Philip J. Jensik

Mutations affecting the SAND domain of DEAF1 cause intellectual disability with severe speech impairment and behavioral problems.

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.

Larval bullfrog skin expresses ENaC despite having no amiloride-blockable transepithelial Na + transport

Amiloride-blockable Na+ transport, measured as an amiloride-blockable short-circuit current (Am-SCC), is mediated by the epithelial Na+ channel (ENaC). Am-SCC is not normally present in bullfrog tadpole skin, but when such skin is cultured with corticoids an amiloride-blockable Na transport appears. Prolactin (PRL) inhibits its corticoid-induced development. Using specific PCR primers for adult frog ENaC and RT-PCR, we investigated whether corticoids can induce all three ENaC subunits, and whether this expression of ENaC subunit(s) can be blocked by adding PRL with the corticoids. We found that (1) the sequences of the RT-PCR products obtained using primers for α-ENaC were identical between larval and adult skins, (2) the mRNAs for all three ENaC subunits were expressed in larval skin under normal conditions despite no amiloride-blockable Na+ transport being detectable, (3) all three subunits were expressed in larval skins whether they were cultured with corticoids (amiloride-blockable Na transport present) or with corticoids supplemented with PRL (no amiloride-blockable Na transport present). An antibody against a peptide from the α-ENaC of adult bullfrog was localized to the apical cells of both larval and adult skins. Since no amiloride-blockable Na transport exists across larval skin under these conditions, these results suggest that ENaC protein was expressed prior to the onset of transport. ENaC may be in the plasma membrane in an inactivated form or, alternatively, within vesicles waiting to be inserted.

Cloned bullfrog skin sodium (fENaC) and xENaC subunits hybridize to form functional sodium channels

Abstract. For many years the adult frog skin has been used as a model system to study transepithelial sodium transport. In other sodium transporting tissues the three homologous subunits of the sodium channel have been cloned. The aim of this study was to clone and characterize the amiloride inhibitable sodium channel (ENaC) in adult bullfrog (Rana catesbeiana) skin. Three transcripts corresponding to the α, β, and γ subunits of ENaC were cloned and sequenced. Co-expression of all three in Xenopus oocytes yielded a functional frog sodium channel (fENaC). Amiloride sensitivity and current voltage relationships suggested that its characteristics were similar to other ENaCs. Subunits from the Xenopus sodium channel (xENaC) and fENaC were combined in all possible triplets. Although functional amiloride inhibitable sodium channels were formed in every case, the amiloride sensitivities were not identical. Subunit combination studies suggested that the α subunit made a major contribution to amiloride sensitivity but interactions of β and γ were also seen. When the amiloride sensitivities of intact skin from adult R. catesbeiana and Xenopus laevis were compared, Rana also had a consistently higher affinity. Comparison of fENaC and xENaC sequences may provide insight into which amino acids beyond those already identified are critical for amiloride binding.

Differential and interactive effects of ligand-bound progesterone receptor A and B isoforms on tyrosine hydroxylase promoter activity.

The classical progesterone receptors (PRs) are expressed in some hypothalamic dopaminergic and brainstem noradrenergic neurones. Progesterone influences prolactin and luteinising hormone release from the anterior pituitary gland, in part by regulating the activity of these catecholaminergic neurones. The present study aimed to determine the effects of PRs on tyrosine hydroxylase (TH) promoter activity. When CAD, SK‐N‐SH and CV‐1 cells were transfected with TH promoter constructs and PR‐A or PR‐B expression vectors, progesterone treatment caused three‐ to six‐fold increases in TH‐9.0 kb promoter activity in PR‐B expressing cells, although only a modest increase or no change in PR‐A expressing cells. Using CAD cells, deletional analysis mapped the site of PR action to the −1403 to −1304 bp region of the TH promoter. Mutational analysis of putative regulatory sequences in this region indicated that multiple DNA elements are required for complete PR‐B transactivation. Electrophoretic mobility shift assays were unable to demonstrate direct PR‐B binding to TH promoter DNA sequences. However, chromatin immunoprecipitation analysis indicated PR‐B was recruited to the TH promoter. Two different PR‐B DNA binding domain mutants had opposing effects on PR‐B‐mediated TH promoter activation. A GS to AA mutation located in the p‐box of the first zinc finger of PR‐B inhibited progesterone transactivation of the TH promoter, whereas a C to A mutation in the zinc finger increased transactivation. PR‐A was able to inhibit PR‐B transactivation in a dose‐dependent manner, although the degree of PR‐A inhibition was dependent on the TH promoter deletion construct. These data indicate that ligand‐bound PR‐B is recruited to DNA elements in the TH promoter and acts as a transcriptional activator of the TH gene, and also that changes in the ratio of PR‐A to PR‐B may affect the ability of progesterone to increase TH expression.

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.

DEAF1 Binds Unmethylated and Variably Spaced CpG Dinucleotide Motifs

DEAF1 is a transcriptional regulator associated with autoimmune and neurological disorders and is known to bind TTCG motifs. To further ascertain preferred DEAF1 DNA ligands, we screened a random oligonucleotide library containing an “anchored” CpG motif. We identified a binding consensus that generally conformed to a repeated TTCGGG motif, with the two invariant CpG dinucleotides separated by 6–11 nucleotides. Alteration of the consensus surrounding the dual CpG dinucleotides, or cytosine methylation of a single CpG half-site, eliminated DEAF1 binding. A sequence within the Htr1a promoter that resembles the binding consensus but contains a single CpG motif was confirmed to have low affinity binding with DEAF1. A DEAF1 binding consensus was identified in the EIF4G3 promoter and ChIP assay showed endogenous DEAF1 was bound to the region. We conclude that DEAF1 preferentially binds variably spaced and unmethylated CpG-containing half-sites when they occur within an appropriate consensus.

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.

Regulation of cytokine-inducible SH2-containing protein (CIS) by ubiquitination and Elongin B/C interaction.

Cytokine-inducible SH2-containing protein (CIS) inhibits prolactin receptor (PRLR) signaling and acts as part of an E3 ubiquitin ligase complex through interactions with Elongin B/C proteins. This study aimed to identify CIS lysine ubiquitination sites and determine roles of ubiquitination and Elongin B/C interactions on CIS protein stability and PRLR signaling inhibition. Site-directed mutations revealed that CIS can be ubiquitinated on all six lysine residues. Elongin B/C interaction box mutation had no influence on CIS ubiquitination. CIS stability was increased by mutation of lysine residues and further enhanced by co-mutation of Elongin B/C interaction domain. CIS inhibition of STAT5B phosphorylation and casein promoter activation was dependent on CIS interactions with Elongin B/C, but not on CIS ubiquitination. These data indicate CIS protein stability is regulated through multiple mechanisms, including ubiquitination and interaction with Elongin B/C proteins, whereas CIS functional inhibition of PRLR signaling is dependent on the Elongin B/C interaction.

Larval bullfrog skin lacks amiloride-blockable epithelial transport because α-ENaC is located within intracellular vesicles in epidermal apical cells and not in the apical plasma membrane

The epithelial Na channel (ENaC) plays an essential role in sodium transport across epithelia such as adult frog skin. Transport across the skin, measured as short-circuit current (SCC), is blocked by amiloride. Bullfrog alpha-ENaC (α-fENaC) is expressed in adult bullfrog skin, and the SCC across this skin is blocked by amiloride. In contrast, an amiloride-blockable SCC is not detected in larval bullfrog skin, even though it expresses α-fENaC. We examined the subcellular localization of α-ENaC in such larval and adult skins. Immunofluorescent and immunoelectron microscopy of apical cells in the larval epidermis revealed α-fENaC localization within intracellular vesicles, but not in the plasma membrane. In contrast, in adult skin α-fENaC was localized to the apical-side membrane and to intracellular vesicles in Stratum granulosum cells. This may support the view that amiloride-blockable SCC is absent from larval skin, but is present in adult skin.

α-ENaC in bullfrog embryo: expression in cement gland, gills and skin

The epithelial sodium channel (ENaC) is involved in Na+ responses such as Na+ absorption and salt taste. The alpha ENaC subunit (α-ENaC) is expressed in the skin of both the adult and larval (tadpole) bullfrog. α-ENaC expression in the developing bullfrog embryo has not been previously investigated. In this study, the expression of α-ENaC at various stages (Sts.) of bullfrog embryonic development is assessed by western blot and immunofluorescence analysis. Bullfrog α-ENaC (α-fENaC) protein was detected by western blot in embryos at Sts. (Gosner/Shumway) 19, 21 and 25. Immunofluorescence studies indicate that α-fENaC was localized to the embryonic cement glands at St. 18 (muscular response), St. 19 (heart beat) and St. 21 (mouth open and/or cornea transparent), to the external gills at St. 21 and to the outermost cell-layer of the skin at St. 25 (operculum complete). The function(s) of ENaC in these embryonic structures remain to be elucidated.