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Summary: Esperanza‐Cebollada E., et al. found a group of 24 microRNAs, to be differentially expressed between two groups of paediatric acute myeloid leukaemia (AML) cases with distinct outcomes. The main target of this microRNA signature is SOCS2, a gene that controls stemness. The results of this study may open doors for further investigation of the role for microRNAs in poor prognostic paediatric AML. Commentary on: Esperanza‐Cebollada et al. A miRNA signature related to stemness identifies high‐risk patients in paediatric acute myeloid leukaemia. Br J Haematol 2023;202:96–110. [ABSTRACT FROM AUTHOR]

Abstract: Thioredoxin-interacting protein (TXNIP) is involved in reactive oxygen species-induced stress responses. In a screen for novel disease genes in murine leukemia virus (MLV)-induced mouse leukemias, we identified Txnip as a frequent target for proviral integration. Ectopic TXNIP expression inhibited the proliferation of myeloid progenitor cells. TXNIP transcript and protein levels were significantly elevated in human AML blasts of certain patients, particularly those harboring translocation t(8;21). Nucleotide sequencing revealed no abnormalities in the TXNIP coding region in AML. These findings suggest that deregulated TXNIP expression contributes to MLV-induced murine leukemia as well as human AML. [Copyright &y& Elsevier]

Many microRNAs (miRNAs) target mRNAs involved in processes aberrant in tumorigenesis, such as proliferation, survival, and differentiation. In particular, the let-7 miRNA family has been proposed to function in tumor suppression, because reduced expression of Iet-7 family members is common in non-small cell lung cancer (NSCLC). Here, we show that Iet-7 functionally inhibits non-small cell tumor development. Ectopic expression of let-7g in K-RasG12D-expressing murine lung cancer cells induced both cell cycle arrest and cell death. In tumor xenografts, we observed significant growth reduction of both murine and human non-small cell lung tumors when overexpression of let-7g was induced from lentiviral vectors. In let-7g expressing tumors, reductions in Ras family and HMGA2 protein levels were detected. Importantly, Iet-7g-mediated tumor suppression was more potent in lung cancer cell lines harboring oncogenic K-Ras mutations than in lines with other mutations. Ectopic expression of K-RasG12D largely rescued Iet-7g mediated tumor suppression, whereas ectopic expression of HMGA2 was less effective. Finally, in an autochthonous model of NSCLC in the mouse, let-7g expression substantially reduced lung tumor burden. [ABSTRACT FROM AUTHOR]

Retroviral insertion mutagenesis has recently received much attention because of its adverse effects in the application of retroviral vector-based gene therapy, resulting in leukemia in certain patients. At the same time, retroviral mutagenesis in mice is being considered a powerful forward genetic strategy to identify disease genes involved in cancer. The publication of the mouse genome sequence and the development of high-throughput genomic approaches have given a further boost to this rapidly evolving field. The increasing numbers of new potential oncogenes identified in retroviral screens have given a valuable basis for a better understanding of cancer related pathways in mice. Important challenges that now lie ahead of us are (i) to determine the relevance and causal relationship of these genes with various types of human cancer (ii) to develop strategies to identify tumor suppressor genes on a large scale, (iii) to place the disease genes into regulatory networks to better understand their role in the complex pathogenesis of cancer, and (iv) to determine their value for diagnosis refinement and therapeutic target intervention in human disease. In this review, we will give a brief update of the current state-of-the-art and thoughts concerning these issues. We will specifically focus on the value of employing retroviral insertion mutagenesis in mice and gene expression profiling in man in the context of acute myeloid leukemia.Molecular Therapy (2007) 15, 13–19. doi:10.1038/sj.mt.6300040 [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) is a heterogeneous group of diseases in which chromosomal aberrations, small insertions or deletions, or point mutations in certain genes have profound consequences for prognosis. However, the majority of AML patients present without currently known genetic defects. Retroviral insertion mutagenesis in mice has become a powerful tool for identifying new disease genes involved in the pathogenesis of leukemia and lymphoma. Here we have used the Grafi-l.4 strain of murine leukemia virus, which causes predominantly AML, in a screen to identify novel genes involved in the pathogenesis of this disease. We report 79 candidate disease genes in common integration sites (CISs) and 15 genes whose family members previously were found to be affected in other studies. The majority of the identified sequences (60%) were not found in lymphomas and monocytic leukemias in previous screens, suggesting a specific involvement in AML. Although most of the virus integrations occurred in or near the 5′ or 3′ ends of the genes, suggesting deregulation of gene expression as a consequence of virus integration, 18 CISs were located exclusively within the genes, conceivably causing gene disruption. [ABSTRACT FROM AUTHOR]

Refractive error (RE) and myopia are complex polygenic conditions with the majority of genome-wide associated genetic variants in non-exonic regions. Given this, and the onset during childhood, gene-regulation is expected to play an important role in its pathogenesis. This prompted us to explore beyond traditional gene finding approaches. We performed a genetic association study between variants in non-coding RNAs and enhancers, and RE and myopia. We obtained single-nucleotide polymorphisms (SNPs) in microRNA (miRNA) genes, miRNA-binding sites, long non-coding RNAs genes (lncRNAs) and enhancers from publicly available databases: miRNASNPv2, PolymiRTS, VISTA Enhancer Browser, FANTOM5 and lncRNASNP2. We investigated whether SNPs overlapping these elements were associated with RE and myopia leveraged from a large GWAS meta-analysis (N = 160,420). With genetic risk scores (GRSs) per element, we investigated the joint effect of associated variants on RE, axial length (AL)/corneal radius (CR), and AL progression in an independent child cohort, the Generation R Study (N = 3638 children). We constructed a score for biological plausibility per SNP in highly confident miRNA-binding sites and enhancers in chromatin accessible regions. We found that SNPs in two miRNA genes, 14 enhancers and 81 lncRNA genes in chromatin accessible regions and 54 highly confident miRNA-binding sites, were in RE and myopia-associated loci. GRSs from SNPs in enhancers were significantly associated with RE, AL/CR and AL progression. GRSs from lncRNAs were significantly associated with all AL/CR and AL progression. GRSs from miRNAs were not associated with any ocular biometric measurement. GRSs from miRNA-binding sites showed suggestive but inconsistent significance. We prioritized candidate miRNA binding sites and candidate enhancers for future functional validation. Pathways of target and host genes of highly ranked variants included eye development (BMP4, MPPED2), neurogenesis (DDIT4, NTM), extracellular matrix (ANTXR2, BMP3), photoreceptor metabolism (DNAJB12), photoreceptor morphogenesis (CHDR1), neural signaling (VIPR2) and TGF-beta signaling (ANAPC16). This is the first large-scale study of non-coding RNAs and enhancers for RE and myopia. Enhancers and lncRNAs could be of large importance as they are associated with childhood myopia. We provide a confident blueprint for future functional validation by prioritizing candidate miRNA binding sites and candidate enhancers. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) are critical regulators of gene expression. As miRNAs are frequently deregulated in many human diseases, including cancer and immunological disorders, it is important to understand their biological functions. Typically, miRNA-encoding genes are transcribed by RNA Polymerase II and generate primary transcripts that are processed by RNase III-endonucleases DROSHA and DICER into small RNAs of approximately 21 nucleotides. All miRNAs are loaded into Argonaute proteins in the RNA-induced silencing complex (RISC) and act as post-transcriptional regulators by binding to the 3′- untranslated region (UTR) of mRNAs. This seed-dependent miRNA binding inhibits the translation and/or promotes the degradation of mRNA targets. Surprisingly, recent data presents evidence for a target-mediated decay mechanism that controls the level of specific miRNAs. In addition, several non-canonical miRNA-containing genes have been recently described and unexpected functions of miRNAs have been identified. For instance, several miRNAs are located in the nucleus, where they are involved in the transcriptional activation or silencing of target genes. These epigenetic modifiers are recruited by RISC and guided by miRNAs to specific loci in the genome. Here, we will review non-canonical aspects of miRNA biology, including novel regulators of miRNA expression and functions of miRNAs in the nucleus. [ABSTRACT FROM AUTHOR]

Osteolineage cell‐derived extracellular vesicles (EVs) play a regulatory role in hematopoiesis and have been shown to promote the ex vivo expansion of human hematopoietic stem and progenitor cells (HSPCs). Here, we demonstrate that EVs from different human osteolineage sources do not have the same HSPC expansion promoting potential. Comparison of stimulatory and non‐stimulatory osteolineage EVs by next‐generation sequencing and mass spectrometry analyses revealed distinct microRNA and protein signatures identifying EV‐derived candidate regulators of ex vivo HSPC expansion. Accordingly, the treatment of umbilical cord blood‐derived CD34+ HSPCs with stimulatory EVs‐altered HSPC transcriptome, including genes with known roles in cell proliferation. An integrative bioinformatics approach, which connects the HSPC gene expression data with the candidate cargo in stimulatory EVs, delineated the potentially targeted biological functions and pathways during hematopoietic cell expansion and development. In conclusion, our study gives novel insights into the complex biological role of EVs in osteolineage cell‐HSPC crosstalk and promotes the utility of EVs and their cargo as therapeutic agents in regenerative medicine. [ABSTRACT FROM AUTHOR]

Myelopoiesis is a complex process driven by essential transcription factors, including C/EBPα, PU.1, RUNX1, KLF4 and IRF8. Together, these factors are critical for the control of myeloid progenitor cell expansion and lineage determination in the development of granulocytes and monocytes/macrophages. MicroRNAs (miRNAs) are expressed in a cell type and lineage specific manner. There is increasing evidence that miRNAs fine-tune the expression of hematopoietic lineage-specific transcription factors and drive the lineage decisions of hematopoietic progenitor cells. In this review, we discuss recently discovered self-activating and feed-back mechanisms in which transcription factors and miRNAs interact during myeloid cell development. Furthermore, we delineate how some of these mechanisms are affected in acute myeloid leukemia (AML) and how disrupted transcription factor-miRNA interplays contribute to leukemogenesis. [ABSTRACT FROM AUTHOR]

Acute myeloid leukemia (AML) results from multiple genetic and epigenetic aberrations, many of which remain unidentified. Frequent loss of large chromosomal regions marks haplo-insufficiency as one of the major mechanisms contributing to leukemogenesis. However, which haplo-insufficient genes (HIGs) are involved in leukemogenesis is largely unknown and powerful experimental strategies aimed at their identification are currently lacking. Here, we present a new approach to discover HIGs, using retroviral integration mutagenesis in mice in which methylated viral integration sites and neighbouring genes were identified. In total we mapped 6 genes which are flanked by methylated viral integration sites (mVIS). Three of these, i.e., Lrmp, Hcls1 and Prkrir, were up regulated and one, i.e., Ptp4a3, was down regulated in the affected tumor. Next, we investigated the role of PTP4A3 in human AML and we show that PTP4A3 expression is a negative prognostic indicator, independent of other prognostic parameters. In conclusion, our novel strategy has identified PTP4A3 to potentially have a role in AML, on one hand as a candidate HIG contributing to leukemogenesis in mice and on the other hand as a prognostic indicator in human AML. [ABSTRACT FROM AUTHOR]

Mounting data show that MIR139 is commonly silenced in solid cancer and hematological malignancies. MIR139 acts as a critical tumor suppressor by tuning the cellular response to different types of stress, including DNA damage, and by repressing oncogenic signaling pathways. Recently, novel insights into the mechanism of MIR139 silencing in tumor cells have been described. These include epigenetic silencing, inhibition of POL-II transcriptional activity on gene regulatory elements, enhanced expression of competing RNAs and post-transcriptional regulation by the microprocessor complex. Some of these MIR139-silencing mechanisms have been demonstrated in different types of cancer, suggesting that these are more general oncogenic events. Reactivation of MIR139 expression in tumor cells causes inhibition of tumor cell expansion and induction of cell death by the repression of oncogenic mRNA targets. In this review, we discuss the different aspects of MIR139 as a tumor suppressor gene and give an overview on different transcriptional mechanisms regulating MIR139 in oncogenic stress and across different types of cancer. The novel insights into the expression regulation and the tumor-suppressing activities of MIR139 may pave the way to new treatment options for cancer. [ABSTRACT FROM AUTHOR]

We report a system for Cre-regulated expression of RNA interference in vivo. Expression, cassettes comprise selectable and FACS-sortable markers in tandem with additional marker genes and shRNAs in the antisense orientation. The cassettes are flanked by tandem LoxP sites arranged so that Cre expression inverts the marker-shRNA construct, allowing its regulated expression (and, at the same time, deletes the original selection/marker genes). The cassettes can be incorporated into retroviral or lentiviral vectors and delivered to cells in culture or used to generate transgenic mice. We describe cassettes incorporating various combinations of reporter genes, miRNA-based RNAi (including two shRNA constructs at once), and oncogenes and demonstrate the delivery of effective RNA interference in cells in culture, efficient transduction into hematopoietic stem cells with cell-type-specific knockdown in their progeny, and rapid generation of regulated shRNA knockdown in transgenic mice. These vector systems allow regulated combinatorial manipulation (both overexpression and loss of function) of gene expression in multiple systems in vitro and in viva. [ABSTRACT FROM AUTHOR]

Exhaustion is a dysfunctional state of cytotoxic CD8+ T cells (CTL) observed in chronic infection and cancer. Current in vivo models of CTL exhaustion using chronic viral infections or cancer yield very few exhausted CTL, limiting the analysis that can be done on these cells. Establishing an in vitro system that rapidly induces CTL exhaustion would therefore greatly facilitate the study of this phenotype, identify the truly exhaustion-associated changes and allow the testing of novel approaches to reverse or prevent exhaustion. Here we show that repeat stimulation of purified TCR transgenic OT-I CTL with their specific peptide induces all the functional (reduced cytokine production and polyfunctionality, decreased in vivo expansion capacity) and phenotypic (increased inhibitory receptors expression and transcription factor changes) characteristics of exhaustion. Importantly, in vitro exhausted cells shared the transcriptomic characteristics of the gold standard of exhaustion, CTL from LCMV cl13 infections. Gene expression of both in vitro and in vivo exhausted CTL was distinct from T cells anergy. Using this system, we show that Tcf7 promoter DNA methylation contributes to TCF1 downregulation in exhausted CTL. Thus this novel in vitro system can be used to identify genes and signaling pathways involved in exhaustion and will facilitate the screening of reagents that prevent/reverse CTL exhaustion. Author summary: In this manuscript, we describe an in vitro method that rapidly establishes large numbers of exhausted CD8+ T cells. The exhaustion of CTL induced by this method has been fully validated by multiple approaches (cytokine production, polyfunctionality, cytotoxicity, in vivo proliferation, inhibitory receptors, transcription factors, RNAseq and DNA methylation). This method will facilitate not only the study of T cell exhaustion but also the screening of drugs. As proof of point, we use this method to show that TCF-1 downregulation in terminally exhausted T cells is accompanied by Tcf7 DNA promoter methylation and show that a transmethylase inhibitor can prevent TCF-1 downregulation. Our method presents a critical resource for the study of CTL exhaustion and the screening of drugs and interventions. [ABSTRACT FROM AUTHOR]

Neutrophilic differentiation is dependent on CCAAT enhancer-binding protein a (C/EBPα), a transcription factor expressed in multiple organs including the bone marrow. Using functional genomic technologies in combination with clustered regularly-interspaced short palindromic repeat (CRISPR)/CRISPR-associated protein 9 genome editing and in vivo mouse modeling, we show that CEBPA is located in a 170-kb topological-associated domain that contains 14 potential enhancers. Of these, 1 enhancer located +42 kb from CEBPA is active and engages with the CEBPA promoter in myeloid cells only. Germ line deletion of the homologous enhancer in mice in vivo reduces Cebpa levels exclusively in hematopoietic stem cells (HSCs) and myeloid-primed progenitor cells leading to severe defects in the granulocytic lineage, without affecting any other Cebpa-expressing organ studied. The enhancer-deleted progenitor cells lose their myeloid transcription program and are blocked indifferentiation. Deletion of the enhancer also causes loss of HSC maintenance. We conclude that a single +42-kb enhancer is essential for CEBPA expression in myeloid cells only. [ABSTRACT FROM AUTHOR]

Interstrand crosslinks (ICLs) are toxic DNA lesions that cause severe genomic damage during replication, especially in Fanconi anemia pathway-deficient cells. This results in progressive bone marrow failure and predisposes to acute myeloid leukemia (AML). The molecular mechanisms responsible for these defects are largely unknown. Using Ercddeficient mice, we show that Trp53 is responsible for ICL-induced bone marrow failure and that loss of Trp53is leukemogenic in this model. In addition, Erccl-deficient myeloid progenitors gain elevated levels of miR-139-3p and miR-199a-3p with age. These microRNAs exert opposite effects on hematopoiesis. Ectopic expression of miR-139-3p strongly inhibited proliferation of myeloid progenitors, whereas inhibition of miR-139-3p activity restored defective proliferation of Erccl-deficient progenitors. Conversely, the inhibition of miR-199a-3p functions aggravated the myeloid proliferation defect in the Ercddeficient model, whereas its enforced expression enhanced proliferation of progenitors. Importantly, miR-199a-3p caused AML in a pre-leukemic mouse model, supporting its role as an onco-microRNA. Target genes include HuRlor miR-139-3p and Prdx6, Runxl, and Suz12 for miR-199a-3p. The latter genes have previously been implicated as tumor suppressors in de novo and secondary AML. These findings show that, in addition to TRP53-controlled mechanisms, miR-139-3p and miR-199a-3p are involved in the defective hematopoietic function of ICL-repair deficient myeloid progenitors. [ABSTRACT FROM AUTHOR]

MicroRNAs (miRNAs) have the potential to regulate cellular differentiation programs; however, miRNA deficiency in primary hematopoietic stem cells (HSCs) results in HSC depletion in mice, leaving the question of whether miRNAs play a role in early-lineage decisions unanswered. To address this issue, we deleted Dicerl, which encodes an essential RNase III enzyme for miRNA biogenesis, in murine CCAAT/enhancer-binding protein a (C/EBPA)-positive myeloid-cornmitted progenitors in vivo. In contrast to the results in HSCs, we found that miRNA depletion affected neither the number of myeloid progenitors nor the percentage of C/EBPA-positive progenitor cells. Analysis of gene-expression profiles from wild-type and D/cert-deficient granulocyte-macrophage progenitors (GMPs) revealed that 20 miRNA families were active in GMPs. Of the derepressed miRNA targets in Dicer1-nu\\ GMPs, 27% are normally exclusively expressed in HSCs or are specific for multipotent progenitors and erythropoiesis, indicating an altered gene-expression landscape. D/cer7-deficient GMPs were defective in myeloid development in vitro and exhibited an increased replating capacity, indicating the regained self-renewal potential of these cells. In mice, Dicerl deletion blocked monocytic differentiation, depleted macrophages, and caused myeloid dysplasia with morphologic features of Pelger-Huët anomaly. These results provide evidence for a miRNA-controiled switch for a cellular program of self-renewal and expansion toward myeloid differentiation in GMPs [ABSTRACT FROM AUTHOR]

The article presents a study which characterizes MicroRNA (miRNA) expression patterns in stages of normal human neutrophilic development. It states that the study collected granulocytes in different stages of granulocytic differentiation, isolated RNA from them and profiled their miRNA expression. Furthermore, It is revealed through principal component analysis that there are sharp distinctions of expressed miRNAs between the granulocytic maturation stages.

MicroRNAs (miRNAs) are pivotal for regulation of hematopoiesis but their critical targets remain largely unknown. Here, we show that ectopic expression of miR-17, -20,-93 and -106, all AAAGUGC seed-containing miRNAs, increases proliferation, colony outgrowth and replating capacity of myeloid progenitors and results in enhanced P-ERK levels. We found that these miRNAs are endogenously and abundantly expressed in myeloid progenitors and down-regulated in mature neutrophils. Quantitative proteomics identified sequestosome 1 (SQSTM1), an ubiquitin-binding protein and regulator of autophagy-mediated protein degradation, as a major target for these miRNAs in myeloid progenitors. In addition, we found increased expression of Sqstm1 transcripts during CSF3-induced neutrophil differentiation of 32D-CSF3R cells and an inverse correlation of SQSTM1 protein levels and miR-106 expression in AML samples. ShRNA-mediated silencing of Sqstm1 phenocopied the effects of ectopic miR-17/20/93/106 expression in hematopoietic progenitors in vitro and in mice. Further, SQSTM1 binds to the ligand-activated colony-stimulating factor 3 receptor (CSF3R) mainly in the late endosomal compartment, but not in LC3 positive autophagosomes. SQSTM1 regulates CSF3R stability and ligand-induced mitogen-activated protein kinase signaling. We demonstrate that AAAGUGC seed-containing miRNAs promote cell expansion, replating capacity and signaling in hematopoietic cells by interference with SQSTM1-regulated pathways. [ABSTRACT FROM AUTHOR]

Raf kinase inhibitory protein (RKIP) negatively regulates the MAP kinase (MAPK), G protein-coupled receptor kinase-2, and NF-κB signalling cascades. RKIP has been implicated as a metastasis suppressor for prostate cancer, but the mechanism is not known. Here, we show that RKIP inhibits invasion by metastatic breast cancer cells and represses breast tumour cell intravasation and bone metastasis in an orthotopic murine model. The mechanism involves inhibition of MAPK, leading to decreased transcription of LIN28 by Myc. Suppression of LIN28 enables enhanced let-7 processing in breast cancer cells. Elevated let-7 expression inhibits HMGA2, a chromatin remodelling protein that activates pro-invasive and pro-metastatic genes, including Snail. LIN28 depletion and let-7 expression suppress bone metastasis, and LIN28 restores bone metastasis in mice bearing RKIP-expressing breast tumour cells. These results indicate that RKIP suppresses invasion and metastasis in part through a signalling cascade involving MAPK, Myc, LIN28, let-7, and downstream let-7 targets. RKIP regulation of two pluripotent stem cell genes, Myc and LIN28, highlights the importance of RKIP as a key metastasis suppressor and potential therapeutic agent. [ABSTRACT FROM AUTHOR]

Summary: miR-17∼92, miR-106b∼25, and miR-106a∼363 belong to a family of highly conserved miRNA clusters. Amplification and overexpression of miR-17∼92 is observed in human cancers, and its oncogenic properties have been confirmed in a mouse model of B cell lymphoma. Here we show that mice deficient for miR-17∼92 die shortly after birth with lung hypoplasia and a ventricular septal defect. The miR-17∼92 cluster is also essential for B cell development. Absence of miR-17∼92 leads to increased levels of the proapoptotic protein Bim and inhibits B cell development at the pro-B to pre-B transition. Furthermore, while ablation of miR-106b∼25 or miR-106a∼363 has no obvious phenotypic consequences, compound mutant embryos lacking both miR-106b∼25 and miR-17∼92 die at midgestation. These results provide key insights into the physiologic functions of this family of microRNAs and suggest a link between the oncogenic properties of miR-17∼92 and its functions during B lymphopoiesis and lung development. [Copyright &y& Elsevier]

The hematopoietic system provides an attractive model for studying growth factor-controlled expansion and differentiation of cells in relation to receptor routing and its consequences for signal transduction. Suppressor of cytokine signaling (SOCS) proteins regulate receptor signaling partly via their ubiquitin ligase (E3)-recruiting SOCS box domain. Whether SOCS proteins affect signaling through modulating intracellular trafficking of receptors is unknown. Here, we show that a juxtamembrane lysine residue (K632) of the granulocyte colony-stimulating factor receptor (G-CSFR) plays a key role in receptor routing and demonstrate that the effects of SOCS3 on G-CSF signaling to a major extent depend on this lysine. Mutation of K632 causes accumulation of G-CSFR in early endosomes and leads to sustained activation of signal transducer and activator of transcription 5 and ERK, but not protein kinase B. Myeloid progenitors expressing G-CSFR mutants lacking K632 show a perturbed proliferation/differentiation balance in response to G-CSF. This is the first demonstration of SOCS-mediated ubiquitination and routing of a cytokine receptor and its impact on maintaining an appropriate signaling output. [ABSTRACT FROM AUTHOR]

The chicken anemia virus-derived protein Apoptin induces apoptosis specifically in human tumor and transformed cells and not in normal, untransformed cells. The cell killing activity correlates with a predominantly nuclear localization of Apoptin in tumor cells, whereas in normal cells, it is detected mainly in cytoplasmic structures. To explore the role of nuclear localization for Apoptin-induced cell death in tumor cells, we employed a mutagenesis strategy. First, we demonstrated that the C terminus of Apoptin contains a bipartite-type nuclear localization signal. Strikingly, further investigation showed that Apoptin contains two different domains that induce apoptosis independently, and for both domains, we found a strong correlation between localization and killing activity. Using inhibitors, we ruled out the involvement of de novo gene transcription and translation and further showed that Apoptin itself does not have any significant transcriptional repression activity, suggesting that Apoptin exerts its effects in the nucleus by some other method. To determine whether nuclear localization is sufficient to enable Apoptin to kill normal, untransformed cells, we expressed fulllength Apoptin fused to a heterologous nuclear localization signal in these cells. However, despite its nuclear localization, no apoptosis was induced, which suggests that nuclear localization per se is not sufficient for Apoptin to become active. These studies increase our understanding of the molecular pathway of Apoptin and may also shed light on the mechanism of cellular transformation. [ABSTRACT FROM AUTHOR]

Investigates the function of specific sequences of bullous pemphigoid antigen 180 (BP180) to its incorporation in hemidesmosomes by cytoplasmic domain regulated by the β4 integrin subunit. Expression of chimeric proteins; Determination of sequence within the cytoplasmic domain of β4; Cytoplasmic components of hemidesmosomes.

Correction to: The EMBO Journal (2007) 26, 1782–1793. doi:10.1038/sj.emboj.7601640 [ABSTRACT FROM AUTHOR]