SEDLIN forms homodimers: characterisation of SEDLIN mutations and their interactions with transcription factors MBP1, PITX1 and SF1

BACKGROUND SEDLIN, a 140 amino acid subunit of the Transport Protein Particle (TRAPP) complex, is ubiquitously expressed and interacts with the transcription factors c-myc promoter-binding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1). SEDLIN mutations cause X-linked spondyloepiphyseal dysplasia tarda (SEDT). METHODOLOGY/PRINCIPAL FINDINGS We investigated the effects of 4 missense (Asp47Tyr, Ser73Leu, Phe83Ser and Val130Asp) and the most C-terminal nonsense (Gln131Stop) SEDT-associated mutations on interactions with MBP1, PITX1 and SF1 by expression in COS7 cells. Wild-type SEDLIN was present in the cytoplasm and nucleus and interacted with MBP1, PITX1 and SF1; the SEDLIN mutations did not alter these subcellular localizations or the interactions. However, SEDLIN was found to homodimerize, and the formation of dimers between wild-type and mutant SEDLIN would mask a loss in these interactions. A mammalian SEDLIN null cell-line is not available, and the interactions between SEDLIN and the transcription factors were therefore investigated in yeast, which does not endogenously express SEDLIN. This revealed that all the SEDT mutations, except Asp47Tyr, lead to a loss of interaction with MBP1, PITX1 and SF1. Three-dimensional modelling studies of SEDLIN revealed that Asp47 resides on the surface whereas all the other mutant residues lie within the hydrophobic core of the protein, and hence are likely to affect the correct folding of SEDLIN and thereby disrupt protein-protein interactions. CONCLUSIONS/SIGNIFICANCE Our studies demonstrate that SEDLIN is present in the nucleus, forms homodimers and that SEDT-associated mutations cause a loss of interaction with the transcription factors MBP1, PITX1 and SF1.This work was supported by the Oliver Bird Fund (Studentship No. RHE/00029/G), The Nuffield Foundation (J.J.), Arthritis Research Campaign (Grant ID 16438) (M.A.N. and R.V.T.), European Community Framework 7 programme grant TREAT-OA (HEALTH-F2-2008-00) (M.A.N. and R.V.T.) and the Medical Research Council (J.J., M.A.N. and R.V.T.). J.J. was an Oliver Bird funded PhD student

Mice deleted for cell division cycle 73 gene develop parathyroid and uterine tumours:model for the hyperparathyroidism-jaw tumour syndrome

The hyperparathyroidism-jaw tumour (HPT-JT) syndrome is an autosomal dominant disorder characterized by occurrence of parathyroid tumours, often atypical adenomas and carcinomas, ossifying jaw fibromas, renal tumours and uterine benign and malignant neoplasms. HPT-JT is caused by mutations of the cell division cycle 73 (CDC73) gene, located on chromosome 1q31.2 and encodes a 531 amino acid protein, parafibromin. To facilitate in vivo studies of Cdc73 in tumourigenesis we generated conventional (Cdc73+/−) and conditional parathyroid-specific (Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre) mouse models. Mice were aged to 18-21 months and studied for survival, tumour development and proliferation, and serum biochemistry, and compared to age-matched wild-type (Cdc73+/+ and Cdc73+/+/PTH-Cre) littermates. Survival of Cdc73+/− mice, when compared to Cdc73+/+ mice was reduced (Cdc73+/−=80%; Cdc73+/+=90% at 18 months of age, Pfourfold higher than that in parathyroid glands of wild-type littermates (P<0.0001). Cdc73+/−, Cdc73+/L/PTH-Cre and Cdc73L/L/PTH-Cre mice had higher mean serum calcium concentrations than wild-type littermates, and Cdc73+/− mice also had increased mean serum parathyroid hormone (PTH) concentrations. Parathyroid tumour development, and elevations in serum calcium and PTH, were similar in males and females. Cdc73+/− mice did not develop bone or renal tumours but female Cdc73+/− mice, at 18 months of age, had uterine neoplasms comprising squamous metaplasia, adenofibroma and adenomyoma. Uterine neoplasms, myometria and jaw bones of Cdc73+/− mice had increased proliferation rates that were 2-fold higher than in Cdc73+/+ mice (P<0.05). Thus, our studies, which have established mouse models for parathyroid tumours and uterine neoplasms that develop in the HPT-JT syndrome, provide in vivo models for future studies of these tumours

De-Suppression of Mesenchymal Cell Identities and Variable Phenotypic Outcomes Associated with Knockout of Bbs1

Bardet–Biedl syndrome (BBS) is an archetypal ciliopathy caused by dysfunction of primary cilia. BBS affects multiple tissues, including the kidney, eye and hypothalamic satiety response. Understanding pan-tissue mechanisms of pathogenesis versus those which are tissue-specific, as well as gauging their associated inter-individual variation owing to genetic background and stochastic processes, is of paramount importance in syndromology. The BBSome is a membrane-trafficking and intraflagellar transport (IFT) adaptor protein complex formed by eight BBS proteins, including BBS1, which is the most commonly mutated gene in BBS. To investigate disease pathogenesis, we generated a series of clonal renal collecting duct IMCD3 cell lines carrying defined biallelic nonsense or frameshift mutations in Bbs1, as well as a panel of matching wild-type CRISPR control clones. Using a phenotypic screen and an unbiased multi-omics approach, we note significant clonal variability for all assays, emphasising the importance of analysing panels of genetically defined clones. Our results suggest that BBS1 is required for the suppression of mesenchymal cell identities as the IMCD3 cell passage number increases. This was associated with a failure to express epithelial cell markers and tight junction formation, which was variable amongst clones. Transcriptomic analysis of hypothalamic preparations from BBS mutant mice, as well as BBS patient fibroblasts, suggested that dysregulation of epithelial-to-mesenchymal transition (EMT) genes is a general predisposing feature of BBS across tissues. Collectively, this work suggests that the dynamic stability of the BBSome is essential for the suppression of mesenchymal cell identities as epithelial cells differentiate

SEDLIN Forms Homodimers: Characterisation of SEDLIN Mutations and Their Interactions with Transcription Factors MBP1, PITX1 and SF1

BACKGROUND: SEDLIN, a 140 amino acid subunit of the Transport Protein Particle (TRAPP) complex, is ubiquitously expressed and interacts with the transcription factors c-myc promoter-binding protein 1 (MBP1), pituitary homeobox 1 (PITX1) and steroidogenic factor 1 (SF1). SEDLIN mutations cause X-linked spondyloepiphyseal dysplasia tarda (SEDT). METHODOLOGY/PRINCIPAL FINDINGS: We investigated the effects of 4 missense (Asp47Tyr, Ser73Leu, Phe83Ser and Val130Asp) and the most C-terminal nonsense (Gln131Stop) SEDT-associated mutations on interactions with MBP1, PITX1 and SF1 by expression in COS7 cells. Wild-type SEDLIN was present in the cytoplasm and nucleus and interacted with MBP1, PITX1 and SF1; the SEDLIN mutations did not alter these subcellular localizations or the interactions. However, SEDLIN was found to homodimerize, and the formation of dimers between wild-type and mutant SEDLIN would mask a loss in these interactions. A mammalian SEDLIN null cell-line is not available, and the interactions between SEDLIN and the transcription factors were therefore investigated in yeast, which does not endogenously express SEDLIN. This revealed that all the SEDT mutations, except Asp47Tyr, lead to a loss of interaction with MBP1, PITX1 and SF1. Three-dimensional modelling studies of SEDLIN revealed that Asp47 resides on the surface whereas all the other mutant residues lie within the hydrophobic core of the protein, and hence are likely to affect the correct folding of SEDLIN and thereby disrupt protein-protein interactions. CONCLUSIONS/SIGNIFICANCE: Our studies demonstrate that SEDLIN is present in the nucleus, forms homodimers and that SEDT-associated mutations cause a loss of interaction with the transcription factors MBP1, PITX1 and SF1

Role of sedlin in spondyloepiphyseal dysplasia tarda

EThOS - Electronic Theses Online ServiceGBUnited Kingdo

Interactions between SEDLIN, MBP1, PITX1 and SF1 using the yeast two-hybrid assay.

<p>Yeast cells, which do not have endogenous expression of SEDLIN, were used to investigate the interactions of wild-type or mutant SEDLIN (Asp47Tyr, Ser73Leu, Phe83Ser, Val130Asp and Gln131Stop) with wild-type SEDLIN, MBP1, PITX1 or SF1. The yeast reporter strain AH109 was used, and p53 and the SV40 large T antigen, which are known to interact <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010646#pone.0010646-Iwabuchi1" target="_blank">[27]</a>, were used as a positive control. The yeasts were transformed with the vectors containing: (A) wild-type SEDLIN in pGADT7-AD (AD-WT) and either wild-type or mutant SEDLINs in pGBKT7-BD (BD-WT, BD-Asp47Tyr, BD-Ser73Leu, BD-Phe83Ser, BD-Val130Asp or BD-Gln131Stop). (B) wild-type and mutant AD-SEDLINs or BD-SEDLIN and each of the transcription factors BD-MBP1, AD-PITX1, AD-SF1. Yeast growth was monitored for 48 hrs after spotting and incubation at 30°C using either double drop out, DDO (Leu^-Trp^-), media as a control or quaternary drop out, QDO (Leu^-Trp^-Ade^-His^-), media in which the growth is dependent on the physical interaction between BD-SEDLIN and AD-transcription factors, or AD-SEDLIN and the BD-transcription factor. The wild-type SEDLIN interacts with wild-type SEDLIN and all of the mutant SEDLIN proteins, consistent with the proposed model for the formation of homodimers. However, the MBP1, PITX1 and SF1 fusion proteins, which interact with the wild-type SEDLIN, interacted only with the mutant Asp47Tyr SEDLIN, but not with the mutant Ser73Leu, Phe83Ser, Val130Asp and Gln131Stop SEDLINs.</p

Interactions between SEDLIN, and MBP1, PITX1 and SF1.

<p>Co-immunoprecipitation studies using COS7 cells demonstrated interactions between wild-type SEDLIN, mutant SEDLINs (Asp47Tyr, Ser73Leu, Phe83Ser, Val130Asp and Gln131Stop, only data from Ser73Leu SEDLIN shown) and MBP1, PITX1 and SF1. (A) cMyc-MBP1 co-transfected with wild-type or mutant HA-SEDLIN; (B) cMyc-PITX1 co-transfected with wild-type or mutant HA-SEDLIN; (C) cMyc-SF1 co-transfected with wild-type or mutant HA-SEDLIN; and (D) empty cMyc vector co-transfected with HA-SEDLIN or empty HA vector co-transfected with cMyc-MBP1, cMyc-PITX1 or cMyc-SF1 constructs. Lysates were incubated with either anti-cMyc polyclonal antibody (M) or anti-HA polyclonal antibody (H), or without any antibody as a negative control (−) and immunoprecipitated with Protein G-Sepharose beads. Protein complexes were eluted and resolved on SDS-PAGE followed by Western blot analysis using an antibody to the cMyc epitope for MBP1, PITX1 and SF1, and to the HA epitope for SEDLIN. Five percent of the lysate (input (I)) that was used for the immunoprecipitation was electrophoresed in parallel with the immunoprecipitated lysates. Wild-type SEDLIN co-immunoprecipitated MBP1, PITX1 and SF1 and the SEDLIN mutations (Asp47Tyr, Ser73Leu, Phe83Ser, Val130Asp and Gln131Stop) did not disrupt these interactions (representative data for mutant Ser73Leu SEDLIN shown).</p

Schematic representation of genomic organization of the SEDL gene, illustrating the 44 identified mutations.

<p>The human <i>SEDL</i> gene consists of six exons that span approximately 22 Kb of genomic DNA and encode a 140 amino acid protein. The 420bp coding region (open boxes) is organised into 4 exons (exon 3 to exon 6) and 3 introns (indicated by a line, not to scale). Non-coding exons (filled boxes) consist of exons 1 and 2, the 5′ portion of exon 3 and the 3′ portion of exon 6. The sizes of the exons, and the translation Start (ATG) and Stop (TGA) codons in exon 3 and 6, respectively, are indicated. The locations of the 44 mutations are indicated, and these consist of: 7 nonsense, 4 missense, 10 splice site, 1 insertion, 15 intraexonic deletions, and 7 deletions that encompassed introns and exons. The four missense mutations (Asp47Tyr, Ser73Leu, Phe83Ser and Val130Asp) and one nonsense mutation (Gln131Stop) were selected for further functional studies (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010646#pone-0010646-g003" target="_blank">Figures 3</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010646#pone-0010646-g004" target="_blank">4</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010646#pone-0010646-g006" target="_blank">6</a> and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010646#pone-0010646-g007" target="_blank">7</a>).</p

Three-dimensional model of SEDLIN showing locations of residues involved in the mutations studied.

<p>The model is based on a published model of mouse Sedlin <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0010646#pone.0010646-Jang1" target="_blank">[2]</a>. (A) A ribbon model of Sedlin showing the alpha helices in red, beta strands in blue and the residues involved in the missense mutations (Asp47Tyr, Ser73Leu, Phe83Ser, and Val130Asp) in yellow. The bold line indicates the 10 C-terminal amino acids that would be deleted by the nonsense SEDLIN mutation (Gln131Stop) shown in yellow. The dashed circle indicates the hydrophobic core of Sedlin. The Ser119 and Ser124 residues that were predicted to be phosphorylation sites (NetPhos2.0, MotifScan and ELM databases) are indicated in green. (B) An ∼45° rotated view of the ribbon model of Sedlin showing the SEDT-associated mutated residues Asp47 and Ser73 (yellow), in SEDT, which together with the residues Tyr60, Thr63, His67 and Gln91 (light blue) aid in forming the hydrophobic groove (indicated by dotted bar line). The alpha helices are shown in red, beta strands in blue, and the linker region in grey.</p