Structure of SALL4 zinc finger domain reveals link between AT-rich DNA binding and Okihiro syndrome

Abstract

Spalt-like 4 (SALL4) maintains vertebrate embryonic stem cell identity and is required for the development of multiple organs, including limbs. Mutations in SALL4 are associated with Okihiro syndrome, and SALL4 is also a known target of thalidomide. SALL4 protein has a distinct preference for AT-rich sequences, recognised by a pair of zinc fingers at the C-terminus. However, unlike many characterised zinc finger proteins, SALL4 shows flexible recognition with many different combinations of AT-rich sequences being targeted. SALL4 interacts with the NuRD corepressor complex which potentially mediates repression of AT-rich genes. We present a crystal structure of SALL4 C-terminal zinc fingers with an AT-rich DNA sequence, which shows that SALL4 uses small hydrophobic and polar side chains to provide flexible recognition in the major groove. Missense mutations reported in patients that lie within the C-terminal zinc fingers reduced overall binding to DNA but not the preference for AT-rich sequences. Furthermore, these mutations altered association of SALL4 with AT-rich genomic sites, providing evidence that these mutations are likely pathogenic.

A long journey to oyster stem cell markers.CgSox2 andCgPOU2 synergic DNA binding and NODE proteins

“…This kinetic of disassembly-reassembly to larger bodies is accompanied by chromatin displacement, which is particularly visible when large bodies form (Fig. 4E, F), as shown elsewhere (Watson et al, 2023), must require cargo transport inside the nucleus and thus energy. Regarding the composition of these bodies, it is quite possible that aggregates are made exclusively or mostly of Cg SalL since otherwise a tremendously large supply of endogenous protein(s) would be required to match the overexpressed Cg SalL protein.…”

“…DAPI-staining showed that formation of these large bodies displaced chromatin (Fig. 4E-F) (see Watson et al, 2023). Sections of confocal image stacks from cells expressing Cg Sox2 and Cg SalL revealed ring structures that actually corresponded to the dots observed above.…”

“…SalL4 is a regulator in the transcriptional network in stem cell maintenance that contains 3 zinc finger regions referred to as ZFC1, 2 and 4. SalL4 mediates its activities through the NODE complex activity (Kagias et al, 2013; Tanimura et al, 2013) but also alternatively, through the direct binding of ZFC4 to AT-rich DNA regions (Watson et al, 2023).…”

“…A unique Sal-Like protein exists in mollusk, with 3 conserved zinc finger regions (Table S3) while the rest of the sequence is much less conserved. Interestingly, the two AAs of the ZFC4 domain (T919 and N922), which are essential to SalL4 DNA binding (Watson et al, 2023), are conserved among the bilaterians (Table S3B). Note that SalL proteins are not found in less evolved animals such as the cnidarians, ctenophores, placozoans and sponges for which we aligned unrelated Zinc finger protein sequences that show some degree of homology with SalL4 ZnF4 (Table S3B) but otherwise no homology.…”

Targeting transcription factors through an IMiD independent zinc finger domain

“…Side chains of the second zinc finger of both molecules also provided four and five additional interactions with the DNA backbone, respectively, including Lys896-Y4, Arg905-Y3, Lys910-X4, Lys914-X5, and His916-Y2. Recently, the crystal structure of human SALL4 ZFC4 (856-930) (Ru, Koga et al, 2022) and murine SALL4 ZFC4 (870-940) (Watson, Pantier et al, 2023) were published. Consistent with the report from Ru et al (Ru et al, 2022), our crystallographic findings of human SALL4 ZFC4 (864-929) bound to its consensus DNA binding target revealed an asymmetric unit that contained one molecule of the duplex DNA and two molecules of ZFC4.…”