9 of DNA (in magenta). (C) Q369-Ade8 interaction. The 2Fo-Fc electron density, contoured at 1σ above the mean, is shown in gray. (D) Structural superimposition of Q369-Ade (in blue for Q369, magenta for Ade, and gray for Thy) and Q369-5caC (in cyan; PDB: 4R2R). (E) The side chains of Q369 and R366 displaced shifts from binding of 5caC (in cyan) to Ade (in magenta). (F–J) Structure of Q369R in complex with the G:C base pair. (F) Schematic representation of the ZF2 with Q369R mutant. (G) The three arginines (R372, R369, and R366) align with three adjacent guanines along the DNA major groove. (H) R369-Gua8 interaction. (I) Structural superimposition of Q369-Ade (in blue) and R369-Gua (in green for R369 and magenta for Gua). (J) R372-Gua7 interaction. (K–N) Structure of Q369H in complex with the A:T base pair. (K) Schematic representation of the ZF2 (Q369H mutant) and ZF3 in recognition of the corresponding triplets. (L) H369-Ade8 interaction. (M) H397-Gua5 interaction. (N) Structural superimposition of Q369-Ade (in cyan) and H369-Ade (yellow for H369, magenta for Ade8).

Figure 3.

Binding affinities of normal WT1 the three Q369 mutants with oligos containing varied forms of cytosine. (A–D) Oligos fully methylated at both CpG sites (M/M), unmethylated at both sites (C/C), or methylated in only the bottom strand at both sites (C/M). (E–H) Oligos modified in only the top strand at both sites with 5mC (M), C, 5hmC, 5fC or 5caC. 5mC was present in the bottom strand at both sites in all cases. (I–L) Oligos modified in only the top strand at the 3′ triplet. All other sites contained 5mC (M).

Figure 4.

Structural analysis of oxidized cytosine binding in the 3′ triplet. (A–F) Structure of Q369R in complex with 5caC DNA. (A) Schematic representation of the ZF2 with Q369R mutant. (B) The R369 positively charged guanidinium group stacks on the negatively charged carboxylate of 5caCyt 8. (C) The 5caC carboxylate goup H-bonds with water molecules. (D) R369 H-bond with Gua9. (E) The movement of the side chain of R366 from normal WT1 (Q369) to the mutant R369. (F) R366 H-bonds with Gua9 and the adjacent Thy10. (G–L) Structure of Q369H in complex with 5caC DNA. (G) Schematic representation of the ZF2 with Q369H mutant. (H) The three positively changed residues (R366, H369, and R372) span four bases. (I) The H369 imidazole ring stacks on the 5caC carboxylate group. (J) H369 is too far removed to form H-bonds with Gua9. (K) R366 forms H-bonds with both Gua9 and Thy10. (L) The 5caC carboxylate goup H-bonds with water molecules. (M) Superimposing the Q369 (in gray) and Q369H (in blue) structures in complex with 5caC DNA. (N) Superimposing the Q369H (in blue) and Q369R (in cyan) structures in complex with 5caC DNA. (O) The main-chain conformations at position 369 are virtually identical, and the side chain conformations are very similar with only small rotational differences in torsion angles among Q369, H369, and R369. (P-Q) Structure of Q369H in complex with 5fC DNA. A water molecule mediates the interaction between H369 and Gua9 (P). Both 5fC (panel Q) and 5caC (panel L) exhibit an intrabase hydrogen bond between their formyl or carboxyl oxygen atoms, respectively, and the adjacent cytosine N4 exocyclic amine nitrogen atom.