DAD1 Gene Research Articles

New variants of the DAD1 and OXA1L genes are associated with asthma and atopy in an adult population

Asthma is a complex disease characterized by reversible and intermittent airway obstruction that has shown a high prevalence and unacceptable mortality in adults. In recent years, several genome-wide association studies (GWAS) have identified variants linked to asthma susceptibility. The DAD1 gene is known for regulating programmed cell death, and OXA1L is described for its involvement in mitochondrial biogenesis and oxidative phosphorylation. The present study aimed to identify variants in the DAD1 and OXA1L genes and to evaluate the association with asthma and atopy markers in adults. 1,084 individuals were divided into mild to moderate asthma, severe asthma, and participants with no asthma (controls). Association analyses were performed using a multivariate logistic regression model adjusted for sex, age, body mass index (BMI), smoking, forced expiratory volume in 1 s (FEV1), and component ancestry master (PC1) using PLINK 1.9 software. This study identified new variants in the DAD1 and OXA1L genes that had never been described before. The C allele of rs200470407 in OXA1L was negatively associated with poor asthma control (OR: 0.32; p-value 0.049) and increased IL-13 (p-value < 0.0001). The alternative allele of rs1681577 was associated with severe asthma (OR: 2.23; p-value 0.01), pulmonary obstruction (OR: 4.12; p-value 0.046), and eosinophilia (OR: 2.42; p-value < 0.001). Our findings demonstrate that variants in the DAD1 and OXA1L genes are linked to asthma and atopy in Brazilian adults.

Konjenital Bilateral Vas Deferens Yokluğu Olan Türk Hastalarda Genomik Kopya Sayısı Varyasyonları Analizi

Aim: Congenital Bilateral Absence of the Vas Deferens (CBAVD) is a developmental abnormality that causes infertility in males. According to the literature, up to 88% of CBAVD cases have at least one pathogenic Cystic Fibrosis Transmembrane Conductance Regulator gene (CFTR) mutation. However, based on our previous data, this rate was 15.90% in Turkish patients with CBAVD. We aimed to identify genomic copy number variations (CNV) and candidate genomic regions which could related to the CBAVD in Turkish population.Methods: CNV analysis was performed in 19 Turkish CBAVD patients normal karyotypes and a wild type CFTR genotype. We suggested that the DAD1 gene may be a candidate gene related to CBAVD by reviewing online databases and analyzing CNV findings. Sanger sequencing of the DAD1 gene exons was performed in 22 patients.Results: We identified 11 CNVs that most likely related with the disease in nine of 19 (47.3%) patients. As the most common CNV, 14q11.2 deletions were detected in there (15.79%) of the patients. There was only DAD1 gene in the sharing genomic region of two of the 14q11.2 deletions. No sequence variation was detected in the DAD1 gene of the patients.Conclusion: The 14q11.2 chromosomal region and the DAD1 gene may be associated with CBAVD. Further studies are needed to indentify the contribution of CNVs and DAD1 gene to CBAVD etiology.

A role of the CTCF binding site at enhancer Eα in the dynamic chromatin organization of the Tcra-Tcrd locus.

The regulation of T cell receptor Tcra gene rearrangement has been extensively studied. The enhancer Eα plays an essential role in Tcra rearrangement by establishing a recombination centre in the Jα array and a chromatin hub for interactions between Vα and Jα genes. But the mechanism of the Eα and its downstream CTCF binding site (here named EACBE) in dynamic chromatin regulation is unknown. The Hi-C data showed that the EACBE is located at the sub-TAD boundary which separates the Tcra–Tcrd locus and the downstream region including the Dad1 gene. The EACBE is required for long-distance regulation of the Eα on the proximal Vα genes, and its deletion impaired the Tcra rearrangement. We also noticed that the EACBE and Eα regulate the genes in the downstream sub-TAD via asymmetric chromatin extrusion. This study provides a new insight into the role of CTCF binding sites at TAD boundaries in gene regulation.

Changes in the promoter of a defender against apoptotic cell death gene affect its expression in upland cotton

AbstractThe DAD1 (defender against apoptotic cell death) gene is a negative regulator of programmed cell death. It plays important roles in plant growth, development, environmental adaptation, and aging. We examined whether and how the expression of DAD1 gene is affected by the promoter changes in the allotetraploid upland cotton (Gossypium hirsutum L.). We compared the expression status of the genes in the leaves of an elite cultivar and a wild variety of G. hirsutum, and cloned and analyzed a 1.3‐kb promoter region of the genes in two cotton types. Results revealed that the expression of a subgenomic homoeolog of DAD1 (DAD1 At ) was significantly upregulated in leaves of the cultivated cotton when compared with that in a wild cotton. In addition, we identified 18 variations, including 14 single nucleotide polymorphisms and four indels between the promoters of the two cotton types. We detected at least 10 of 18 variations in the promoters related to possible cis‐regulatory elements. Further transient tobacco assays using heterogeneous DAD1 At promoter::GUS fusion showed that the activity of the DAD1 At promoter in the cultivated cotton was stronger than that in the wild cotton, which suggested a dynamic change in the DAD1 At promoter during genetic divergence of the two cottons. This study provides an example of how gene expression could be affected by specific changes in the gene promoter.

Molecular characterization of a defender against apoptotic cell death 1 gene (CfDAD1) from the mollusk Chlamys farreri

The defender against apoptotic cell death 1 (DAD1) was a negative regulator to inhibit the apoptosis process. In the present study, a DAD1 gene (designated as CfDAD1) was identified inZhikong scallop Chlamys farreri. Its full-length cDNA sequence of CfDAD1 contained a 342 bp open reading frame (ORF), which encoded a mature protein of 113 amino acids. A DAD domain was revealed from the deduced protein sequence of CfDAD1. The mRNA transcripts of CfDAD1 could be detectable in all the investigated tissues, including hemocytes, muscle, mantle, gill, hepatopancreas and gonad, among which the maximum expression level were found in hemocytes. Being infected with Vibrio splendidus, Staphylococcus aureus or Yarrowia lipolytica, the mRNA expression levels of CfDAD1 in hemocytes were all significantly up-regulated. Moreover, the apoptosis level of CfDAD1-suppressed group was significant higher than those of control groups. All these results indicated that CfDAD1 was efficient negative regulator of apoptosis and involved in the innate immune responses of scallop.

Polymorphisms in the DAD1 and OXA1L genes are associated with asthma and atopy in a South American population

Atopic asthma, which is characterized by the chronic inflammation and morbidity of airways, is a disease of great complexity, and multiple genetic and environmental factors are involved in its etiology. In the first genome-wide association study (GWAS) conducted in Brazil for asthma, a positive association was found between atopic asthma and a variant (rs1999071), which is located between the DAD1 and OXA1L genes, although neither gene has previously been reported to be associated with asthma or allergies. The DAD1 gene is involved in the regulation of programmed cell death, and OXA1L is involved in biogenesis and mitochondrial oxidative phosphorylation. This study aimed to evaluate how polymorphisms in DAD1 and OXA1L are associated with asthma and markers of atopy in individuals from the Salvador cohort of the SCAALA (Social Change Asthma and Allergy in Latin America) program. The DNA of 1220 individuals was genotyped using the Illumina 2.5 Human Omni Bead chip. Logistic regression analyses were performed with PLINK 1.9 software to verify the association between DAD1 and OXA1L polymorphisms and asthma and atopic markers, adjusted for sex, age, helminth infections and ancestry markers, using an additive model. The DAD1 and OXA1L genes were associated with some of the evaluated phenotypes, such as asthma, skin prick test (SPT), specific IgE for aeroallergens, and Th1/Th2-type cytokine production. Using qPCR, as well as in silico gene expression analysis, we have demonstrated that some of the polymorphisms in both genes are able to affect their respective gene expression levels. In addition, DAD1 was over-expressed in asthmatic patients when compared with controls. Thus, our findings demonstrate that variants in both the DAD1 and OXA1L genes may affect atopy and asthma in a Latin American population with a high prevalence of asthma.

Improved peripheral T cell expression of T-cell receptor (TCR)-α transgenes in TCRα/Dad1 locus-like "two gene" reporter systems (136.14)

Abstract The mouse T-cell receptor (TCR)-a; locus control region (LCR) resides in between the Ca; exons and the Dad1 gene. We reported the "single cell level" activity of the TCRa LCR in the context of linked heterologous single reporter transgenes randomly integrated into the genome of mice. We found that the LCR supports the normal pattern of TCRa expression in the thymus. However, abnormal ectopic expression and T cell transcript reductions were observed in the periphery. These data indicated that important cis-acting elements (not associated with the LCR) are required for strict cell type-specificity, and peripheral T cell maintenance of TCRa expression. Here we describe data from experiments with transgenic mice bearing constructs in which the LCR is flanked by two transcription units (as it is in the genome by TCRa [5'] and Dad1 [3']). In the first system, two heterologous reporter genes flank the LCR. The other system is an 80-kb TCRa/Dad1 Bacterial Artificial Chromosome (BAC) fragment in which a Va promoter driven reporter is inserted upstream of the Ca exons, followed by the TCRa LCR and the Dad1 gene locus. We find that peripheral T cells maintain expression of the TCRa reporter gene in the BAC construct. Surprisingly, in both "two-gene" reporter systems, ectopic, non-T cell expression of the TCRa reporter is virtually eliminated. The data suggest that important cis-acting elements maintaining normal TCRa expression patterns in peripheral T cells reside in the Dad 1 gene locus. Supported by NIH grants AI053050 (to BDO), RR03037 (to the Hunter College Gene Center) and UL1RR024996 (to the Clinical and Translation Science Center at Weill Cornell Medical College).

CTCF‐independent, but not CTCF‐dependent, elements significantly contribute to the insulation capacity of a locus control region

Locus control regions (LCR) provide high-level, position-independent expression to a transgene in chromatin. They achieve this through DNA sequences supporting activities that enhance transcription and somehow protect transgene expression from integration site-dependent position effects. The former activity has been mapped to classical enhancer DNA sequences of most LCRs. In contrast, the elements supporting the latter capacity that suppresses position effects are less well understood. Insulator/Boundary sequences are thought to play a role in separating regulatory influence in the genome and support enhancer blocking and/or chromatin barrier activity. The LCR in the mouse T cell receptor (TCR)- 1 □/Dad1 gene locus contains a CTCF-dependent and multiple CTCF-independent enhancer blocking regions whose roles in LCR activity are unknown. Using randomly integrated reporter transgenes in mice, we find that the CTCF region plays a very minor role in LCR function. In contrast, we report the in vivo function of two additional downstream CTCF-independent elements within the LCR. Internal deletion of either of these elements significantly impairs LCR activity. Our results reveal that the position effect suppression capacity of an LCR arises largely from an array of CTCF-independent elements, some of which share characteristics with barrier-type insulators. Support: NIH #AI-053050 and NSF #MCB-0236964

Analysis of the DECREASED APICAL DOMINANCE genes of petunia in the control of axillary branching.

Control of branch development is a major determinant of architecture in plants. Branching in petunia (Petunia hybrida) is controlled by the DECREASED APICAL DOMINANCE (DAD) genes. Gene functions were investigated by plant grafting, morphology studies, double-mutant characterization, and gene expression analysis. Both dad1-1 and dad3 increased branching mutants can be reverted to a near-wild-type phenotype by grafting to a wild-type or a dad2 mutant root stock, indicating that both genes affect the production of a graft-transmissible substance that controls branching. Expression of the DAD1 gene in the stems of grafted plants, detected by quantitative reverse transcription-polymerase chain reaction correlates with the branching phenotype of the plants. The dad2-1 mutant cannot be reverted by grafting, indicating that this gene acts predominantly in the shoot of the plant. Double-mutant analysis indicates that the DAD2 gene acts in the same pathway as the DAD1 and DAD3 genes because the dad1-1dad2-1 and dad2-1dad3 double mutants are indistinguishable from the dad2-1 mutant. However, the dad1-1dad3 double mutant has an additive phenotype, with decreased height of the plants, delayed flowering, and reduced germination rates compared to the single mutants. This result, together with the observation that the dad1-1 and dad3 mutants cannot be reverted by grafting to each other, suggests that the DAD1 and DAD3 genes act in the same pathway, but not in a simple stepwise fashion.

Quantitative Expression of Growth Regulatory Genes in Matched, Eutopic and Ectopic Endometrial Cells from Women with Endometriosis

Quantitative Expression of Growth Regulatory Genes in Matched, Eutopic and Ectopic Endometrial Cells from Women with Endometriosis

Programmed cell death (PCD) processes begin extremely early in Alstroemeria petal senescence.

• In the Liliaceous species Alstroemeria, petal senescence is characterized by wilting and inrolling, terminating in abscission 8-10d after flower opening. • In many species, flower development and senescence involves programmed cell death (PCD). PCD in Alstroemeria petals was investigated by light (LM) and transmission electron microscopy (TEM) (to study nuclear degradation and cellular integrity), DNA laddering and the expression programme of the DAD-1 gene. • TEM showed nuclear and cellular degradation commenced before the flowers were fully open and that epidermal cells remained intact whilst the mesophyll cells degenerated completely. DNA laddering increased throughout petal development. Expression of the ALSDAD-1 partial cDNA was shown to be downregulated after flower opening. • We conclude that some PCD processes are started extremely early and proceed throughout flower opening and senescence, whereas others occur more rapidly between stages 4-6 (i.e. postanthesis). The spatial distribution of PCD across the petals is discussed. Several molecular and physiological markers of PCD are present during Alstroemeria petal senescence.

Function and factor interactions of a locus control region element in the mouse T cell receptor-alpha/Dad1 gene locus.

Locus control regions (LCRs) refer to cis-acting elements composed of several DNase I hypersensitive sites, which synergize to protect transgenes from integration-site dependent effects in a tissue-specific manner. LCRs have been identified in many immunologically important gene loci, including one between the TCRdelta/TCRalpha gene segments and the ubiquitously expressed Dad1 gene. Expression of a transgene under the control of all the LCR elements is T cell specific. However, a subfragment of this LCR is functional in a wide variety of tissues. How a ubiquitously active element can participate in tissue-restricted LCR activity is not clear. In this study, we localize the ubiquitously active sequences of the TCR-alpha LCR to an 800-bp region containing a prominent DNase hypersensitive site. In isolation, the activity in this region suppresses position effect transgene silencing in many tissues. A combination of in vivo footprint examination of this element in widely active transgene and EMSAs revealed tissue-unrestricted factor occupancy patterns and binding of several ubiquitously expressed transcription factors. In contrast, tissue-specific, differential protein occupancies at this element were observed in the endogenous locus or full-length LCR transgene. We identified tissue-restricted AML-1 and Elf-1 as proteins that potentially act via this element. These data demonstrate that a widely active LCR module can synergize with other LCR components to produce tissue-specific LCR activity through differential protein occupancy and function and provide evidence to support a role for this LCR module in the regulation of both TCR and Dad1 genes.

The DEFECTIVE IN ANTHER DEHISCIENCE gene encodes a novel phospholipase A1 catalyzing the initial step of jasmonic acid biosynthesis, which synchronizes pollen maturation, anther dehiscence, and flower opening in Arabidopsis.

The Arabidopsis mutant defective in anther dehiscence1 (dad1) shows defects in anther dehiscence, pollen maturation, and flower opening. The defects were rescued by the exogenous application of jasmonic acid (JA) or linolenic acid, which is consistent with the reduced accumulation of JA in the dad1 flower buds. We identified the DAD1 gene by T-DNA tagging, which is characteristic to a putative N-terminal transit peptide and a conserved motif found in lipase active sites. DAD1 protein expressed in Escherichia coli hydrolyzed phospholipids in an sn-1-specific manner, and DAD1-green fluorescent protein fusion protein expressed in leaf epidermal cells localized predominantly in chloroplasts. These results indicate that the DAD1 protein is a chloroplastic phospholipase A1 that catalyzes the initial step of JA biosynthesis. DAD1 promoter::beta-glucuronidase analysis revealed that the expression of DAD1 is restricted in the stamen filaments. A model is presented in which JA synthesized in the filaments regulates the water transport in stamens and petals.

Inhibition of N-Linked Glycosylation Causes Apoptosis in Hamster BHK21 Cells

The tsBN7 cell line is one of the temperature-sensitive mutants for cell proliferation derived from hamster BHK21 cell line. It has a mutation in the DAD1 gene and enters apoptosis at the restrictive temperature of 39°C. The defect of Dad1p causes a loss of N-linked glycosylation; therefore, it was thought that an inhibition of N-linked glycosylation induced apoptosis.However, tunicamycin, a potent inhibitor of N-linked glycosylation, had not caused apoptosis in wild-type BHK21 cells. In order to clarify this discrepancy, wild-type BHK21 cells treated with tunicamycin and tsBN7 cells incubated at 39.5°C were examined by the annexin V staining and TUNEL methods. Both methods showed that tunicamycin induces apoptosis in wild-type BHK21 cells, similar to the defect of Dad1p. Thus, we concluded that loss of N-linked glycosylation causes apoptosis.

A novel human processed gene, DAD-R, maps to 12p12 and is expressed in several organs

A cDNA of a processed gene of human DAD-1 (defender against apoptotic cell death) was cloned from the human neuroblastoma cell line SH-SY5Y. The genomic sequence of this novel processed gene, DAD-R, lacked introns and was flanked by 8 bp terminal repeats. RT-PCR showed that the transcript is expressed predominantly in testis, ovaries, pancreas, lung and skeletal muscle. DAD-R has several possible initiation codons, one of them producing an open reading frame comprising 75% of the DAD-1 gene. We determined the chromosomal localization of DAD-R as 12p11.2–12p12.1, an area linked to familial synpolydactyly and frequently amplified in a variety of cancers, including those of testis, ovaries, pancreas and lungs.

Molecular cloning of a homologue of dad-1 gene in citrus: distinctive expression during fruit development

A cDNA homologue to the human defender against apoptotic death gene ( dad-1), which is involved in programmed cell death, was isolated from satsuma mandarin ( Citrus unshiu Marc.) fruit. It (Citdad-1-1) was 345 bp long, with a deduced protein sequence of 115 amino acids. Southern hybridization suggests that dad-1-related sequences are present as a small gene family in the citrus genome. Expression of Citdad-1-1 was progressively down-regulated in leaves as they matured, but not in juice sac/segment epidermis (edible part) towards fruit ripening. The role of dad-1 during citrus development is also discussed.

Enhancer-Blocking Activity Within the DNase I Hypersensitive Site 2 to 6 Region Between the TCR α and Dad1 Genes

AbstractAlthough tightly linked, the TCR α and δ genes are expressed specifically in T lymphocytes, whereas the Dad1 gene is ubiquitously expressed. Between TCR α and Dad1 are eight DNase I hypersensitive sites (HS). HS1 colocalizes with the TCR α enhancer (Eα) and is T cell-specific; HS2, -3, -4, -5, and -6 map downstream of HS1 and are tissue-nonspecific. The region spanning HS2–6 was reported to display chromatin-opening activity and to confer copy number-dependent and integration site-independent transgene expression in transgenic mice. Here, we demonstrate that HS2–6 also displays enhancer-blocking activity, as it can block an enhancer from activating a promoter when located between the two in a chromatin-integrated context, and can do so without repressing either the enhancer or the promoter. Multiple enhancer-blocking elements are arrayed across HS2–6. We show that HS2–6 by itself does not activate transcription in chromatin context, but can synergize with an enhancer when located upstream of an enhancer and promoter. We propose that HS2–6 primarily functions as an insulator or boundary element that may be critical for the autonomous regulation of the TCR α and Dad1 genes.

Abnormalities of developmental cell death in Dad1-deficient mice.

Dad1, the defender against apoptotic cell death, comprises the oligosaccharyltransferase complex and is well conserved among eukaryotes. In hamster BHK21-derived tsBN7 cells, loss of Dad1 causes apoptosis which cannot be prevented by Bcl-2. To determine the role of Dad1 function in vivo, we prepared by gene targeting, mice harbouring a disrupted Dad1 gene. Homozygous mutants died shortly after they were implanted with the characteristic features of apoptosis. In an in vitro blastocyst culture system, Dad1-null cells displayed abnormalities which were comparable to those obtained in vivo. However, oligosaccharyltransferase activity was apparently retained even after the Dad1-null cells were destined to die. Some live-born heterozygous mutants displayed soft-tissue syndactyly. Mild thymic hypoplasia was also indicated in heterozygotes. These results suggest the involvement of the Dad1 gene in the acquisition of a common syndactyly phenotype, as well as in the control of programmed cell death during development.

DAD1 Is Required for the Function and the Structural Integrity of the Oligosaccharyltransferase Complex

Asparagine-linked glycosylation is a highly conserved protein modification reaction that occurs in all eukaryotic organisms. The oligosaccharyltransferase (OST), which has its active site exposed on the luminal face of the endoplasmic reticulum (ER), catalyzes the transfer of preassembled high mannose oligosaccharides onto certain asparagine residues of nascent polypeptides. The mammalian OST complex was initially thought to be composed of three transmembrane proteins, ribophorin I (RI), ribophorin II (RII), and OST48. Most recently, a small integral membrane protein of 12 kDa called DAD1 has been identified as an additional member of the mammalian OST complex. A point mutation in the DAD1 gene is responsible for the temperature-sensitive phenotype of a baby hamster kidney-derived cell line (tsBN7) that undergoes apoptosis at the non-permissive temperature. Furthermore, the mutant protein DAD1 is not detectable in tsBN7 cells 6 h after shifting the cells to the non-permissive temperature. This temperature-sensitive cell line offered unique opportunities to study the effects caused by the loss of one OST subunit on the other three subunits and also on N-linked glycosylation. Western blot analysis of cell lysates showed that after 6 h at the non-permissive temperature, steady-state levels of the ribophorins were reduced by about 50%, and OST48 was barely detectable. On the other hand, steady-state levels of other components of the rough ER, such as the alpha-subunits of the TRAP (translocon-associated membrane protein) and the Sec61 complex, which are components of the translocation apparatus, are not affected by the instability of the OST subunits. Furthermore, N-glycosylation of the ribophorins was seriously affected 6 h after shifting the cells to the non-permissive temperature, and after 12 h they were synthesized only in the non-glycosylated form. As may be expected, this defect in the OST complex at the non-permissive temperature caused also the underglycosylation of a secretory glycoprotein. We concluded that degradation of DAD1 at the non-permissive temperature not only affects the stability of OST48 and the ribophorins but also results in the functional inactivation of the OST complex.

Interactions among Subunits of the Oligosaccharyltransferase Complex

The mammalian oligosaccharyltransferase (OST) is an oligomeric complex composed of three membrane proteins of the endoplasmic reticulum: ribophorin I (RI), ribophorin II (RII), and OST48. In addition, sequence homology between the Ost2 subunit of the yeast OST complex and Dad1 (defender against apoptotic death) suggests that Dad1 may represent a fourth subunit of the mammalian OST complex. In attempts to elucidate the structural organization of this complex, we have studied the interactions among its subunits. Using the yeast two-hybrid system, we have shown that the luminal domains of RI and RII (RIL and RIIL, respectively) interacted with the luminal domain of OST48 (OST48L), but no direct interaction was observed between RIL and RIIL. These results were confirmed by biochemical assays. Deletion analyses using the yeast two-hybrid system showed that subdomain of RIL or RIIL adjacent to the respective transmembrane domains interacted with OST48L. Of the three equal length subdomains of OST48L, the one at the N terminus and the one next to the transmembrane domain interacted with RIL. None of these three subdomains of OST48L interacted with RIIL. The yeast two-hybrid assay also revealed affinity between the cytoplasmically located N-terminal region of Dad1 and the short cytoplasmic tail of OST48, thus placing Dad1 firmly into the OST complex. In addition, we found a homotypic interaction between the cytoplasmic domains of RI, which may play a role in the formation of the oligomeric array formed by components of the translocation machinery.