Identification of a Nuclear Export Signal and Protein Interaction Domains in Deformed Epidermal Autoregulatory Factor-1 (DEAF-1)

Abstract

Deformed epidermal autoregulatory factor-1 (DEAF-1) is a DNA-binding protein required for embryonic development and linked to clinical depression and suicidal behavior in humans. Although primarily nuclear, cytoplasmic localization of DEAF-1 has been observed, and this suggests the presence of a nuclear export signal (NES). Using a series of fluorescent fusion proteins, an NES with a novel spacing of leucines (LXLX 6 LLX 5 LX 2 L) was identified near the COOH-terminal MYND domain at amino acids 454 -476. The NES was leptomycin B-sensitive and mutation of the leucine residues decreased or eliminated nuclear export activity. In vitro pull downs and an in vivo fluorescent protein interaction assay identified a DEAF-1/DEAF-1 protein interaction domain within the NES region. DNA binding had been previously mapped to a positively charged surface patch in the novel DNA binding fold called the "SAND" domain. A second protein-protein interaction domain was identified at amino acids 243-306 that contains the DNA-binding SAND domain and also an adjacent zinc binding motif and a monopartite nuclear localization signal (NLS). Deletion of these adjacent sequences or mutation of the conserved cysteines or histidine in the zinc binding motif not only inhibits protein interaction but also eliminates DNA binding, demonstrating that DEAF-1 protein-protein interaction is required for DNA recognition. The identification of an NES and NLS provides a basis for the control of DEAF-1 subcellular localization and function, whereas the requirement of protein-protein interaction by the SAND domain appears to be unique among this class of transcription factors.Deformed epidermal autoregulatory factor-1 (DEAF-1) 1 was first identified in Drosophila as a DNA-binding protein and potential regulator of the homeotic gene Deformed (1). The human, rat, and monkey homologs of Drosophila DEAF-1 (dDEAF-1) were previously called "nuclear DEAF-1 related" (NUDR) because of the limited protein similarity (46%) to dDEAF-1 (2). Genomic sequencing projects have confirmed a single gene in metazoan genomes with the overall structure of dDEAF-1, therefore we have adopted the DEAF-1 designation for all orthologs of the gene. DEAF-1 proteins are structurally defined as having both a DNA binding SAND domain (Sp100, AIRE-1, NucP41/75, DEAF-1) (3-5) and a carboxyl-terminal zinc finger motif called the MYND domain (myeloid translocation protein 8, Nervy, DEAF-1) (6, 7). Functional domains outside of these regions of DEAF-1 have yet to be extensively characterized.DEAF-1 appears to be an important factor in development and cancer. Loss of function mutations in DEAF-1 produce early embryonic arrest or segmentation defects in Drosophila, whereas overexpression of DEAF-1 can disrupt eye and wing development (8). DEAF-1 mRNA is widely expressed during mouse embryogenesis with elevated levels in several tissues, including the central nervous system and the dorsal root ganglia (9). DEAF-1 interacts with LMO4 and NLI (10), transcriptional regulators that mediate embryonic p...

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Functional analysis of SAND mutations in AIRE supports dominant inheritance of the G228W mutation

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