Ectopic Mutant Research Articles

IDH1 mutation impairs antiviral response and potentiates oncolytic virotherapy in glioma

IDH1 mutations frequently occur early in human glioma. While IDH1 mutation has been shown to promote gliomagenesis via DNA and histone methylation, little is known regarding its regulation in antiviral immunity. Here, we discover that IDH1 mutation inhibits virus-induced interferon (IFN) antiviral responses in glioma cells. Mechanistically, D2HG produced by mutant IDH1 enhances the binding of DNMT1 to IRF3/7 promoters such that IRF3/7 are downregulated, leading to impaired type I IFN response in glioma cells, which enhances the susceptibility of gliomas to viral infection. Furthermore, we identify DNMT1 as a potential biomarker predicting which IDH1mut gliomas are most likely to respond to oncolytic virus. Finally, both D2HG and ectopic mutant IDH1 can potentiate the replication and oncolytic efficacy of VSVΔ51 in female mouse models. These findings reveal a pivotal role for IDH1 mutation in regulating antiviral response and demonstrate that IDH1 mutation confers sensitivity to oncolytic virotherapy.

Mutations of the aminoacyl-tRNA-synthetases SARS and WARS2 are implicated in the etiology of autosomal recessive intellectual disability.

Intellectual disability (ID) is the hallmark of an extremely heterogeneous group of disorders that comprises a wide variety of syndromic and non-syndromic phenotypes. Here, we report on mutations in two aminoacyl-tRNA synthetases that are associated with ID in two unrelated Iranian families. In the first family, we identified a homozygous missense mutation (c.514G>A, p.Asp172Asn) in the cytoplasmic seryl-tRNA synthetase (SARS) gene. The mutation affects the enzymatic core domain of the protein and impairs its enzymatic activity, probably leading to reduced cytoplasmic tRNASer concentrations. The mutant protein was predicted to be unstable, which could be substantiated by investigating ectopic mutant SARS in transfected HEK293T cells. In the second family, we found a compound heterozygous genotype of the mitochondrial tryptophanyl-tRNA synthetase (WARS2) gene, comprising a nonsense mutation (c.325delA, p.Ser109Alafs*15), which very likely entails nonsense-mediated mRNA decay and a missense mutation (c.37T>G, p.Trp13Gly). The latter affects the mitochondrial localization signal of WARS2, causing protein mislocalization. Including AIMP1, which we have recently implicated in the etiology of ID, three genes with a role in tRNA-aminoacylation are now associated with this condition. We therefore suggest that the functional integrity of tRNAs in general is an important factor in the development and maintenance of human cognitive functions.

Enlargement of the Axial Length and Altered Ultrastructural Features of the Sclera in a Mutant Lumican Transgenic Mouse Model.

Lumican (LUM) is a candidate gene for myopia in the MYP3 locus. In this study, a mutant lumican (L199P) transgenic mouse model was established to investigate the axial length changes and ultrastructural features of the sclera. The mouse model was established by pronuclear microinjection. Transgenic mice and wild-type B6 mice were killed at eight weeks of age. Gene expression levels of LUM and collagen type I (COL1) in the sclera were analyzed by quantitative real-time polymerase chain reaction (qPCR), and the protein levels were assessed by Western blot analysis. Ocular axial lengths were measured on the enucleated whole eye under a dissecting microscope. Ultrastructural features of collagen fibrils in the sclera were examined with transmission electron microscopy (TEM). Lumican and collagen type I were both elevated at the transcriptional and protein levels. The mean axial length of eyes in the transgenic mice was significantly longer than that in the wild-type mice (3,231.0 ± 11.2 μm (transgenic group) vs 3,199.7 ± 11.1 μm (controls), p<0.05 =). Some ultrastructural changes were observed in the sclera of the transgenic mice under TEM, such as evident lamellar disorganizations and abnormal inter-fibril spacing. The average collagen fibril diameter was smaller than that in their wild-type counterparts. These results indicate that the ectopic mutant lumican (L199P) may induce enlargement of axial lengths and abnormal structures and distributions of collagen fibrils in mouse sclera. This transgenic mouse model can be used for the mechanistic study of myopia.

RNA-sequencing of Cercospora beticola DMI-sensitive and -resistant isolates after treatment with tetraconazole identifies common and contrasting pathway induction

Cercospora beticola causes Cercospora leaf spot of sugar beet. Cercospora leaf spot management measures often include application of the sterol demethylation inhibitor (DMI) class of fungicides. The reliance on DMIs and the consequent selection pressures imposed by their widespread use has led to the emergence of resistance in C. beticola populations. Insight into the molecular basis of tetraconazole resistance may lead to molecular tools to identify DMI-resistant strains for fungicide resistance management programs. Previous work has shown that expression of the gene encoding the DMI target enzyme (CYP51) is generally higher and inducible in DMI-resistant C. beticola field strains. In this study, we extended the molecular basis of DMI resistance in this pathosystem by profiling the transcriptional response of two C. beticola strains contrasting for resistance to tetraconazole. A majority of the genes in the ergosterol biosynthesis pathway were induced to similar levels in both strains with the exception of CbCyp51, which was induced several-fold higher in the DMI-resistant strain. In contrast, a secondary metabolite gene cluster was induced in the resistance strain, but repressed in the sensitive strain. Genes encoding proteins with various cell membrane fortification processes were induced in the resistance strain. Site-directed and ectopic mutants of candidate DMI-resistance genes all resulted in significantly higher EC50 values than the wild-type strain, suggesting that the cell wall and/or membrane modified as a result of the transformation process increased resistance to tetraconazole. Taken together, this study identifies important cell membrane components and provides insight into the molecular events underlying DMI resistance in C. beticola.

Abstract POSTER-BIOL-1349: The oncogenic role of DICER1 RNase IIIb domain mutations in ovarian sertoli-leydig cell tumors

Abstract DICER1, an endoribonuclease required for microRNA (miRNA) biogenesis, is essential for embryogenesis and development of many organs including ovary. Germline truncating mutations in DICER1 are seen in families with the pleuropulmonary blastoma-family tumour and dysplasia syndrome. These individuals have a propensity to develop ovarian Sertoli-Leydig cell tumors (SLCTs), a rare class of sex-cord stromal cell tumors that occur in young female. We have recently identified hotspot somatic mutations in RNase IIIb domain (simplified as ‘hotspot mutations’ thereafter) of DICER1 in half of SLCTs. These hotspot mutations impaired IIIb cleavage activity of DICER1 in in vitro cleavage assay and caused a global abolishment of 5p-miRNAs and a partial reduction of 3p-miRNAs in mouse embryonic stems cells expressing ectopic mutant DICER1. However, whether the miRNA biogenesis defect occurs in tumor samples and how DICER1 hotspot mutations promote tumorigenesis are largely unknown. By reviewing the histological features of ovarian SLCTs, we observed a strong correlation between DICER1 hotspot mutation and ovairain SLCTs with retiform pattern, which is often malignant with worse prognosis. Using Illumina small RNA sequencing technology and Exiqon miRNA realtime qPCR, we reported that the global expression of 5p-miRNAs was dramatically reduced in ovarian SLCTs carrying DICER1 hotspot mutations compared to their counterparts without DICER1 hotspot mutation. Although the 3p-miRNAs were largely unaffected, a subset of 3p-miRNAs was significantly increased in ovarian SLCTs with DICER1 hotspot mutations. To address the impact of DICER1 mutation on cell fate, we examined the gene expression profile in SLCTs using Illumina DASL whole transcriptome analysis followed by validation with Nanostring nCounter system. The miRNA production defect was associated with deregulation of genes governing cell fate determination in ovary, such as CYP19A1 (aromatase), FOXL2, FST, FGF9 and FGFR2, and genes that are targets of let-7, such as HMGA2, DICER1, ARID3A and CDC25A. Accordingly, knockin of DICER1 hotspot mutation in SVOG3e cells (immortalized human granulosa cells) led to altered miRNA biogenesis and deregulated expression of genes crucial for ovarian gonadal genesis and cellular transformation. SVOG3e Cells expressing DICER1 hotspot mutation grew faster than those with wildtype DICER1, which was at least partially due to loss of let-7 expression because inhibition of let-7 function by a miRNA sponge also accelerated cell growth. Furthermore, we also showed that loss of function of RNase IIIa domain of DICER1 had similar effect on cell proliferation as wildtype DICER1. Taken together, our data revealed that the hotspot somatic mutations of DICER1 in RNase IIIb domain cause systemic loss of 5p-miRNAs and selective upregulation of some 3p-miRNAs to drive pseudo-differentiation of testicular elements in ovary and cause oncogenic transformation. Thus, our current study sheds lights on the oncogenic potential of DICER1 hotspot mutations in tumor development and sets the stage for developing effective treatment method for malignant ovarian SLCTs. Citation Format: Yemin Wang, Jiamin Chen, Winnie Yang, Janine Senz, Michael S. Anglesio, Blake Gilks, Gregg B. Morin, David G. Huntsman. The oncogenic role of DICER1 RNase IIIb domain mutations in ovarian sertoli-leydig cell tumors [abstract]. In: Proceedings of the 10th Biennial Ovarian Cancer Research Symposium; Sep 8-9, 2014; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2015;21(16 Suppl):Abstract nr POSTER-BIOL-1349.

Codanin-1 Binds to Key Erythroid Genes and Its Knockdown Coupled with Ectopic Mutant Expression Recapitulates the Congenital Dyserythropoietic Anemia Type I (CDA I) Phenotype

Congenital dyserythropoietic anemia type I (CDA I) is an autosomal recessive disorder marked by specific morphological abnormalities of erythroblasts in the bone marrow, resulting in ineffective erythropoiesis and anemia. CDA I principally affects the erythroid lineage, while other non-erythroid hematopoietic lineages are unaffected. Light microscopy of bone marrow shows erythroid hyperplasia and binucleated erythroblasts connected by internuclear bridges. Electron microscopy of bone marrow demonstrates erythroblasts with spongy heterochromatin and invagination of the nuclear membrane. The gene responsible for causing CDA I, CDAN1, encodes for a ubiquitous protein, codanin-1, with unknown function. The goal of our study is to understand how codanin-1 regulates erythropoiesis and how codanin-1 defects result in CDA I.To determine codanin-1 expression pattern during erythroid differentiation, we analyzed codanin-1 RNA and protein levels in erythroid primary cells and cell lines. RNA-seq conducted in erythroid-induced human primary CD34+ cells revealed codanin-1 RNA expression is maintained in erythroid lineage. Similarly, immunoblotting shows codanin-1 protein is expressed at high levels in human erythroleukemia cell line stimulated down the erythroid lineage. Codanin-1 has been identified as a chromatin-binding protein. Thus, we conducted ChIP-seq analysis to determine the location of DNA binding sites for codanin-1 in K562 cells. The ChIP-seq results identified 2534 binding sites for codanin-1. Interestingly, many of these sites belong to key erythroid genes. Furthermore, 28% of these DNA binding sites overlap with GATA-1 bound sites.Because putative loss of function mutations in CDAN1 result in CDA I, we studied the effects of codanin-1 gene silencing on erythroid differentiation. shRNA-mediated knockdown reduced codanin-1 mRNA by 50% and protein expression levels by 90%. To assess the effects of codanin-1 perturbation on erythroid differentiation, hemoglobin synthesis was quantified using Drabkin’s reagent in which the absorbance measured at 540 nm is proportional to hemoglobin content in the sample. Erythroid-induced knockdown cells displayed decreased hemoglobin content when compared to non-targeting control shRNA (0.20 versus 0.48, measured in 3 separate experiments; p<0.01). In order to assess binuclearity of the knockdown cells, we stained cytospin slides with Wright Giemsa to assess nuclear morphology. Codanin-1 knockdown cells showed increased binuclearity when compared to control shRNA cells (6% versus 1%, measured in 3 separate experiments; p<0.01). Furthermore, we quantified changes in gene expression induced by codanin-1 gene silencing. Quantitative PCR showed that knockdown of codanin-1 decreased expression of key erythroid genes.To establish a robust CDA I cell system, the first well-characterized CDAN1 mutation (R1042W) was studied because codanin-1 deletion mutants are embryonic lethal in mice and do not occur in humans. Endogenous codanin-1 knockdown cells were transduced with mutant or wild type codanin-1 and induced down the erythroid lineage. Because CDA I is characterized by abnormal heterochromatin appearance, we conducted electron microscopy studies to examine the ultrastructural features. Studies show that knockdown of endogenous codanin-1 coupled with ectopic expression of mutant codanin-1 results in recapitulation of the CDA I phenotype in the K562 cell line model.Because CDA I is almost exclusively restricted to the erythroid lineage, understanding its pathophysiology has the potential to identify critical pathways in erythropoiesis. Here we show that the full phenotype of CDA I is recapitulated upon not only the loss of function of the codanin-1 protein but also on the expression of the mutant form of codanin-1. Our analysis demonstrates that codanin-1 binds to important erythroid loci, suggesting that codanin-1 affects the expression of erythroid genes. Further work will focus on studying codanin-1 function in human primary erythroid cells and in animal models. DisclosuresNo relevant conflicts of interest to declare.

Fusarium graminearum Tri12p Influences Virulence to Wheat and Trichothecene Accumulation

The gene Tri12 encodes a predicted major facilitator superfamily protein suggested to play a role in export of trichothecene mycotoxins produced by Fusarium spp. It is unclear, however, how the Tri12 protein (Tri12p) may influence trichothecene sensitivity and virulence of the wheat pathogen Fusarium graminearum. In this study, we establish a role for Tri12 in toxin accumulation and sensitivity as well as in pathogenicity toward wheat. Tri12 deletion mutants (tri12) are reduced in virulence and result in decreased trichothecene accumulation when inoculated on wheat compared with the wild-type strain or an ectopic mutant. Reduced radial growth of tri12 mutants on trichothecene biosynthesis induction medium was observed relative to the wild type and the ectopic strains. Diminished trichothecene accumulation was observed in liquid medium cultures inoculated with tri12 mutants. Wild-type fungal cells grown under conditions that induce trichothecene biosynthesis develop distinct subapical swelling and form large vacuoles. A strain expressing Tri12p linked to green fluorescent protein shows localization of the protein consistent with the plasma membrane. Our results indicate Tri12 plays a role in self-protection and influences toxin production and virulence of the fungus in planta.

Oncogenic KRAS-induced epiregulin overexpression contributes to aggressive phenotype and is a promising therapeutic target in non-small-cell lung cancer

KRAS mutations are one of the most common driver mutations in non-small-cell lung cancer (NSCLC) and finding druggable target molecules to inhibit oncogenic KRAS signaling is a significant challenge in NSCLC therapy. We recently identified epiregulin (EREG) as one of several putative transcriptional targets of oncogenic KRAS signaling in both KRAS-mutant NSCLC cells and immortalized bronchial epithelial cells expressing ectopic mutant KRAS. In the current study, we found that EREG is overexpressed in NSCLCs harboring KRAS, BRAF or EGFR mutations compared with NSCLCs with wild-type KRAS/BRAF/EGFR. Small interfering RNAs (siRNAs) targeting mutant KRAS, but not an siRNA targeting wild-type KRAS, significantly reduced EREG expression in KRAS-mutant and EREG-overexpressing NSCLC cell lines. In these cell lines, EREG expression was downregulated by MEK and ERK inhibitors. Importantly, EREG expression significantly correlated with KRAS expression or KRAS copy number in KRAS-mutant NSCLC cell lines. Further expression analysis using 89 NSCLC specimens showed that EREG was predominantly expressed in NSCLCs with pleural involvement, lymphatic permeation or vascular invasion and in KRAS-mutant adenocarcinomas. In addition, multivariate analysis revealed that EREG expression is an independent prognostic marker and EREG overexpression in combination with KRAS mutations was associated with an unfavorable prognosis for lung adenocarcinoma patients. In KRAS-mutant and EREG overexpressing NSCLC cells, siRNA-mediated EREG silencing inhibited anchorage-dependent and -independent growth and induced apoptosis. Our findings suggest that oncogenic KRAS-induced EREG overexpression contributes to an aggressive phenotype and could be a promising therapeutic target in oncogenic KRAS-driven NSCLC.

Meiotic silencing in the homothallic fungus Gibberella zeae

The homothallic ascomycete fungus Gibberella zeae is an important pathogen on major cereal crops. The objective of this study was to determine whether meiotic silencing occurs in G. zeae. Cytological studies demonstrated that GFP and RFP-fusion proteins were not detected during meiosis, both in heterozygous outcrosses and homozygous selfings. The deletion of rsp-1, a homologue used for studies on meiotic silencing of Neurospora crassa, triggered abnormal ascospores from selfing, but outcrosses between the mutant and wild-type strain resulted in some ascospores with mutant phenotype (low occurrence of ascus dominance). When the ectopic mutants that carried an additional copy of rsp-1 were selfed, they primarily produced ascospores with normal shape but a few ascospores (0.23 %) were abnormal, in which both endogenous and ectopically integrated genes contained numerous point mutations. The ectopic mutants showed low occurrence of ascus dominance in outcrosses with strains that carried the wild-type allele. Approximately 10 % of ascospores were abnormal but all of the single-ascospore isolates produced normal-shaped ascospores from selfing. However, no ascus dominance was observed when the mutants were outcrossed with a sad-1 deletion mutant, which lacks the putative RNA-dependent RNA polymerase essential for meiotic silencing in N. crassa. All results were consistent with those generated from an additional gene, roa, required for ascospore morphogenesis. This study demonstrated that G. zeae possesses a functional meiotic silencing mechanism which is triggered by unpaired DNA, as in N. crassa.

Requirement of ATM-Dependent Monoubiquitylation of Histone H2B for Timely Repair of DNA Double-Strand Breaks

The cellular response to DNA double-strand breaks (DSBs) is mobilized by the protein kinase ATM, which phosphorylates key players in the DNA damage response (DDR) network. A major question is how ATM controls DSB repair. Optimal repair requires chromatin relaxation at damaged sites. Chromatin reorganization is coupled to dynamic alterations in histone posttranslational modifications. Here, we show that in human cells, DSBs induce monoubiquitylation of histone H2B, a modification that is associated in undamaged cells with transcription elongation. We find that this process relies on recruitment to DSB sites and ATM-dependent phosphorylation of the responsible E3 ubiquitin ligase: the RNF20-RNF40 heterodimer. H2B monoubiquitylation is required for timely recruitment of players in the two major DSB repair pathways-nonhomologous end-joining and homologous recombination repair-and optimal repair via both pathways. Our data and previous data suggest a two-stage model for chromatin decondensation that facilitates DSB repair.

A block in lineage differentiation of immortal human mammary stem / progenitor cells by ectopically-expressed oncogenes

Introduction:Emerging evidence suggests a direct role of cancer stem cells (CSCs) in the development of breast cancer. In vitro cellular models that recapitulate properties of CSCs are therefore highly desirable. We have previously shown that normal human mammary epithelial cells (hMECs) immortalized with human telomerase reverse transcriptase (hTERT) possess properties of mammary stem / progenitor cells.Materials and Methods:In the present study, we used this cell system to test the idea that other known hMEC-immortalizing oncogenes (RhoA, HPVE6, HPVE7, p53 mutant, and treatment with γ-radiation), share with hTERT, the ability to maintain mammary stem / progenitor cells.Results:The results presented here demonstrate that similar to hMECs immortalized with hTERT, all hMEC cell lines immortalized using various oncogenic strategies express stem / progenitor cell markers. Furthermore, analyses using 2D and 3D culture assays demonstrate that all the immortal cell lines retain their ability to self-renew and to differentiate along the luminal lineage. Remarkably, the stem / progenitor cell lines generated using various oncogenic strategies exhibit a block in differentiation along the myoepithelial lineage, a trait that is retained on hTERT-immortalized stem / progenitors. The inability to differentiate along the myoepithelial lineage could be induced by ectopic mutant p53 expression in hTERT-immortalized hMEC.Conclusions:Our studies demonstrate that stem / progenitor cell characteristics of hMECs are maintained upon immortalization by using various cancer-relevant oncogenic strategies. Oncogene-immortalized hMECs show a block in their ability to differentiate along the myoepithelial lineage. Abrogation of the myoepithelial differentiation potential by a number of distinct oncogenic insults suggests a potential explanation for the predominance of luminal and rarity of myoepithelial breast cancers.

Canonical Wnt signaling is antagonized by noncanonical Wnt5a in hepatocellular carcinoma cells

Backgroundβ-catenin mutations that constitutively activate the canonical Wnt signaling have been observed in a subset of hepatocellular carcinomas (HCCs). These mutations are associated with chromosomal stability, low histological grade, low tumor invasion and better patient survival. We hypothesized that canonical Wnt signaling is selectively activated in well-differentiated, but repressed in poorly differentiated HCCs. To this aim, we characterized differentiation status of HCC cell lines and compared their expression status of Wnt pathway genes, and explored their activity of canonical Wnt signaling.ResultsWe classified human HCC cell lines into "well-differentiated" and "poorly differentiated" subtypes, based on the expression of hepatocyte lineage, epithelial and mesenchymal markers. Poorly differentiated cell lines lost epithelial and hepatocyte lineage markers, and overexpressed mesenchymal markers. Also, they were highly motile and invasive. We compared the expression of 45 Wnt pathway genes between two subtypes. TCF1 and TCF4 factors, and LRP5 and LRP6 co-receptors were ubiquitously expressed. Likewise, six Frizzled receptors, and canonical Wnt3 ligand were expressed in both subtypes. In contrast, canonical ligand Wnt8b and noncanonical ligands Wnt4, Wnt5a, Wnt5b and Wnt7b were expressed selectively in well- and poorly differentiated cell lines, respectively. Canonical Wnt signaling activity, as tested by a TCF reporter assay was detected in 80% of well-differentiated, contrary to 14% of poorly differentiated cell lines. TCF activity generated by ectopic mutant β-catenin was weak in poorly differentiated SNU449 cell line, suggesting a repressive mechanism. We tested Wnt5a as a candidate antagonist. It strongly inhibited canonical Wnt signaling that is activated by mutant β-catenin in HCC cell lines.ConclusionDifferential expression of Wnt ligands in HCC cells is associated with selective activation of canonical Wnt signaling in well-differentiated, and its repression in poorly differentiated cell lines. One potential mechanism of repression involved Wnt5a, acting as an antagonist of canonical Wnt signaling. Our observations support the hypothesis that Wnt pathway is selectively activated or repressed depending on differentiation status of HCC cells. We propose that canonical and noncanonical Wnt pathways have complementary roles in HCC, where the canonical signaling contributes to tumor initiation, and noncanonical signaling to tumor progression.

Protein kinase C mediates mutant N-Ras–induced developmental abnormalities in normal human erythroid cells

RAS mutations are one of the most frequent molecular abnormalities associated with myeloid leukemia and preleukemia, yet there is a poor understanding of how they contribute to the pathogenesis of these conditions. Here, we describe the consequences of ectopic mutant N-Ras (N-Ras*) expression on normal human erythropoiesis. We show that during early (erythropoietin [EPO]-independent) erythropoiesis, N-Ras* promoted the amplification of a phenotypically primitive but functionally defective subpopulation of CD34(+) erythroblasts. N-Ras* also up-regulated the expression of megakaryocyte antigens on human erythroblasts. Although early erythroblasts expressing N-Ras* were able to respond to erythropoietin and generate mature progeny, this occurred with greatly reduced efficiency, probably explaining the poor colony growth characteristics of these cells. We further report that this oncogene promoted the expression and activation of protein kinase C (PKC) and that the effects of N-Ras* on erythropoiesis could be abrogated or attenuated by inhibition of PKC. Similarly, the effects of this oncogene could be partially mimicked by treatment with PKC agonist. Together, these data suggest that expression of N-Ras* is able to subvert the normal developmental cues that regulate erythropoiesis by activating PKC. This gives rise to phenotypic and functional abnormalities commonly observed in preleukemia, suggesting a direct link between RAS mutations and the pathogenesis of preleukemia.

Post‐transcriptional Silencing of the Tomatinase Gene in Fusarium oxysporum f. sp lycopersici

Abstract Fusarium oxysporum f. sp. lycopersici produces an extracellular enzyme tomatinase, which has been known to detoxify α‐tomatine, a preformed inhibitor of fungal growth in tomato plants. To understand the role of the tomatinase in pathogenicity, we performed transformation‐mediated disruption of the tomatinase gene in F. oxysporum f. sp. lycopersici. Mutants obtained showed increase in sensitivity to α‐tomatine and decrease in tomatinase activity in vitro. Inoculation assay showed reduced pathogenicity of the mutants on tomato plants, suggesting an important role of tomatinase in pathogenicity of this fungus. However, Southern blot analysis revealed that the resident tomatinase gene of the mutants was not disrupted but the gene replacement vector was ectopically integrated into the genome of the mutants. To clarify the mechanisms involved, we examined one of the ectopic insertion mutants at both DNA and RNA levels. No methylation of the resident tomatinase gene was detected. Northern blot analysis revealed low level of the tomatinase gene expression and also the presence of antisense RNA in the ectopic mutant. Thus, post‐transcriptional gene silencing seems to be involved in the suppression of tomatinase in the ectopic mutant.

Coexpression of Wild-Type Tyrosinase Enhances Maturation of Temperature-Sensitive Tyrosinase Mutants

Tyrosinase is a type I membrane glycoprotein whose activity is essential for melanin synthesis. Loss of function mutations in tyrosinase is the cause of oculocutaneous albinism 1. In the milder oculocutaneous albinism 1B form in which mutant proteins retain residual activity, the severity of albinism depends on the type of mutations expressed in the melanocyte. In this study, we show that coexpression of wild-type protein with temperature-sensitive tyrosinase mutants corrects the mutant conformation defect in an activity-dependent manner. Exit from the endoplasmic reticulum and complex carbohydrate processing in the Golgi was promoted when temperature-sensitive tyrosinase mutants were ectopically expressed in host melanocytes carrying wild-type protein even at the nonpermissive temperature. Incubation of transfected melanocytes with DOPA (the cofactor and substrate for tyrosinase), or tyrosine (the substrate), further enhanced processing of ectopic mutant proteins. The analysis of glycosylation-deficient mutants revealed regions in tyrosinase with high, low, and intermediate dependency on glycans for maturation. We concluded that the presence of tyrosinase activity enhances the maturation of temperature-sensitive and glycosylation-deficient forms of tyrosinase. The results may explain the variation in pigmentation and the development of pigment later in life in patients carrying different mutant alleles of oculocutaneous albinism 1B.

Inhibition of HIF-1- and wild-type p53-stimulated transcription by codon Arg175 p53 mutants with selective loss of functions

Overexpression of ectopic mutant p53 represses wild-type p53-stimulated transcription, known as a dominant negative effect. On the other hand, overexpression of wild-type p53 can repress transcription stimulated by several transcription factors, including hypoxia-inducible factor-1 (HIF-1). Using a panel of well-characterized Arg175 p53 mutants we found that only mutants (Tyr175, Trp175, Asp175 and Phe175) which have completely lost their ability to transactivate repress wild-type p53-stimulated Bax, p21 and PG13 promoter constructs. In contrast, Asn175, Gln175, Leu175 and Pro175 mutants which partially retained transactivating functions did not exert dominant negative effects against PG13 and p21 promoter constructs. However, these latter mutants failed to activate Bax and, instead, exerted a dominant negative effect on a Bax-Luc promoter construct. We conclude that a dominant negative effect is promoter selective as a consequence of selective loss of transactivating function. Albeit less potent than wild-type p53, all Arg175 p53 mutants retained partial ability to repress HIF-1-stimulated transcription. We propose that transrepression and the dominant negative effect have similar mechanisms and may involve competition with transcription factors (wild-type p53, HIF-1, etc.) for cofactors such as p300. Thus, a p53(22/23) mutant, which is deficient in p300 binding, did not exert dominant negative effects. Like transrepression, the dominant negative effect required overexpression of mutant p53 and, therefore, is not dominant. In the presence of a wild-type p53 allele, levels of endogenous mutant p53 protein were low in heterozygous cells. Endogenous mutant p53 became overexpressed only after loss of the second p53 allele. Therefore, endogenous mutant p53s are unable to display a dominant negative effect. This explains why loss of the second p53 allele is required to eliminate p53 functions in cancer cells.

Identification of a contractile deficit in adult cardiac myocytes expressing hypertrophic cardiomyopathy-associated mutant troponin T proteins.

The direct effects of expressing hypertrophic cardiomyopathy-associated (HCM-associated) mutant troponin T (TnT) proteins on the force generation of single adult cardiac myocytes have not been established. Replication-defective recombinant adenovirus vectors were generated for gene transfer of HCM-associated I79N and R92Q mutant cardiac TnT cDNAs into fully differentiated adult cardiac myocytes in primary culture. We tested the hypothesis that the mutant TnT proteins would be expressed and incorporated into the cardiac sarcomere and would behave as dominant-negative proteins to directly alter calcium-activated force generation at the level of the single cardiac myocyte. Interestingly, under identical experimental conditions, the ectopic expression of the mutant TnTs was significantly less ( approximately 8% of total) than that obtained with expression of wild-type TnT ( approximately 35%) in the myocytes. Confocal imaging of immunolabeled TnT showed a regular periodic pattern of localization of ectopic mutant TnT that was not different than that in normal controls, suggesting that mutant TnT incorporation had no deleterious effects on sarcomeric architecture. Direct measurements of isometric force production in single cardiac myocytes demonstrated marked desensitization of submaximal calcium-activated tension, with unchanged maximum tension generation in mutant TnT-expressing myocytes compared with control myocytes. Collectively, these results demonstrate an impaired expression of the mutant protein and a disabling of cardiac contraction in the submaximal range of myoplasmic calcium concentrations. Our functional results suggest that development of new pharmacological, chemical, or genetic approaches to sensitize the thin-filament regulatory protein system could ameliorate force deficits associated with expression of I79N and R92Q in adult cardiac myocytes.