The SWI/SNF Subunit INI1 Contains an N-Terminal Winged Helix DNA Binding Domain that Is a Target for Mutations in Schwannomatosis

Abstract

SWI/SNF complexes use the energy of ATP hydrolysis to remodel chromatin. In mammals they play a central role in regulating gene expression during differentiation and proliferation. Mutations in SWI/SNF subunits are among the most frequent gene alterations in cancer. The INI1/hSNF5/SMARCB1 subunit is mutated in both malignant rhabdoid tumor, a highly aggressive childhood cancer, and schwannomatosis, a tumor-predisposing syndrome characterized by mostly benign tumors of the CNS. Here, we show that mutations in INI1 that cause schwannomatosis target a hitherto unidentified N-terminal winged helix DNA binding domain that is also present in the BAF45a/PHF10 subunit of the SWI/SNF complex. The domain is structurally related to the SKI/SNO/DAC domain, which is found in a number of metazoan chromatin-associated proteins.

Figure 1

Structure of the N-Terminal Domain of INI1 (A) Schematic representation of the domain organization of INI1 and BAF45a/PHF10. (B) Superposition of the backbone traces of residues 10–110 of the 20 lowest-energy structures of the INI1 N-terminal domain (left) and a ribbon diagram of a representative structure (right). (C) Comparison of the structures of, from left to right, the INI1 N-terminal domain, the DHD domain of SnoN, and Mbp1.

Figure 2

Sequence Conservation in the INI1 and BAF45a Domains (A) Sequence alignment of the N-terminal winged helix domains of INI1 homologs from representative species. The secondary structure of the domain is shown above the sequences, with sheets and helices indicated by arrows and cylinders. Species abbreviations: Hs, Homo sapiens; Gg, Gallus gallus; Xl, Xenopus laevis; Dr, Danio rerio; Ce, Caenorhabditis elegans. The numbering is for the human protein. Residues mutated in familial schwannomatosis are indicated by green triangles. Residues mutated in sporadic disease are indicated with gray triangles. Residues that undergo large changes in chemical shift upon DNA binding are colored red. (B) Structure-based alignment of the homologous domain in BAF45a proteins from the same species. The sequence and secondary structure of the INI1 domain are shown below the alignment. (C) Cartoon representation of the INI1 N-terminal domain, with the side chains of highly conserved residues shown as sticks.

Figure 3

Binding of the INI1 Domain to dsDNA (A) ¹H,¹⁵N-BEST-TROSY spectra of the INI1 domain free (black) and bound to a dsDNA oligonucleotide (red). Residues that undergo particular large changes in chemical shift upon DNA binding are indicated. (B) Cartoon representation of the INI1 N-terminal domain, with the side chains of residues that undergo large changes in chemical shift shown as sticks and colored red. (C) Cartoon representation of PCG2, the Magnaporthe oryzae ortholog of Mbp1, bound to its target DNA sequence.

Figure 4

Residues Mutated in Familial Schwannomatosis Close-up views of the INI1 N-terminal domain showing the positions of Pro14 (A), Pro48 (B) and Arg53 (C). The mutated residues are indicated with an asterisk.