Deformed Epidermal Autoregulatory Factor-1 Research Articles

Expansion and mechanistic insights into de novo DEAF1 variants in DEAF1-associated neurodevelopmental disorders.

De novo deleterious and heritable biallelic mutations in the DNA binding domain (DBD) of the transcription factor deformed epidermal autoregulatory factor 1 (DEAF1) result in a phenotypic spectrum of disorders termed DEAF1-associated neurodevelopmental disorders (DAND). RNA-sequencing using hippocampal RNA from mice with conditional deletion of Deaf1 in the central nervous system indicate that loss of Deaf1 activity results in the altered expression of genes involved in neuronal function, dendritic spine maintenance, development, and activity, with reduced dendritic spines in hippocampal regions. Since DEAF1 is not a dosage-sensitive gene, we assessed the dominant negative activity of previously identified de novo variants and a heritable recessive DEAF1 variant on selected DEAF1-regulated genes in 2 different cell models. While no altered gene expression was observed in cells over-expressing the recessive heritable variant, the gene expression profiles of cells over-expressing de novo variants resulted in similar gene expression changes as observed in CRISPR-Cas9-mediated DEAF1-deleted cells. Altered expression of DEAF1-regulated genes was rescued by exogenous expression of WT-DEAF1 but not by de novo variants in cells lacking endogenous DEAF1. De novo heterozygous variants within the DBD of DEAF1 were identified in 10 individuals with a phenotypic spectrum including autism spectrum disorder, developmental delays, sleep disturbance, high pain tolerance, and mild dysmorphic features. Functional assays demonstrate these variants alter DEAF1 transcriptional activity. Taken together, this study expands the clinical phenotypic spectrum of individuals with DAND, furthers our understanding of potential roles of DEAF1 on neuronal function, and demonstrates dominant negative activity of identified de novo variants.

Human Lymph Node Stromal Cells Have the Machinery to Regulate Peripheral Tolerance during Health and Rheumatoid Arthritis.

Background: In rheumatoid arthritis (RA) the cause for loss of tolerance and anti-citrullinated protein antibody (ACPA) production remains unidentified. Mouse studies showed that lymph node stromal cells (LNSCs) maintain peripheral tolerance through presentation of peripheral tissue antigens (PTAs). We hypothesize that dysregulation of peripheral tolerance mechanisms in human LNSCs might underlie pathogenesis of RA. Method: Lymph node (LN) needle biopsies were obtained from 24 RA patients, 23 individuals positive for RA-associated autoantibodies but without clinical disease (RA-risk individuals), and 14 seronegative healthy individuals. Ex vivo human LNs from non-RA individuals were used to directly analyze stromal cells. Molecules involved in antigen presentation and immune modulation were measured in LNSCs upon interferon γ (IFNγ) stimulation (n = 15). Results: Citrullinated targets of ACPAs were detected in human LN tissue and in cultured LNSCs. Human LNSCs express several PTAs, transcription factors autoimmune regulator (AIRE) and deformed epidermal autoregulatory factor 1 (DEAF1), and molecules involved in citrullination, antigen presentation, and immunomodulation. Overall, no clear differences between donor groups were observed with exception of a slightly lower induction of human leukocyte antigen-DR (HLA-DR) and programmed cell death 1 ligand (PD-L1) molecules in LNSCs from RA patients. Conclusion: Human LNSCs have the machinery to regulate peripheral tolerance making them an attractive target to exploit in tolerance induction and maintenance.

Impaired memory and marble burying activity in deformed epidermal autoregulatory factor 1 (Deaf1) conditional knockout mice

Deleterious mutations within the DNA binding domain of the transcription factor deformed epidermal autoregulatory factor 1 (DEAF1) result in a phenotypic spectrum of neurodevelopmental disorders including intellectual disabilities and autism spectrum disorders. While whole animal deletion of Deaf1 in mice is lethal, mice with conditional disruption of the gene in neuronal precursor cells can display memory deficits and increased anxiety-like behavior. This study aimed to further characterize learning and memory alterations and assess changes in marble burying activity and hippocampal size in mice with conditional deletion of Deaf1. Mice lacking DEAF1 in the CNS (NKO) displayed reduced memory in both contextual fear conditioning and a 3-day massed trials Morris water maze paradigm. NKO mice had reduced marble burying activity in full cage marble burying tests. Using a half-cage marble test, NKO mice again buried fewer marbles and spent significantly more time on the side of the cage away from the marbles compared to control animals. The area of the dorsal hippocampus of NKO mice was decreased compared to control and animals with a single Deaf1 allele. These results continue to establish the importance of DEAF1 in cognitive behavior and provide new evidence that DEAF1 regulates hippocampal morphology.

The Transcription Factor Deaf1 Modulates Engrailed-1 Expression to Regulate Skin Appendage Fate

The Transcription Factor Deaf1 Modulates Engrailed-1 Expression to Regulate Skin Appendage Fate

Functional analysis of novel DEAF1 variants identified through clinical exome sequencing expands DEAF1-associated neurodevelopmental disorder (DAND) phenotype.

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, as 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.

Evidence for genetic regulation of mRNA expression of the dosage-sensitive gene retinoic acid induced-1 (RAI1) in human brain.

RAI1 (retinoic acid induced-1) is a dosage-sensitive gene that causes Smith-Magenis syndrome (SMS) when mutated or deleted and Potocki-Lupski Syndrome (PTLS) when duplicated, with psychiatric features commonly observed in both syndromes. How common genetic variants regulate this gene, however, is unknown. In this study, we found that RAI1 mRNA expression in Chinese prefrontal and temporal cortex correlate with genotypes of common single nucleotide polymorphisms (SNPs) located in the RAI1 5′-upstream region. Using genotype imputation, “R2-Δ2” analysis, and data from the RegulomeDB database, we identified SNPs rs4925102 and rs9907986 as possible regulatory variants, accounting for approximately 30–40% of the variance in RAI1 mRNA expression in both brain regions. Specifically, rs4925102 and rs9907986 are predicted to disrupt the binding of retinoic acid RXR-RAR receptors and the transcription factor DEAF1 (Deformed epidermal autoregulatory factor-1), respectively. Consistent with these predictions, we observed binding of RXRα and RARα to the predicted RAI1 target in chromatin immunoprecipitation assays. Retinoic acid is crucial for early development of the central neural system, and DEAF1 is associated with intellectual disability. The observation that a significant portion of RAI1 mRNA expression is genetically controlled raises the possibility that common RAI1 5′-region regulatory variants contribute more generally to psychiatric disorders.

The structure of an LIM-only protein 4 (LMO4) and Deformed epidermal autoregulatory factor-1 (DEAF1) complex reveals a common mode of binding to LMO4.

LIM-domain only protein 4 (LMO4) is a widely expressed protein with important roles in embryonic development and breast cancer. It has been reported to bind many partners, including the transcription factor Deformed epidermal autoregulatory factor-1 (DEAF1), with which LMO4 shares many biological parallels. We used yeast two-hybrid assays to show that DEAF1 binds both LIM domains of LMO4 and that DEAF1 binds the same face on LMO4 as two other LMO4-binding partners, namely LIM domain binding protein 1 (LDB1) and C-terminal binding protein interacting protein (CtIP/RBBP8). Mutagenic screening analysed by the same method, indicates that the key residues in the interaction lie in LMO4LIM2 and the N-terminal half of the LMO4-binding domain in DEAF1. We generated a stable LMO4LIM2-DEAF1 complex and determined the solution structure of that complex. Although the LMO4-binding domain from DEAF1 is intrinsically disordered, it becomes structured on binding. The structure confirms that LDB1, CtIP and DEAF1 all bind to the same face on LMO4. LMO4 appears to form a hub in protein-protein interaction networks, linking numerous pathways within cells. Competitive binding for LMO4 therefore most likely provides a level of regulation between those different pathways.

DEAF1 Is a Pellino1-interacting Protein Required for Interferon Production by Sendai Virus and Double-stranded RNA*

Double-stranded (ds) RNA of viral origin, a ligand for Melanoma Differentiation-associated gene 5 (MDA5) and Toll-Like Receptor 3 (TLR3), induces the TANK-Binding Kinase 1 (TBK1)-dependent phosphorylation and activation of Interferon Regulatory Factor 3 (IRF3) and the E3 ubiquitin ligase Pellino1, which are required for interferon β (IFNβ) gene transcription. Here, we report that Pellino1 interacts with the transcription factor Deformed Epidermal Autoregulatory Factor 1 (DEAF1). The interaction is independent of the E3 ligase activity of Pellino1, but weakened by the phosphorylation of Pellino1. We show that DEAF1 binds to the IFNβ promoter and to IRF3 and IRF7, that it is required for the transcription of the IFNβ gene and IFNβ secretion in MEFs infected with Sendai virus or transfected with poly(I:C). DEAF1 is also needed for TLR3-dependent IFNβ production. Taken together, our results identify DEAF1 as a novel component of the signal transduction network by which dsRNA of viral origin stimulates IFNβ production.

Reduced DEAF1 function during type 1 diabetes inhibits translation in lymph node stromal cells by suppressing Eif4g3

The transcriptional regulator deformed epidermal autoregulatory factor 1 (DEAF1) has been suggested to play a role in maintaining peripheral tolerance by controlling the transcription of peripheral tissue antigen genes in lymph node stromal cells (LNSCs). Here, we demonstrate that DEAF1 also regulates the translation of genes in LNSCs by controlling the transcription of the poorly characterized eukaryotic translation initiation factor 4 gamma 3 (Eif4g3) that encodes eIF4GII. Eif4g3 gene expression was reduced in the pancreatic lymph nodes of Deaf1-KO mice, non-obese diabetic mice, and type 1 diabetes patients, where functional Deaf1 is absent or diminished. Silencing of Deaf1 reduced Eif4g3 expression, but increased the expression of Caspase 3, a serine protease that degrades eIF4GII. Polysome profiling showed that reduced Eif4g3 expression in LNSCs resulted in the diminished translation of various genes, including Anpep, the gene for aminopeptidase N, an enzyme involved in fine-tuning antigen presentation on major histocompatibility complex (MHC) class II. Together these findings suggest that reduced DEAF1 function, and subsequent loss of Eif4g3 transcription may affect peripheral tissue antigen (PTA) expression in LNSCs and contribute to the pathology of T1D.

Deformed Epidermal Autoregulatory Factor-1 (DEAF1) Interacts with the Ku70 Subunit of the DNA-Dependent Protein Kinase Complex

Deformed Epidermal Autoregulatory Factor 1 (DEAF1) is a transcription factor linked to suicide, cancer, autoimmune disorders and neural tube defects. To better understand the role of DEAF1 in protein interaction networks, a GST-DEAF1 fusion protein was used to isolate interacting proteins in mammalian cell lysates, and the XRCC6 (Ku70) and the XRCC5 (Ku80) subunits of DNA dependent protein kinase (DNA-PK) complex were identified by mass spectrometry, and the DNA-PK catalytic subunit was identified by immunoblotting. Interaction of DEAF1 with Ku70 and Ku80 was confirmed to occur within cells by co-immunoprecipitation of epitope-tagged proteins, and was mediated through interaction with the Ku70 subunit. Using in vitro GST-pulldowns, interaction between DEAF1 and the Ku70 subunit was mapped to the DEAF1 DNA binding domain and the C-terminal Bax-binding region of Ku70. In transfected cells, DEAF1 and Ku70 colocalized to the nucleus, but Ku70 could not relocalize a mutant cytoplasmic form of DEAF1 to the nucleus. Using an in vitro kinase assay, DEAF1 was phosphorylated by DNA-PK in a DNA-independent manner. Electrophoretic mobility shift assays showed that DEAF1 or Ku70/Ku80 did not interfere with the DNA binding of each other, but DNA containing DEAF1 binding sites inhibited the DEAF1-Ku70 interaction. The data demonstrates that DEAF1 can interact with the DNA-PK complex through interactions of its DNA binding domain with the carboxy-terminal region of Ku70 that contains the Bax binding domain, and that DEAF1 is a potential substrate for DNA-PK.

Interactome Mapping of the Phosphatidylinositol 3-Kinase-Mammalian Target of Rapamycin Pathway Identifies Deformed Epidermal Autoregulatory Factor-1 as a New Glycogen Synthase Kinase-3 Interactor

The phosphatidylinositol 3-kinase-mammalian target of rapamycin (PI3K-mTOR) pathway plays pivotal roles in cell survival, growth, and proliferation downstream of growth factors. Its perturbations are associated with cancer progression, type 2 diabetes, and neurological disorders. To better understand the mechanisms of action and regulation of this pathway, we initiated a large scale yeast two-hybrid screen for 33 components of the PI3K-mTOR pathway. Identification of 67 new interactions was followed by validation by co-affinity purification and exhaustive literature curation of existing information. We provide a nearly complete, functionally annotated interactome of 802 interactions for the PI3K-mTOR pathway. Our screen revealed a predominant place for glycogen synthase kinase-3 (GSK3) A and B and the AMP-activated protein kinase. In particular, we identified the deformed epidermal autoregulatory factor-1 (DEAF1) transcription factor as an interactor and in vitro substrate of GSK3A and GSK3B. Moreover, GSK3 inhibitors increased DEAF1 transcriptional activity on the 5-HT1A serotonin receptor promoter. We propose that DEAF1 may represent a therapeutic target of lithium and other GSK3 inhibitors used in bipolar disease and depression.

Functional analysis of the osteoarthritis susceptibility–associated GDF5 regulatory polymorphism

Single-nucleotide polymorphism (SNP) rs143383 (T to C) in the 5'-untranslated region (5'-UTR) of GDF5 has recently been reported to be associated with osteoarthritis (OA) susceptibility, with lower expression of the risk-associated T allele observed in vitro and in vivo. The in vivo studies were performed on cartilage tissue from OA patients. The present study was undertaken to expand the analysis of the effect of this SNP on GDF5 allelic expression to more joint tissue types, to investigate for cis and trans factors that interact with the SNP, and to examine novel cis-acting GDF5 regulatory polymorphisms. Tissue samples were collected from OA patients undergoing joint replacement of the hip or knee. Nucleic acid was extracted, and, using rs143383 and an assay that discriminates and quantifies allelic expression, the relative amount of GDF5 expression from the T and C alleles was measured. Additional common variants in the GDF5 transcript sequence were interrogated as potential regulatory elements using allelic expression and luciferase reporter assays, and electrophoretic mobility shift assays were used to search for trans factors binding to rs143383. We observed a consistent allelic expression imbalance of GDF5 in all tissues tested, implying that the functional effect mediated by rs143383 on GDF5 expression is joint-wide. We identified a second polymorphism, located in the 3'-UTR of GDF5, that influenced allelic expression of the gene independent of rs143383. Finally, we observed differential binding of deformed epidermal autoregulatory factor 1 (DEAF-1) to the 2 alleles of rs143383. These findings show that the OA susceptibility mediated by polymorphism in GDF5 is not restricted to cartilage, emphasizing the need to consider the disease as involving the whole joint. The existence of an additional cis-acting regulatory polymorphism highlights the complexity of the regulation of expression of this important OA susceptibility locus. DEAF-1 is a trans-acting factor that merits further investigation as a potential tool for modulating GDF5 expression.

Variation in neighbouring genes of the dopaminergic and serotonergic systems affects feather pecking behaviour of laying hens

Feather pecking is a behavioural disorder of laying hens and has serious animal welfare and economic implications. One of the several aetiological hypotheses proposes that the disorder results from redirected exploratory behaviour. Variation in the gene encoding the dopamine D4 receptor (DRD4) has been shown to be associated with exploratory behaviour in several species, including in a passerine bird species. We therefore considered DRD4 as a candidate gene for feather pecking. We have annotated DRD4 in the chicken genome and have re-sequenced it in 140 animals belonging to: experimental layer lines divergently selected for high and low propensity to feather pecking; the unselected founder population; and two commercial lines with low and high propensity to feather pecking. We have identified two sub-haplotypes of DRD4 that are highly significantly associated with feather pecking behaviour in the experimental (P = 7.30 x 10(-7)) as well as in the commercial lines (P = 2.78 x 10(-6)). Linkage disequilibrium (LD) extends into a neighbouring gene encoding deformed epidermal autoregulatory factor 1 (DEAF1). The product of DEAF1 regulates the transcription of the gene encoding the serotonin (5-hydroxytryptamine) 1A receptor. Thus, DEAF1 represents another candidate gene for feather pecking. Re-sequencing of five animals homozygous for the 'low-pecking' sub-haplotype and of six animals homozygous for the 'high-pecking' sub-haplotype delineated an LD block of 14 833 bases spanning the two genes. None of the variants in the LD block is obviously functional. However, the haplotype information will be useful to select against the propensity to feather pecking in chicken and to elucidate the functional implications of the variants.

DEAF-1 regulates immunity gene expression in Drosophila

Immunity genes are activated in the Drosophila fat body by Rel and GATA transcription factors. Here, we present evidence that an additional regulatory factor, deformed epidermal autoregulatory factor-1 (DEAF-1), also contributes to the immune response and is specifically important for the induction of two genes encoding antimicrobial peptides, Metchnikowin (Mtk) and Drosomycin (Drs). The systematic mutagenesis of a minimal Mtk 5' enhancer identified a sequence motif essential for both a response to LPS preparations in S2 cells and activation in the larval fat body in response to bacterial infection. Using affinity chromatography coupled to multidimensional protein identification technology (MudPIT), we identified DEAF-1 as a candidate regulator. DEAF-1 activates the expression of Mtk and Drs promoter-luciferase fusion genes in S2 cells. SELEX assays and footprinting data indicate that DEAF-1 binds to and activates Mtk and Drs regulatory DNAs via a TTCGGBT motif. The insertion of this motif into the Diptericin (Dpt) regulatory region confers DEAF-1 responsiveness to this normally DEAF-1-independent enhancer. The coexpression of DEAF-1 with Dorsal, Dif, and Relish results in the synergistic activation of transcription. We propose that DEAF-1 is a regulator of Drosophila immunity.

Post‐translational modifications of Deformed Epidermal Autoregulatory Factor‐1

Deformed Epidermal Autoregulatory Factor 1 (DEAF‐1) is a transcriptional regulator that is important for embryonic development in mice, and is linked to depression and cancer in humans. The localization of DEAF‐1 and its interaction with other proteins have been shown to affect DEAF‐1 functions. To identify post‐translational modifications of DEAF‐1 that may affect function, purified proteins from HEK293T cells were subjected to liquid chromatography‐mass spectrometry. Three phosphorylated amino acids and a methylated arginine within the DNA binding domain were identified. In vitro mutagenesis and functional assays were performed to determine if these sites could influence known activities of DEAF‐1, including: DNA binding, transcriptional regulation, localization, apoptosis, and protein‐protein interactions. In vitro assays were used to confirm cyclin dependent kinase 5 (CDK5) as the modifying enzyme of at least two of the phosphorylation sites and protein arginine methyltransferase 6 and 8 (PRMT6 and PRMT8) were found to methylate DEAF‐1. How these protein modifications of DEAF‐1 regulate its functions are being explored.

Cell-Specific Repressor or Enhancer Activities of Deaf-1 at a Serotonin 1A Receptor Gene Polymorphism

The serotonin-1A (5-HT1A) receptor is the primary somatodendritic autoreceptor that inhibits the activity of serotonergic raphe neurons and is also expressed in nonserotonergic cortical and limbic neurons. Alterations in 5-HT1A receptor levels are implicated in mood disorders, and a functional C(-1019)G 5-HT1A promoter polymorphism has been associated with depression, suicide, and panic disorder. We examined the cell-specific activity of identified transcription factors, human nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR)/Deaf-1 and Hes5, at the 5-HT1A C(-1019) site. In serotonergic raphe RN46A cells, Deaf-1 and Hes5 repressed the 5-HT1A receptor gene at the C(-1019)-allele but not the G(-1019)-allele. However, in nonserotonergic cells that express 5-HT1A receptors (septal SN48, neuroblastoma SKN-SH, and neuroblastoma/glioma NG108-15 cells), Deaf-1 enhanced 5-HT1A promoter activity at the C(-1019)-allele but not the G-allele, whereas Hes5 repressed in all cell types. The enhancer activity of Deaf-1 was orientation independent and competed out Hes5 repression. To test whether Deaf-1 activity is intrinsic, the activity of a Gal4DBD (DNA binding domain)-Deaf-1 fusion protein at a heterologous Gal4 DNA element was examined. Gal4DBD-Deaf-1 repressed transcription in RN46A cells but enhanced transcription in SN48 cells, indicating that these opposite activities are intrinsic to Deaf-1. Repressor or enhancer activities of Deaf-1 or Gal4DBD-Deaf-1 were blocked by histone deacetylase inhibitor trichostatin A. Thus, the intrinsic activity of Deaf-1 at the 5-HT1A promoter is opposite in presynaptic versus postsynaptic neuronal cells and requires deacetylation. Cell-specific regulation by Deaf-1 could underlie region-specific alterations in 5-HT1A receptor expression in different mood disorders.

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

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.

Characterization of a nuclear deformed epidermal autoregulatory factor-1 (DEAF-1)-related (NUDR) transcriptional regulator protein.

A monkey kidney cDNA that encodes a nuclear regulatory factor was identified by expression and affinity binding to a synthetic retinoic acid response element (RARE) and was used to isolate human placental and rat germ cell cDNAs by hybridization. The cDNAs encode a 59-kDa protein [nuclear DEAF-1-related (NUDR)] which shows sequence similarity to the Drosophila Deformed epidermal autoregulatory factor-1 (DEAF-1), a nonhomeodomain cofactor of embryonic Deformed gene expression. Similarities to other proteins indicate five functional domains in NUDR including an alanine-rich region prevalent in developmental transcription factors, a domain found in the promyelocytic leukemia-associated SP100 proteins, and a zinc finger homology domain associated with the AML1/MTG8 oncoprotein. Although NUDR mRNA displayed a wide tissue distribution in rats, elevated levels of protein were only observed in testicular germ cells, developing fetus, and transformed cell lines. Nuclear localization of NUDR was demonstrated by immunocytochemistry and by a green fluorescent protein-NUDR fusion protein. Site-directed mutagenesis of a nuclear localization signal resulted in cytoplasmic localization of the protein and eliminated NUDR-dependent transcriptional activation. Recombinant NUDR protein showed affinity for the RARE in mobility shifts; however it was efficiently displaced by retinoic acid receptor (RAR)/retinoid X receptor (RXR) complexes. In transient transfections, NUDR produced up to 26-fold inductions of a human proenkephalin promoter-reporter plasmid, with minimal effects on the promoters for prodynorphin or thymidine kinase. Placement of a RARE on the proenkephalin promoter increased NUDR-dependent activation to 41-fold, but this RARE-dependent increase was not transferable to a thymidine kinase promoter. Recombinant NUDR protein showed minimal binding affinity for proenkephalin promoter sequences, but was able to select DNA sequences from a random oligonucleotide library that had similar core-binding motifs (TTCG) as those recognized by DEAF-1. This motif is also present between the half-sites of several endogenous RAREs. The derived consensus- binding motif recognized by NUDR (TTCGGGNNTTTCCGG) was confirmed by mobility shift and deoxyribonuclease I (DNase I) protection assays; however, the consensus sequence was also unable to confer NUDR-dependent transcriptional activation to the thymidine kinase promoter. Our data suggests that NUDR may activate transcription independently of promoter binding, perhaps through protein-protein interaction with basal transcription factors, or by activation of secondary factors. The sequence and functional similarities between NUDR and DEAF-1 suggest that NUDR may also act as a cofactor to regulate the transcription of genes during fetal development or differentiation of testicular cells.

Characterization of a Nuclear Deformed Epidermal Autoregulatory Factor-1 (DEAF-1)-Related (NUDR) Transcriptional Regulator Protein

Characterization of a Nuclear Deformed Epidermal Autoregulatory Factor-1 (DEAF-1)-Related (NUDR) Transcriptional Regulator Protein

DEAF-1, a novel protein that binds an essential region in a Deformed response element.

A 120 bp homeotic response element that is regulated specifically by Deformed in Drosophila embryos contains a single binding site for Deformed protein. However, a 24 bp sub-element containing this site does not constitute a Deformed response element. Specific activation requires a second region in the 120 bp element, which presumably contains one or more binding sites for Deformed cofactors. We have isolated a novel protein from Drosophila nuclear extracts which binds specifically to a site in this second region. This protein, which we call DEAF-1 (Deformed epidermal autoregulatory factor-1), contains three conserved domains. One of these includes a cysteine repeat motif that is similar to a motif found in Drosophila Nervy and the human MTG8 proto-oncoprotein, and another matches a region of Drosophila Trithorax. Mutations in the response element designed to improve binding to DEAF-1 in vitro resulted in increased embryonic expression. Conversely, small mutations designed to diminish binding to DEAF-1 resulted in reduced expression of the element. Thus, DEAF-1 is likely to contribute to the functional activity, and perhaps to the homeotic specificity, of this response element. Consistent with this hypothesis, we have discovered DEAF-1 binding sites in other Deformed response elements.