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...

Recently, we identified in two individuals with intellectual disability (ID) different de novo mutations in DEAF1, which encodes a transcription factor with an important role in embryonic development. To ascertain whether these mutations in DEAF1 are causative for the ID phenotype, we performed targeted resequencing of DEAF1 in an additional cohort of over 2,300 individuals with unexplained ID and identified two additional individuals with de novo mutations in this gene. All four individuals had severe ID with severely affected speech development, and three showed severe behavioral problems. DEAF1 is highly expressed in the CNS, especially during early embryonic development. All four mutations were missense mutations affecting the SAND domain of DEAF1. Altered DEAF1 harboring any of the four amino acid changes showed impaired transcriptional regulation of the DEAF1 promoter. Moreover, behavioral studies in mice with a conditional knockout of Deaf1 in the brain showed memory deficits and increased anxiety-like behavior. Our results demonstrate that mutations in DEAF1 cause ID and behavioral problems, most likely as a result of impaired transcriptional regulation by DEAF1.

In this article, the authors reported on four individuals with intellectual disability, severely affected speech development, behavioral problems, and missense mutations affecting the SAND domain of DEAF1. Functional studies showing a loss of function of DEAF1 and behavioral studies in a conditional knockout mouse provided additional support for causality of the DEAF1 mutations in these four reported individuals.

ATP activates an apical-to-basolateral nonselective cation current across the skin of larval bullfrogs (Rana catesbeiana) with similarities to currents carried by some P2X receptors. A functional P2X receptor was cloned from tadpole skin RNA that encodes a 409-amino acid protein with highest protein homology to cP2X(8). RT-PCR showed that this transcript was found in skin, heart, eye, brain, and skeletal muscle of tadpoles but not in skin, brain, or heart of adults. After transcribed RNA from this clone was injected into Xenopus oocytes, application of ATP activated a transient current similar to other P2X receptors and the ATP-activated transient in short-circuit current (I(sc)) across intact skin. The agonists 2-methylthio-ATP and adenosine-5'-O-(thiotriphoshate) also activated transient currents. alpha,beta-Methylene-ATP and ADP were poor agonists of this receptor. Suramin and pyridoxal phosphate 6-azophenyl-2',4'-disulfonic acid tetrasodium (PPADS) were potent antagonists, and PPADS showed an irreversible blockade of this receptor to agonist activation. Under external Na(+)-free, Ca(2+)/Mg(2+)-free conditions (N-methyl-D-glucamine replacement, 0.5 mM EGTA), ATP activated a steadily increasing inward current. Fluorescence microscopy showed that propidium was entering the cells, suggesting that a relatively large pore size was formed under zero divalent conditions. This clone has some characteristics consistent with previously described ATP-activated I(sc) in the tadpole skin. Because the clone is not found in adult skin, it may have some exclusive role in the tadpole such as sensory reception by the skin or triggering apoptosis at metamorphosis.

Deformed epidermal autoregulatory factor-1 (DEAF1), a transcription factor essential for central nervous system and early embryonic development, has recently been implicated in a series of intellectual disability related neurodevelopmental anomalies termed, in this study, DEAF1-associated neurodevelopmental disorder (DAND). We identified six potentially deleterious DEAF1 variants in a cohort of individuals with DAND via clinical exome sequencing (CES) and in-silico analysis, including two novel de novo variants: missense variant c.634G>A p.Gly212Ser in the SAND domain and deletion variant c.913_915del p.Lys305del in the NLS domain, as well as c.676C>T p.Arg226Trp, c.700T>A p.Trp234Arg, c.737G>C p.Arg246Thr, and c.791A>C p.Gln264Pro. Luciferase reporter, immunofluorescence staining and electrophoretic mobility shift assays revealed that these variants had decreased transcriptional repression activity at the DEAF1 promoter and reduced affinity to consensus DEAF1 DNA binding sequences. In addition, c.913_915del p.K305del localized primarily to the cytoplasm and interacted with wild-type DEAF1. Our results demonstrate that variants located within the SAND or NLS domains significantly reduce DEAF1 transcriptional regulatory activities and are thus, likely to contribute to the underlying clinical concerns in DAND patients. These findings illustrate the importance of experimental characterization of variants with uncertain significance identified by CES to assess their potential clinical significance and possible use in diagnosis.