Dominant-negative Allele Research Articles

Retrospective outcomes in patients with BCR-ABL positive Acute Lymphoid Leukemia without Allogeneic stem cell transplantation at the Houston Methodist

S176 Introduction: Ph-like acute lymphoblastic leukemia (ALL) is characterized by a variety of rearrangements and other genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We recently identified recurring rearrangements of the erythropoietin receptor (EPOR) into the immunoglobulin heavy (IGH) or kappa (IGK) chain loci. The goal of this study was to examine the role of these alterations in kinase signaling and lymphoid transformation.Methods:Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of EPO. EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Luciferase-marked xenografts of human EPORrearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo. Results: Eight cases out 154 Ph-like ALL (5.2%) harbored rearrangements of EPOR into either the IGH or IGK loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR, resulting in overexpression of a C-terminal truncated receptor that retains the phosphorylation site required for STAT5 activation, but lacking multiple residues required for negative EPOR regulation. Notably, the location of the truncation sites is similar to those arising from inherited mutations in primary familial congenital polycythemia. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Xenograft leukemic cells from EPOR-rearranged cases exhibited high levels of mutant EPOR on the cell surface, constitutive pSTAT5 and noteworthy ex vivo and in vivo sensitivity to the JAK2 inhibitor ruxolitinib. Conclusions: Rearrangements of truncated EPOR into the IGH/IGK chain loci define a new subgroup of Phlike ALL and unexpectedly implicate EPOR signaling as an important factor influencing B-lymphoid malignancies. Finally, these findings suggest that aberrant EPOR signaling is amenable to JAK-STAT5 inhibition in cases harboring these rearrangements.

Runx1 is required for hematopoietic defects and leukemogenesis in Cbfb-MYH11 knock-in mice.

CBFβ-SMMHC, the fusion protein generated by the chromosome 16 inversion fusion gene, CBFB-MYH11, is known to initiate leukemogenesis. However, the mechanism through which CBFβ-SMMHC contributes to leukemia development is not well understood. Previously it was proposed that CBFβ-SMMHC acts by dominantly repressing the transcription factor RUNX1, but we recently showed that CBFβ-SMMHC has activities that are independent of RUNX1 repression. In addition, we showed that a modified CBFβ-SMMHC with decreased RUNX1 binding activity accelerates leukemogenesis. These results raise questions about the importance of RUNX1 in leukemogenesis by CBFβ-SMMHC. To test this, we generated mice expressing Cbfb-MYH11 in a Runx1 deficient background, resulting from either homozygous Runx1 null alleles (Runx1−/−) or a single dominant negative Runx1 allele (Runx1+/lz). We found that loss of Runx1 activity rescued the differentiation defects induced by Cbfb-MYH11 during primitive hematopoiesis. During definitive hematopoiesis, RUNX1 loss also significantly reduced the proliferation and differentiation defects induced by Cbfb-MYH11. Importantly, Cbfb-MYH11 induced leukemia had much longer latency in Runx1+/lz mice than in Runx1 sufficient mice. These data indicate that Runx1 activity is critical for Cbfb-MYH11 induced hematopoietic defects and leukemogenesis.

TSC2/mTORC1 signaling controls Paneth and goblet cell differentiation in the intestinal epithelium.

The intestinal mucosa undergoes a continual process of proliferation, differentiation and apoptosis, which is regulated by multiple signaling pathways. Notch signaling is critical for the control of intestinal stem cell maintenance and differentiation. However, the precise mechanisms involved in the regulation of differentiation are not fully understood. Previously, we have shown that tuberous sclerosis 2 (TSC2) positively regulates the expression of the goblet cell differentiation marker, MUC2, in intestinal cells. Using transgenic mice constitutively expressing a dominant negative TSC2 allele, we observed that TSC2 inactivation increased mTORC1 and Notch activities, and altered differentiation throughout the intestinal epithelium, with a marked decrease in the goblet and Paneth cell lineages. Conversely, treatment of mice with either Notch inhibitor dibenzazepine (DBZ) or mTORC1 inhibitor rapamycin significantly attenuated the reduction of goblet and Paneth cells. Accordingly, knockdown of TSC2 activated, whereas knockdown of mTOR or treatment with rapamycin decreased, the activity of Notch signaling in the intestinal cell line LS174T. Importantly, our findings demonstrate that TSC2/mTORC1 signaling contributes to the maintenance of intestinal epithelium homeostasis by regulating Notch activity.

Rett syndrome like phenotypes in the R255X Mecp2 mutant mouse are rescued by MECP2 transgene.

Rett syndrome (RTT) is a severe neurodevelopmental disorder that is usually caused by mutations in Methyl-CpG-binding Protein 2 (MECP2). Four of the eight common disease causing mutations in MECP2 are nonsense mutations and are responsible for over 35% of all cases of RTT. A strategy to overcome disease-causing nonsense mutations is treatment with nonsense mutation suppressing drugs that allow expression of full-length proteins from mutated genes with premature in-frame stop codons. To determine if this strategy is useful in RTT, we characterized a new mouse model containing a knock-in nonsense mutation (p.R255X) in the Mecp2 locus (Mecp2(R255X)). To determine whether the truncated gene product acts as a dominant negative allele and if RTT-like phenotypes could be rescued by expression of wild-type protein, we genetically introduced an extra copy of MECP2 via an MECP2 transgene. The addition of MECP2 transgene to Mecp2(R255X) mice abolished the phenotypic abnormalities and resulted in near complete rescue. Expression of MECP2 transgene Mecp2(R255X) allele also rescued mTORC1 signaling abnormalities discovered in mice with loss of function and overexpression of Mecp2. Finally, we treated Mecp2(R255X) embryonic fibroblasts with the nonsense mutation suppressing drug gentamicin and we were able to induce expression of full-length MeCP2 from the mutant p.R255X allele. These data provide proof of concept that the p.R255X mutation of MECP2 is amenable to the nonsense suppression therapeutic strategy and provide guidelines for the extent of rescue that can be expected by re-expressing MeCP2 protein.

Cryptic Truncating Rearrangements of the Erythropoietin Receptor in Ph-like Acute Lymphoblastic Leukemia

▪Introduction: BCR-ABL1-like, or Philadelphia-like acute lymphoblastic leukemia (Ph-like ALL), is characterized by a gene expression profile similar to BCR-ABL1-positive ALL, with a broad range of genetic alterations activating cytokine receptor and kinase signaling and poor outcome. We previously reported a rearrangement of EPOR, encoding the erythropoietin receptor, into the immunoglobulin heavy chain locus (IGH). The aims of this study were to define the frequency and genomic architecture of EPOR rearrangements in B-ALL and to examine their role in kinase signaling and lymphoid transformation.Methods: Whole genome and/or transcriptome sequencing was performed on 154 Ph-like ALL cases. Sanger sequencing and fluorescent in situ hybridization were used to confirm and map the EPOR rearrangements. Wild-type or EPOR rearranged alleles were expressed in interleukin-3 (IL-3)-dependent mouse hematopoietic Ba/F3 cells and interleukin-7 (IL-7)-dependent pre-B cells harboring alterations of Arf and/or the dominant negative IKZF1 allele IK6 observed in EPOR-rearranged ALL. Proliferation and signaling were examined in the absence or presence of erythropoietin (EPO). EPOR expression and signaling in cell lines and primary leukemic cells were examined by immunofluorescence, flow cytometry and immunoblotting. Epor-/- fetal liver cells were transduced with empty vector, EPOR wild-type or rearranged alleles and used for erythroid colony forming unit (CFU-E) and erythroid burst-forming unit (BFU-E) assays. Luciferase-marked xenografts of human EPOR-rearranged ALL were established in NOD-SCID-IL2R gamma (NSG) null mice, and signaling, EPO-dependent proliferation and sensitivity to the JAK inhibitor ruxolitinib were assessed ex vivo and in vivo.Results: Eight cases (5.2% of Ph-like ALL) harbored rearrangements of EPOR into either the IGH or immunoglobulin kappa light chain (IGK) loci with two consequences: i) inversion and insertion of EPOR 5’ untranscribed region into the the promoter and enhancer region of IGH/IGK; ii) truncation of the last coding exon of EPOR. Such rearrangements resulted in overexpression of a C-terminal truncated receptor that retained the phosphorylation site required for STAT5 activation, but lacked multiple intracytoplasmic tyrosine residues whose phosphorylation is required for normal negative regulation of the receptor. Notably, the locations of the truncation sites overlap with those arising from inherited mutations in primary familial congenital polycythemia, in which frameshift and nonsense mutations truncate the receptor. A real-time quantitative PCR assay was established to provide a diagnostic tool and to confirm that primary leukemia cells with these EPOR rearrangements overexpress N-terminal exons but lack expression of C-terminal truncated exon eight. The truncated alleles were expressed at higher levels than wild-type EPOR in IL-3-dependent Ba/F3 and IL-7-dependent Arf-/- mouse pre-B cells, and sustained cell proliferation and increased STAT5 phosphorylation following stimulation with exogenous EPO. Expression of wild-type or truncated EPOR in Epor-/- fetal liver cells promoted erythroid differentiation with formation of CFU-E and BFU-E colonies, indicating that truncated receptors sustain erythroid development. Xenografted EPOR-rearranged leukemic cells exhibited high levels of mutant EPOR on the cell surface, constitutive STAT5 phosphorylation and sensitivity to the JAK2 inhibitor ruxolitinib ex vivo and in vivo.Conclusions: We have identified a subset of Ph-like ALL cases characterized by rearrangements of truncated EPOR into the IGH/IGK chain loci. This represents an entirely new mechanism of EPOR deregulation and unexpectedly implicates EPOR signaling as an important factor influencing B-lymphoid malignancies that are amenable to JAK-STAT5 inhibition. Clinical trials testing ruxolitinib in ALL patients with EPOR rearrangements are warranted. DisclosuresNo relevant conflicts of interest to declare.

Properties, regulation, pharmacology, and functions of the K₂p channel, TRESK.

TWIK-related spinal cord K(+) channel (TRESK) is the gene product of KCNK18, the last discovered leak potassium K2P channel gene. Under resting conditions, TRESK is constitutively phosphorylated at two regulatory regions. Protein kinase A (PKA) and microtubule affinity-regulating (MARK) kinases can be applied in experiments to phosphorylate these sites of TRESK expressed in Xenopus oocytes, respectively. Upon generation of a calcium signal, TRESK is dephosphorylated and thereby activated by calcineurin. In this process, the binding of calcineurin to the channel by non-catalytic interacting sites is essential. The phosphorylation/dephosphorylation regulatory process is modified by 14-3-3 proteins. Human, but not murine TRESK is also activated by protein kinase C. TRESK is expressed most abundantly in sensory neurons of the dorsal root ganglia (DRG) and trigeminal ganglia, and the channel modifies certain forms of nociceptive afferentation. In a large pedigree, a dominant negative mutant TRESK allele was found to co-segregate perfectly with migraine phenotype. While this genetic defect may be responsible only for a very small fraction of migraine cases, specific TRESK activation is expected to exert beneficial effect in common forms of the disease.

Nephronophthisis-associated CEP164 regulates cell cycle progression, apoptosis and epithelial-to-mesenchymal transition.

We recently reported that centrosomal protein 164 (CEP164) regulates both cilia and the DNA damage response in the autosomal recessive polycystic kidney disease nephronophthisis. Here we examine the functional role of CEP164 in nephronophthisis-related ciliopathies and concomitant fibrosis. Live cell imaging of RPE-FUCCI (fluorescent, ubiquitination-based cell cycle indicator) cells after siRNA knockdown of CEP164 revealed an overall quicker cell cycle than control cells, although early S-phase was significantly longer. Follow-up FACS experiments with renal IMCD3 cells confirm that Cep164 siRNA knockdown promotes cells to accumulate in S-phase. We demonstrate that this effect can be rescued by human wild-type CEP164, but not disease-associated mutants. siRNA of CEP164 revealed a proliferation defect over time, as measured by CyQuant assays. The discrepancy between accelerated cell cycle and inhibited overall proliferation could be explained by induction of apoptosis and epithelial-to-mesenchymal transition. Reduction of CEP164 levels induces apoptosis in immunofluorescence, FACS and RT-QPCR experiments. Furthermore, knockdown of Cep164 or overexpression of dominant negative mutant allele CEP164 Q525X induces epithelial-to-mesenchymal transition, and concomitant upregulation of genes associated with fibrosis. Zebrafish injected with cep164 morpholinos likewise manifest developmental abnormalities, impaired DNA damage signaling, apoptosis and a pro-fibrotic response in vivo. This study reveals a novel role for CEP164 in the pathogenesis of nephronophthisis, in which mutations cause ciliary defects coupled with DNA damage induced replicative stress, cell death, and epithelial-to-mesenchymal transition, and suggests that these events drive the characteristic fibrosis observed in nephronophthisis kidneys.

Abstract 959: FAS mutations induce therapeutic resistance in non-Hodgkin lymphomas

Abstract Lymphomas are common (1/30 Canadians), but responses to chemotherapy depend on the tumour biology. We sequenced > 300 lymphomas at diagnosis and at relapse, identified FAS as a possible tumour suppressor gene and found that mutant FAS is associated with therapeutic resistance. FAS is the death receptor that initiates the extrinsic apoptotic pathway once activated by FAS ligand (FASL). Our studies indicated that the most common FAS mutation is a dominant negative allele whose product inhibits FAS-mediated apoptosis. This allele induces resistance to doxorubicin in murine lymphoma cells transplanted into syngeneic immune competent C57BL/6 mice but, unexpectedly, the allele has no effect on chemosensitivity in cultured lymphoma lines in vitro. Because FAS signaling is initiated by FASL from neighboring cells, we asked whether chemotherapy induces FAS or FASL expression in immune cells, thereby activating apoptosis in vivo but not in vitro. Indeed, exposure to chemotherapy induced FAS in primary and malignant B cells and increased FASL in benign T cells. Thus, we hypothesize that chemotherapy may induce an anti-tumour immune response mediated by FAS-FASL interactions, and that FAS mutations cause therapeutic resistance by blocking the chemotherapy-induced immune response. We will extend this work by evaluating the role of the immune response in chemotherapy-induced lymphoma apoptosis and by identifying therapies that will target FAS mutant lymphomas. Citation Format: Nathalie Johnson, Denis Gaucher, Ryan Morin, Randy Gascoyne, Joseph Connors, Marco Marra, Jerry Pelletier, Hawley Rigsby, Koren Mann. FAS mutations induce therapeutic resistance in non-Hodgkin lymphomas. [abstract]. In: Proceedings of the 105th Annual Meeting of the American Association for Cancer Research; 2014 Apr 5-9; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2014;74(19 Suppl):Abstract nr 959. doi:10.1158/1538-7445.AM2014-959

A dominant-negative mutation of mouse Lmx1b causes glaucoma and is semi-lethal via LDB1-mediated dimerization [corrected

Mutations in the LIM-homeodomain transcription factor LMX1B cause nail-patella syndrome, an autosomal dominant pleiotrophic human disorder in which nail, patella and elbow dysplasia is associated with other skeletal abnormalities and variably nephropathy and glaucoma. It is thought to be a haploinsufficient disorder. Studies in the mouse have shown that during development Lmx1b controls limb dorsal-ventral patterning and is also required for kidney and eye development, midbrain-hindbrain boundary establishment and the specification of specific neuronal subtypes. Mice completely deficient for Lmx1b die at birth. In contrast to the situation in humans, heterozygous null mice do not have a mutant phenotype. Here we report a novel mouse mutant Icst, an N-ethyl-N-nitrosourea-induced missense substitution, V265D, in the homeodomain of LMX1B that abolishes DNA binding and thereby the ability to transactivate other genes. Although the homozygous phenotypic consequences of Icst and the null allele of Lmx1b are the same, heterozygous Icst elicits a phenotype whilst the null allele does not. Heterozygous Icst causes glaucomatous eye defects and is semi-lethal, probably due to kidney failure. We show that the null phenotype is rescued more effectively by an Lmx1b transgene than is Icst. Co-immunoprecipitation experiments show that both wild-type and Icst LMX1B are found in complexes with LIM domain binding protein 1 (LDB1), resulting in lower levels of functional LMX1B in Icst heterozygotes than null heterozygotes. We conclude that Icst is a dominant-negative allele of Lmx1b. These findings indicate a reassessment of whether nail-patella syndrome is always haploinsufficient. Furthermore, Icst is a rare example of a model of human glaucoma caused by mutation of the same gene in humans and mice.

A modified screening system for loss-of-function and dominant negative alleles of essential MCMV genes.

Inactivation of gene products by dominant negative mutants is a valuable tool to assign functions to yet uncharacterized proteins, to map protein-protein interactions or to dissect physiological pathways. Detailed functional and structural knowledge about the target protein would allow the construction of inhibitory mutants by targeted mutagenesis. Yet, such data are limited for the majority of viral proteins, so that the target gene needs to be subjected to random mutagenesis to identify suitable mutants. However, for cytomegaloviruses this requires a two-step screening approach, which is time-consuming and labor-intensive. Here, we report the establishment of a high-throughput suitable screening system for the identification of inhibitory alleles of essential genes of the murine cytomegalovirus (MCMV). In this screen, the site-specific recombination of a specifically modified MCMV genome was transferred from the bacterial background to permissive host cells, thereby combining the genetic engineering and the rescue test in one step. Using a reference set of characterized pM53 mutants it was shown that the novel system is applicable to identify non-complementing as well as inhibitory mutants in a high-throughput suitable setup. The new cis-complementation assay was also applied to a basic genetic characterization of pM99, which was identified as essential for MCMV growth. We believe that the here described novel genetic screening approach can be adapted for the genetic characterization of essential genes of any large DNA viruses.

Control of virus diseases in soybeans.

Soybean, one of the world's most important sources of animal feed and vegetable oil, can be infected by numerous viruses. However, only a small number of the viruses that can potentially infect soybean are considered as major economic problems to soybean production. Therefore, we consider management options available to control diseases caused by eight viruses that cause, or have the potential to cause, significant economic loss to producers. We summarize management tactics in use and suggest direction for the future. Clearly, the most important tactic is disease resistance. Several resistance genes are available for three of the eight viruses discussed. Other options include use of virus-free seed and avoidance of alternative virus hosts when planting. Attempts at arthropod vector control have generally not provided consistent disease management. In the future, disease management will be considerably enhanced by knowledge of the interaction between soybean and viral proteins. Identification of genes required for soybean defense may represent key regulatory hubs that will enhance or broaden the spectrum of basal resistance to viruses. It may be possible to create new recessive or dominant negative alleles of host proteins that do not support viral functions but perform normal cellular function. The future approach to virus control based on gene editing or exploiting allelic diversity points to necessary research into soybean-virus interactions. This will help to generate the knowledge needed for rational design of durable resistance that will maximize global production.

Mak5 and Ebp2 Act Together on Early Pre-60S Particles and Their Reduced Functionality Bypasses the Requirement for the Essential Pre-60S Factor Nsa1

Ribosomes are the molecular machines that translate mRNAs into proteins. The synthesis of ribosomes is therefore a fundamental cellular process and consists in the ordered assembly of 79 ribosomal proteins (r-proteins) and four ribosomal RNAs (rRNAs) into a small 40S and a large 60S ribosomal subunit that form the translating 80S ribosomes. Most of our knowledge concerning this dynamic multi-step process comes from studies with the yeast Saccharomyces cerevisiae, which have shown that assembly and maturation of pre-ribosomal particles, as they travel from the nucleolus to the cytoplasm, relies on a multitude (>200) of biogenesis factors. Amongst these are many energy-consuming enzymes, including 19 ATP-dependent RNA helicases and three AAA-ATPases. We have previously shown that the AAA-ATPase Rix7 promotes the release of the essential biogenesis factor Nsa1 from late nucleolar pre-60S particles. Here we show that mutant alleles of genes encoding the DEAD-box RNA helicase Mak5, the C/D-box snoRNP component Nop1 and the rRNA-binding protein Nop4 bypass the requirement for Nsa1. Interestingly, dominant-negative alleles of RIX7 retain their phenotype in the absence of Nsa1, suggesting that Rix7 may have additional nuclear substrates besides Nsa1. Mak5 is associated with the Nsa1 pre-60S particle and synthetic lethal screens with mak5 alleles identified the r-protein Rpl14 and the 60S biogenesis factors Ebp2, Nop16 and Rpf1, which are genetically linked amongst each other. We propose that these ’Mak5 cluster’ factors orchestrate the structural arrangement of a eukaryote-specific 60S subunit surface composed of Rpl6, Rpl14 and Rpl16 and rRNA expansion segments ES7L and ES39L. Finally, over-expression of Rix7 negatively affects growth of mak5 and ebp2 mutant cells both in the absence and presence of Nsa1, suggesting that Rix7, at least when excessively abundant, may act on structurally defective pre-60S subunits and may subject these to degradation.

RUNX1 Is Required For Maintenance Of Established T-ALL Blasts

T-cell acute lymphoblastic leukemia (T-ALL) is a clinically aggressive malignancy of immature T cells. Intensive multiagent chemotherapy achieves cure in 80-90% of pediatric patients, but only 40% of adult patients survive beyond 5 years. Data from recent ChIP-seq studies has shown that RUNX1 binds throughout the T-ALL genome at sites co-occupied by known oncogenic transcription factors including TAL1 and NOTCH1. For this reason, it has been suggested that RUNX1 may be part of a transcriptional activation complex that drives an oncogenic gene expression program in this cell context. In contrast, next generation sequencing studies have recently identified heterozygous point mutations throughout the RUNX1 coding region in T-ALL including some which are predicted to encode truncated polypeptides resembling dominant negative alleles, thus raising the possibility that RUNX1 may function rather as a tumor suppressor in this context. In an effort to explore the functional role of RUNX1 in T-ALL, we examined the effect of RUNX1 knockdown in a broad panel of established human T-ALL cell lines and xenograft-expanded patient biopsy samples.Cells transduced with lentiviral shRNAs targeting coding and 3' UTR regions of RUNX1 showed a clear growth disadvantage as compared to either non-transduced cells in the same culture or cells transduced with non-silencing shRNAs in parallel cultures. As well, absolute cell counts of cultures containing only shRUNX1-transduced cells demonstrated dramatically reduced growth rates as compared to either non-transduced or non-silencing shRNA-transduced controls. BrdU incorporation and CFSE dye dilution studies showed that most cell lines exhibited reduced proliferation in response to RUNX1 knock-down, while a subset of lines also showed reduced cell viability. These phenotypes were largely consistent across a panel of over 20 T-ALL cell lines and 4 xenograft-expanded patient samples, including several which harbored either nonsense or missense RUNX1 mutations. These results support the notion that established T-ALL cells are generally dependent on RUNX1 for continued cell growth and, in some cases, also for survival.We also explored candidate RUNX1 target genes which might be responsible for mediating the observed growth/survival phenotypes. Assembling a short list of the “usual suspects” including genes known to regulate growth of T-ALL cells generally (c-MYC, PTEN), to possess a substantial RUNX1 ChIP-seq peak in T-ALL cells (IGF1R, IL7R), or to be bona fide RUNX1 targets in other cell contexts (p21/WAF1, p27/KIP1), we performed western blot or flow cytometric analyses of multiple shRUNX1-transduced cell lines. We noted consistent regulation of some, but not all of these in a manner consistent with RUNX1 positively supporting cell growth.From these results, we conclude that RUNX1 plays a pro-oncogenic role in established T-ALL cells. We surmise that the complement of presumed loss-of-function RUNX1 mutations observed in patient T-ALLs may be indicative of its known roles in regulating normal T cell development such that loss-of-function mutations may lead to differentiation arrest and consequently promote tumor initiation. For the majority of T-ALLs that express wild-type RUNX1 proteins, however, our results suggest that RUNX1 acts functionally to support maintenance of the malignant clone by promoting expression of known oncogenic factors and repressing expression known tumor suppressors. Disclosures:Aster:Cell Signaling Technology: Consultancy; Merck, Inc.: Research Funding; Pfizer, Inc.: Research Funding; Genentech, Inc.: Honoraria.

Variant ALDH2 is associated with accelerated progression of bone marrow failure in Japanese Fanconi anemia patients

AbstractFanconi anemia (FA) is a severe hereditary disorder with defective DNA damage response and repair. It is characterized by phenotypes including progressive bone marrow failure (BMF), developmental abnormalities, and increased occurrence of leukemia and cancer. Recent studies in mice have suggested that the FA proteins might counteract aldehyde-induced genotoxicity in hematopoietic stem cells. Nearly half of the Japanese population carries a dominant-negative allele (rs671) of the aldehyde-catalyzing enzyme ALDH2 (acetaldehyde dehydrogenase 2), providing an opportunity to test this hypothesis in humans. We examined 64 Japanese FA patients, and found that the ALDH2 variant is associated with accelerated progression of BMF, while birth weight or the number of physical abnormalities was not affected. Moreover, malformations at some specific anatomic locations were observed more frequently in ALDH2-deficient patients. Our current data indicate that the level of ALDH2 activity impacts pathogenesis in FA, suggesting the possibility of a novel therapeutic approach.

Ethylene is involved in strawberry fruit ripening in an organ-specific manner.

The fruit of the strawberry Fragaria×ananassa has traditionally been classified as non-climacteric because its ripening process is not governed by ethylene. However, previous studies have reported the timely endogenous production of minor amounts of ethylene by the fruit as well as the differential expression of genes of the ethylene synthesis, reception, and signalling pathways during fruit development. Mining of the Fragaria vesca genome allowed for the identification of the two main ethylene biosynthetic genes, 1-aminocyclopropane-1-carboxylic acid (ACC) synthase and ACC oxidase. Their expression pattern during fruit ripening was found to be stage and organ (achene or receptacle) specific. Strawberry plants with altered sensitivity to ethylene could be employed to unravel the role of ethylene in the ripening process of the strawberry fruit. To this end, independent lines of transgenic strawberry plants were generated that overexpress the Arabidopsis etr1-1 mutant ethylene receptor, which is a dominant negative allele, causing diminished sensitivity to ethylene. Genes involved in ethylene perception as well as in its related downstream processes, such as flavonoid biosynthesis, pectin metabolism, and volatile biosynthesis, were differently expressed in two transgenic tissues, the achene and the receptacle. The different transcriptional responsiveness of the achene and the receptacle to ethylene was also revealed by the metabolic profiling of the primary metabolites in these two organs. The free amino acid content was higher in the transgenic lines compared with the control in the mature achene, while glucose and fructose, and citric and malic acids were at lower levels. In the receptacle, the most conspicuous change in the transgenic lines was the depletion of the tricarboxylic acid cycle intermediates at the white stage of development, most probably as a consequence of diminished respiration. The results are discussed in the context of the importance of ethylene during strawberry fruit ripening.

The Noncircadian Function of the Circadian Clock Gene in the Regulation of Male Fertility

Mice homozygous for a dominant-negative allele of the Clock gene (Clock (Δ19/Δ19)) have slightly but significantly decreased male fertility. The molecular mechanism for this reduction in fertility is unknown. In the present study, we used a small hairpin RNA (shRNA) strategy to specifically knock down the Clock gene expression in the testes of male mice and determined its effect on male fertility. Clock knockdown led to smaller litter size, a lower in vitro fertility rate, lower blastula formation rate, and lower acrosin activity of the knockdown sperm. Locomotor activity analysis of the Clock knockdown mice revealed that Clock knockdown in testes did not alter their circadian rhythm. Taken together, these results provide the first evidence that Clock gene expression in round spermatids is essential for maintaining male reproductivity and suggest that acrosin may be a novel regulatory target of the Clock gene that would regulate the fertilization and early embryonic development to blastula. These findings may provide new clues for development of novel male contraceptive strategies.

Negating the dominant-negative allele: a new treatment paradigm for arrhythmias explored in human induced pluripotent stem cell-derived cardiomyocytes

This editorial refers to ‘Allele-specific RNA interference rescues the long-QT syndrome phenotype in human induced pluripotent stem cell cardiomyocytes’[†][1], by E. Matsa et al. , on page 1078 Congenital long-QT syndrome (LQTS) is a disorder of cardiac repolarization that manifests typically with ventricular arrhythmias such as torsades de pointes in patients with prolonged QT intervals in the ECG and structurally normal hearts. The known mutations affect genes encoding either subunits of cardiac ion channels or proteins involved in their regulation. Matsa and colleagues1 describe how they succeeded in using human induced pluripotent stem cell (hiPSC) technology not only to model a subtype of LQTS (LQT2), but also to evaluate a novel therapeutic approach using human cardiomyocytes derived from patient-specific hiPSCs. The authors generated cardiomyocytes from hiPSCs derived from a patient carrying a heterozygous mutation in the KCNH2 gene, corresponding to an A561T amino acid exchange in the KCNH2 -encoded protein hERG. Four hERG subunits co-assemble to form the pore of the ion channel generating the delayed rectifier potassium current IKr, an important determinant of the cardiac action potential duration. In accordance with the clinical phenotype of the patient, the hiPSC-derived cardiomyocytes displayed prolonged action potentials. The authors further demonstrated that, similar to other LQT2-causing ion channel mutations,2 the A561T mutation results in impaired ion channel trafficking: mutated subunits were not glycosylated properly and thus were retained in a perinuclear compartment instead of being integrated into the plasma membrane. Presumably due to co-assembly, the mutation also interfered with normal trafficking of the wild-type subunits in … [1]: #fn-2

Identification of a histone acetyltransferase as a novel regulator of Drosophila intestinal stem cells

One of the major challenges in stem cell research is to decipher the controlling mechanisms/genes of stem cell homeostasis. Through an RNAi mediated genetic screen of living animals, we identified an evolutionarily conserved histone acetyltransferase Atac2 as a novel regulator of Drosophila intestinal stem cells (ISCs). Expression of Atac2-RNAi or a dominant negative allele of Atac2 generated more ISCs, while excessive Atac2 or a histone deacetylase inhibitor promoted ISC differentiation without affecting ISC survival or lineage specification. These findings extend our knowledge of epigenetic mechanisms in stem cell regulation.

LcrV Mutants That Abolish Yersinia Type III Injectisome Function

LcrV, the type III needle cap protein of pathogenic Yersinia, has been proposed to function as a tether between YscF, the needle protein, and YopB-YopD to constitute the injectisome, a conduit for the translocation of effector proteins into host cells. Further, insertion of LcrV-capped needles from a calcium-rich environment into host cells may trigger the low-calcium signal for effector translocation. Here, we used a genetic approach to test the hypothesis that the needle cap responds to the low-calcium signal by promoting injectisome assembly. Growth restriction of Yersinia pestis in the absence of calcium (low-calcium response [LCR(+)] phenotype) was exploited to isolate dominant negative lcrV alleles with missense mutations in its amber stop codon (lcrV(*327)). The addition of at least four amino acids or the eight-residue Strep tag to the C terminus was sufficient to generate an LCR(-) phenotype, with variant LcrV capping type III needles that cannot assemble the YopD injectisome component. The C-terminal Strep tag appears buried within the cap structure, blocking effector transport even in Y. pestis yscF variants that are otherwise calcium blind, a constitutive type III secretion phenotype. Thus, LcrV(*327) mutants arrest the needle cap in a state in which it cannot respond to the low-calcium signal with either injectisome assembly or the activation of type III secretion. Insertion of the Strep tag at other positions of LcrV produced variants with wild-type LCR(+), LCR(-), or dominant negative LCR(-) phenotypes, thereby allowing us to identify discrete sites within LcrV as essential for its attributes as a secretion substrate, needle cap, and injectisome assembly factor.

Oligonucleotide Models of Telomeric DNA and RNA Form a Hybrid G-quadruplex Structure as a Potential Component of Telomeres

Telomeric repeat-containing RNA, a non-coding RNA molecule, has recently been found in mammalian cells. The detailed structural features and functions of the telomeric RNA at human chromosome ends remain unclear, although this RNA molecule may be a key component of the telomere machinery. In this study, using model human telomeric DNA and RNA sequences, we demonstrated that human telomeric RNA and DNA oligonucleotides form a DNA-RNA G-quadruplex. We next employed chemistry-based oligonucleotide probes to mimic the naturally formed telomeric DNA-RNA G-quadruplexes in living cells, suggesting that the process of DNA-RNA G-quadruplex formation with oligonucleotide models of telomeric DNA and RNA could occur in cells. Furthermore, we investigated the possible roles of this DNA-RNA G-quadruplex. The formation of the DNA-RNA G-quadruplex causes a significant increase in the clonogenic capacity of cells and has an effect on inhibition of cellular senescence. Here, we have used a model system to provide evidence about the formation of G-quadruplex structures involving telomeric DNA and RNA sequences that have the potential to provide a protective capping structure for telomere ends.