Cytoplasmic synthesis of endogenous Alu complementary DNA via reverse transcription and implications in age-related macular degeneration.

Alu retroelements propagate via retrotransposition by hijacking long interspersed nuclear element-1 (L1) reverse transcriptase (RT) and endonuclease activities. Reverse transcription of Alu RNA into complementary DNA (cDNA) is presumed to occur exclusively in the nucleus at the genomic integration site. Whether Alu cDNA is synthesized independently of genomic integration is unknown. Alu RNA promotes retinal pigmented epithelium (RPE) death in geographic atrophy, an untreatable type of age-related macular degeneration. We report that Alu RNA-induced RPE degeneration is mediated via cytoplasmic L1-reverse-transcribed Alu cDNA independently of retrotransposition. Alu RNA did not induce cDNA production or RPE degeneration in L1-inhibited animals or human cells. Alu reverse transcription can be initiated in the cytoplasm via self-priming of Alu RNA. In four health insurance databases, use of nucleoside RT inhibitors was associated with reduced risk of developing atrophic macular degeneration (pooled adjusted hazard ratio, 0.616; 95% confidence interval, 0.493-0.770), thus identifying inhibitors of this Alu replication cycle shunt as potential therapies for a major cause of blindness.

The transcription factor FOXM1 regulates the balance between proliferation and aberrant differentiation in head and neck squamous cell carcinoma.

Sustained expression of FOXM1 is a hallmark of nearly all human cancers including squamous cell carcinomas of the head and neck (HNSCC). HNSCCs partially preserve the epithelial differentiation program, which recapitulates fetal and adult traits of the tissue of tumor origin but is deregulated by genetic alterations and tumor-supporting pathways. Using shRNA-mediated knockdown, we demonstrate a minimal impact of FOXM1 on proliferation and migration of HNSCC cell lines under standard cell culture conditions. However, FOXM1 knockdown in three-dimensional (3D) culture and xenograft tumor models resulted in reduced proliferation, decreased invasion, and a more differentiated-like phenotype, indicating a context-dependent modulation of FOXM1 activity in HNSCC cells. By ectopic overexpression of FOXM1 in HNSCC cell lines, we demonstrate a reduced expression of cutaneous-type keratin K1 and involucrin as a marker of squamous differentiation, supporting the role of FOXM1 in modulation of aberrant differentiation in HNSCC. Thus, our data provide a strong rationale for targeting FOXM1 in HNSCC. © 2019 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Mouse model of postsurgical primary tumor recurrence and regional lymph node metastasis progression in HPV-related head and neck cancer.

HPV-positive head and neck squamous cell carcinoma (HNSCC) is increasingly frequent. Management is particularly debated in the case of postsurgical high-risk features, that is, positive surgical margins and extracapsular spread (ECS). In this increasingly complex emerging framework of HNSCC treatment, representative preclinical models are needed to support future clinical trials and advances in personalized medicine. Here, we present an immunocompetent mouse model based on the implantation of mouse tonsil epithelial HPV16-E6/E7-expressing cancer cells into the submental region of the floor-of-the-mouth. Primary tumors were found to replicate the patterns of human HNSCC local invasion and lymphatic dissemination. To study disease progression after surgery, tumors were removed likely leaving behind residual disease. Surgical resection of tumors was followed by a high rate of local recurrences (>90%) within the first 2-3 weeks. While only 50% of mice had lymph node metastases (LNM) at time of primary tumor excision, all mice with recurrent tumors showed evidence of LNM. To study the consecutive steps of LNM progression and distant metastasis development, LNs from tumor-bearing mice were transplanted into naïve recipient mice. Using this approach, transplanted LNs were found to recapitulate all stages and relevant histological features of regional metastasis progression, including ECS and metastatic spread to the lungs. Altogether, we have developed an immunocompetent HPV-positive HNSCC mouse model of postsurgical local recurrence and regional and distant metastasis progression suitable for preclinical studies.

Mutant p53 promotes epithelial-mesenchymal plasticity and enhances metastasis in mammary carcinomas of WAP-T mice.

To study the postulated mutant p53 (mutp53) "gain of function" effects in mammary tumor development, progression and metastasis, we crossed SV40 transgenic WAP-T mice with mutant p53 transgenic WAP-mutp53 mice. Compared to tumors in monotransgenic WAP-T mice, tumors in bitransgenic WAP-T x WAP-mutp53 mice showed higher tumor grading, enhanced vascularization, and significantly increased metastasis. Bitransgenic tumors revealed a gene signature associated with the oncogenic epithelial-mesenchymal transition pathway (EMT gene signature). In cultures of WAP-T tumor-derived G-2 cancer cells, which are comprised of subpopulations displaying "mesenchymal" and "epithelial" phenotypes, this EMT gene signature was associated with the "mesenchymal" compartment. Furthermore, ectopic expression of mutp53 in G-2 cells sufficed to induce a strong EMT phenotype. In contrast to these in vitro effects, monotransgenic and bitransgenic tumors were phenotypically similar suggesting that in vivo the tumor cell phenotype might be under control of the tumor microenvironment. In support, orthotopic transplantation of G-2 cells as well as of G-2 cells expressing ectopic mutp53 into syngeneic mice resulted in tumors with a predominantly epithelial phenotype, closely similar to that of endogenous primary tumors. We conclude that induction of an EMT gene signature by mutp53 in bitransgenic tumors primarily promotes tumor cell plasticity, that is, the probability of tumor cells to undergo EMT processes under appropriate stimuli, thereby possibly increasing their potential to disseminate and metastasize.

A tetravalent nanovaccine that inhibits growth of HPV-associated head and neck carcinoma via dendritic and T cell activation.

The global incidence of human papillomavirus (HPV) associated head and neck carcinoma is on the rise, in response to this a tetravalent therapeutic vaccine named Qß-HPVag was developed. This vaccine, utilizing virus-like particles (VLPs) loaded with toll-like receptor ligands and chemically coupled to four HPV16-derived peptides, demonstrated strong anti-tumor effects in a murine head and neck cancer model. Qß-HPVag impeded tumor progression, increased infiltration of HPV-specific T cells, and significantly improved survival. The vaccine`s efficacy was associated with immune repolarization in the tumor microenvironment, characterized by expanded activated dendritic cell subsets (cDC1, cDC2, DC3). Notably, mice responding to treatment exhibited a higher percentage of migratory DC3 cells expressing CCR7. These findings suggest promising prospects for optimized VLP-based vaccines in treating HPV-associated head and neck cancer.

Transcription factors link mouse WAP-T mammary tumors with human breast cancer.

Mouse models are important tools to decipher the molecular mechanisms of mammary carcinogenesis and to mimic the respective human disease. Despite sharing common phenotypic and genetic features, the proper translation of murine models to human breast cancer remains a challenging task. In a previous study we showed that in the SV40 transgenic WAP-T mice an active Met-pathway and epithelial-mesenchymal characteristics distinguish low- and high-grade mammary carcinoma. To assign these murine tumors to corresponding human tumors we here incorporated the analysis of expression of transcription factor (TF) coding genes and show that thereby a more accurate interspecies translation can be achieved. We describe a novel cross-species translation procedure and demonstrate that expression of unsupervised selected TFs, such as ELF5, HOXA5 and TFCP2L1, can clearly distinguish between the human molecular breast cancer subtypes--or as, for example, expression of TFAP2B between yet unclassified subgroups. By integrating different levels of information like histology, gene set enrichment, expression of differentiation markers and TFs we conclude that tumors in WAP-T mice exhibit similarities to both, human basal-like and non-basal-like subtypes. We furthermore suggest that the low- and high-grade WAP-T tumor phenotypes might arise from distinct cells of tumor origin. Our results underscore the importance of TFs as common cross-species denominators in the regulatory networks underlying mammary carcinogenesis.

Low-grade and high-grade mammary carcinomas in WAP-T transgenic mice are independent entities distinguished by Met expression.

Mammary carcinomas developing in SV40 transgenic WAP-T mice arise in two distinct histological phenotypes: as differentiated low-grade and undifferentiated high-grade tumors. We integrated different types of information such as histological grading, analysis of aCGH-based gene copy number and gene expression profiling to provide a comprehensive molecular description of mammary tumors in WAP-T mice. Applying a novel procedure for the correlation of gene copy number with gene expression on a global scale, we observed in tumor samples a global coherence between genotype and transcription. This coherence can be interpreted as a matched transcriptional regulation inherited from the cells of tumor origin and determined by the activity of cancer driver genes. Despite common recurrent genomic aberrations, e.g. gain of chr. 15 in most WAP-T tumors, loss of chr. 19 frequently occurs only in low-grade tumors. These tumors show features of "basal-like" epithelial differentiation, particularly expression of keratin 14. The high-grade tumors are clearly separated from the low-grade tumors by strong expression of the Met gene and by coexpression of epithelial (e.g. keratin 18) and mesenchymal (e.g. vimentin) markers. In high-grade tumors, the expression of the nonmutated Met protein is associated with Met-locus amplification and Met activity. The role of Met as a cancer driver gene is supported by the contribution of active Met signaling to motility and growth of mammary tumor-derived cells. Finally, we discuss the independent origin of low- and high-grade tumors from distinct cells of tumor origin, possibly luminal progenitors, distinguished by Met gene expression and Met signaling.

Recombinant p53 displays heterogeneity during isoelectric focusing.

Human recombinant, baculovirus-expressed p53 protein focuses on 2D gels in multiple spots in the narrow pI range. Re-electrophoresis of the individual spots resulted in the appearance of multiple spots. The strings of spots were neither species specific, nor characteristic for baculovirus-expressed p53. Moreover, mutant p53 did not deviate from wild-type p53, indicating that this is an inherent property of p53. Okadaic acid treatment of insect cells, phosphate substitution reaction of purified p53, and individual analysis of all spots by mass spectrometry revealed that only a fraction of the recombinant p53 is phosphorylated. This finding excluded that the individual p53 spots in 2D gels reflect charge isomers generated by phosphorylation, but rather suggest that they are due to conformational flexibility of urea-denatured monomeric p53 molecules or deamidation of asparagine and glutamine residues. The latter possibility was confirmed by NanoLC-ESI MS/MS analysis. Our data provide a putative hint for a novel regulatory level for function and stability of p53, particularly the long-lived mutant p53 overexpressed in diverse tumor types.

Modulation of gene expression in U251 glioblastoma cells by binding of mutant p53 R273H to intronic and intergenic sequences.

Missense point mutations in the TP53 gene are frequent genetic alterations in human tumor tissue and cell lines derived thereof. Mutant p53 (mutp53) proteins have lost sequence-specific DNA binding, but have retained the ability to interact in a structure-selective manner with non-B DNA and to act as regulators of transcription. To identify functional binding sites of mutp53, we established a small library of genomic sequences bound by p53(R273H) in U251 human glioblastoma cells using chromatin immunoprecipitation (ChIP). Mutp53 binding to isolated DNA fragments confirmed the specificity of the ChIP. The mutp53 bound DNA sequences are rich in repetitive DNA elements, which are dispersed over non-coding DNA regions. Stable down-regulation of mutp53 expression strongly suggested that mutp53 binding to genomic DNA is functional. We identified the PPARGC1A and FRMD5 genes as p53(R273H) targets regulated by binding to intronic and intra-genic sequences. We propose a model that attributes the oncogenic functions of mutp53 to its ability to interact with intronic and intergenic non-B DNA sequences and modulate gene transcription via re-organization of chromatin.

Vaccination with a nanoparticle E7 vaccine can prevent tumor recurrence following surgery in a human papillomavirus head and neck cancer model.

High-risk human papillomavirus (HPV) encoding E6/E7-HPV oncogenes are responsible for a subgroup of head and neck squamous-cell carcinoma (HNSCC) and thus therapeutic E7-vaccines may be used to control HPV+HNSCC tumors. Herein we investigated the effects of an optimized nanoparticle-conjugated E7 long-peptide vaccine adjuvanted with CpG (NP-E7LP) in an orthotopic immunocompetent mouse model of HPV+HNSCC which is based on injection of HPV16 E6/E7-expressing mEERL95-cells into the submental space. In absence of surgery, vaccination performed before or after tumor-cell injection decreased tumor growth or prolonged mice survival only marginally, despite the high numbers of vaccine-induced circulating E7-specific IFN-γ-secreting CD8+ T-cells. This contrasts with the high-efficacy of NP-E7LP-vaccination reported in the genital and subcutaneous HPV16-E6/E7-expressing TC-1 models. Our data show that in a direct comparison, NP-E7LP-vaccination fully controlled TC-1, but not mEERL95, tumors subcutaneously growing in the flanks. Immune-cell infiltration was 10-fold higher in TC-1-tumors, than in mEERL95-tumors, suggesting that vaccine-induced CD8+ T-cells can only poorly infiltrate mEERL95-tumors. Indeed, immunofluorescence staining of orthotopic mEERL95-tumors showed that CD3+ T-cells are preferentially located peritumorally. However, when NP-E7LP-vaccination was performed after mEERL95-cell injection, but before resection of primary tumors, no postsurgical recurrence was observed and 100% of the mice survived until the experimental endpoint (day 70) in the NP-E7LP-vaccinated group. In contrast, we observed a 60% recurrence rate and only 35% survival in PBS-vaccinated mice. This suggests that removal of the primary tumor modified the tumor microenvironment, allowing a therapeutic effect of the vaccine-induced anti-tumor response. E7-vaccination combined with surgery may thus benefit patients with HPV+HNSCC.

CD40 Agonist Targeted to Fibroblast Activation Protein α Synergizes with Radiotherapy in Murine HPV-Positive Head and Neck Tumors.

PURPOSE: The incidence of human papillomavirus-associated head and neck squamous cell carcinoma (HPV+-HNSCC) is rising worldwide and although current therapeutic modalities are efficient in the majority of patients, there is a high rate of treatment failures. Thus, novel combination approaches are urgently needed to achieve better disease control in patients with HPV+-HNSCC. We investigated the safety and therapeutic efficacy of a novel fibroblast activation protein (FAP)-targeted CD40 agonist (FAP-CD40) in combination with local hypofractionated radiation in a syngeneic HPV+-HNSCC model. EXPERIMENTAL DESIGN: Using an established orthotopic model, we treated tumor-bearing mice with local hypofractionated radiotherapy (2 × 6 Gy) alone or in combination with a systemic administration of the FAP-CD40 antibody. Following up the mice, we evaluated the changes in the tumor microenvironment (TME) by immunofluorescence, FACS, and NanoString RNA analysis. RESULTS: The suboptimal radiotherapy regimen chosen failed to control tumors in the treated mice. The FAP-CD40 administered in monotherapy transiently controlled tumor growth, whereas the combined therapy induced durable complete responses in more than 80% of the tumor-bearing mice. This notable efficacy relied on the radiotherapy-induced remodeling of the TME and activation of the CD8+ T-cell-cDC1 axis and was devoid of the systemic toxicity frequently associated with CD40-targeted therapy. Moreover, the robust immunologic memory developed effectively prevented tumor relapses, a common feature in patients with HNSCC. CONCLUSIONS: Our study provides proof of concept, as well as mechanistic insights of the therapeutic efficacy of a bispecific FAP-CD40 combined with local radiotherapy in a FAP+-HNSCC model increasing overall survival and inducing long-term antitumor immunity.

Alu complementary DNA is enriched in atrophic macular degeneration and triggers retinal pigmented epithelium toxicity via cytosolic innate immunity.

Long interspersed nuclear element-1 (L1)­mediated reverse transcription (RT) of Alu RNA into cytoplasmic Alu complementary DNA (cDNA) has been implicated in retinal pigmented epithelium (RPE) degeneration. The mechanism of Alu cDNA­induced cytotoxicity and its relevance to human disease are unknown. Here we report that Alu cDNA is highly enriched in the RPE of human eyes with geographic atrophy, an untreatable form of age-related macular degeneration. We demonstrate that the DNA sensor cGAS engages Alu cDNA to induce cytosolic mitochondrial DNA escape, which amplifies cGAS activation, triggering RPE degeneration via the inflammasome. The L1-extinct rice rat was resistant to Alu RNA­induced Alu cDNA synthesis and RPE degeneration, which were enabled upon L1-RT overexpression. Nucleoside RT inhibitors (NRTIs), which inhibit both L1-RT and inflammasome activity, and NRTI derivatives (Kamuvudines) that inhibit inflammasome, but not RT, both block Alu cDNA toxicity, identifying inflammasome activation as the terminal effector of RPE degeneration.

Wild-type p53 enhances efficiency of simian virus 40 large-T-antigen-induced cellular transformation.

Abortive infection of BALB/c mouse embryo fibroblasts differing in p53 gene status (p53(+/+) versus p53(-/)(-)) with simian virus 40 (SV40) revealed a quantitatively and qualitatively decreased transformation efficiency in p53(-/-) cells compared to p53(+/+) cells, suggesting a supportive effect of wild-type (wt) p53 in the SV40 transformation process. SV40 transformation efficiency also was low in immortalized p53(-/-) BALB/c 10-1 cells but could be restored to approximately the level in immortalized p53(+/+) BALB/c 3T3 cells by reconstituting wt p53, but not mutant p53 (mutp53), expression. Stable expression of large T antigen (LT) in p53(+/+) 3T3 cells resulted in full transformation, while LT expression in p53(-/-) 10-1 cells could not promote growth in suspension or in soft agar to a significant extent. The helper effect of wt p53 is mediated by its cooperation with LT and resides in the p53 N terminus, as an N-terminally truncated p53 (DeltaNp53) could not rescue the p53-null phenotype. The p53 N terminus serves as a scaffold for recruiting transcriptional regulators like p300/CBP and Mdm2 into the LT-p53 complex. Consequently, LT affected global and specific gene expression in p53(+/+) cells significantly more than in p53(-/-) cells. Our data suggest that recruitment of transcriptional regulators into the LT-p53 complex may help to modify cellular gene expression in response to the needs of cellular transformation.

Functional endogenous LINE-1 retrotransposons are expressed and mobilized in rat chloroleukemia cells.

LINE-1 (L1) is a highly successful autonomous non-LTR retrotransposon and a major force shaping mammalian genomes. Although there are about 600 000 L1 copies covering 23% of the rat genome, full-length rat L1s (L1Rn) with intact open reading frames (ORFs) representing functional master copies for retrotransposition have not been identified yet. In conjunction with studies to elucidate the role of L1 retrotransposons in tumorigenesis, we isolated and characterized 10 different cDNAs from transcribed full-length L1Rn elements in rat chloroleukemia (RCL) cells, each encoding intact ORF1 proteins (ORF1p). We identified the first functional L1Rn retrotransposon from this pool of cDNAs, determined its activity in HeLa cells and in the RCL cell line the cDNAs originated from and demonstrate that it is mobilized in the tumor cell line in which it is expressed. Furthermore, we generated monoclonal antibodies directed against L1Rn ORF1 and ORF2-encoded recombinant proteins, analyzed the expression of L1-encoded proteins and found ORF1p predominantly in the nucleus. Our results support the hypothesis that the reported explosive amplification of genomic L1Rn sequences after their transcriptional activation in RCL cells is based on L1 retrotransposition. Therefore, L1 activity might be one cause for genomic instability observed during the progression of leukemia.

Mutant p53(R270H) gain of function phenotype in a mouse model for oncogene-induced mammary carcinogenesis.

In human breast cancer, mutations in the p53 gene are associated with poor prognosis. However, analysis of patient data so far did not clarify, whether missense point mutations in the p53 gene, in addition to causing loss of wild-type p53 function, also confer a gain of function phenotype to the encoded mutant p53. As heterogeneity of patient material and data might obscure a clear answer, we studied the effects of a coexpressed mutant p53(R270H) in transgenic mice in which SV40 early proteins initiate the development of mammary adenocarcinoma (WAP-T mice). In such tumors the endogenous wild-type p53 is functionally compromised by complex formation with SV40 T-antigen, thereby constituting a loss of wild-type p53 function situation that allowed analysis of the postulated gain of function effects of mutant p53(R270H). We found that mutant p53(R270H) in bi-transgenic mice enhanced the transition from intraepithelial neoplasia to invasive carcinoma, resulting in a higher frequency of invasive carcinoma per gland and per mouse, a more severe tumor phenotype, and more frequent pulmonary metastasis. Surprisingly, mutant p53(R270H) in this system does not increase genomic instability. Therefore, other postulated gain of function activities of mutant p53 must be responsible for the effects described here.

Cellular Barcoding Identifies Clonal Substitution as a Hallmark of Local Recurrence in a Surgical Model of Head and Neck Squamous Cell Carcinoma.

Local recurrence after surgery for head and neck squamous cell carcinoma (HNSCC) remains a common event associated with a dismal prognosis. Improving this outcome requires a better understanding of cancer cell populations that expand from postsurgical minimal residual disease (MRD). Therefore, we assessed clonal dynamics in a surgical model of barcoded HNSCC growing in the submental region of immunodeficient mice. Clonal substitution and massive reduction of clonal heterogeneity emerged as hallmarks of local recurrence, as the clones dominating in less heterogeneous recurrences were scarce in their matched primary tumors. These lineages were selected by their ability to persist after surgery and competitively expand from MRD. Clones enriched in recurrences exhibited both private and shared genetic features and likely originated from ancestors shared with clones dominating in primary tumors. They demonstrated high invasiveness and epithelial-to-mesenchymal transition, eventually providing an attractive target for obtaining better local control for these tumors.

Epigenetic mechanisms affect mutant p53 transgene expression in WAP-mutp53 transgenic mice.

We describe the construction and phenotypic characterization of 23 whey acidic protein (WAP)-mutp53 transgenic mouse lines. The mutp53-expressing lines showed a mosaic expression pattern for the transgenes, leading to a heterogeneous yet mouse line-specific expression pattern for mutp53 upon induction. Only few lines were obtained, in which the majority of the induced mammary epithelial cells expressed the mutp53 transgene, most of the transgenic lines did not express mutp53, or expressed the transgene in less than 2% of the induced mammary epithelial cells. Hormone requirements for mutp53 transgene expression from the WAP-promoter differed in high and low expressing lines, being low in high expressing lines, and even lower in multiparous mutp53 mice, where persistent expression of the transgene occurred. Repeated induction of mutp53 expression through repeated parturition resulted in the formation of expanding mutp53-expressing foci within the mammary alveolar epithelium. The data suggest that epigenetic mechanisms play a role in modulating the expression of the mutp53 transgene. To support this idea, we crossed a nonexpressing WAP-mutp53 line with a strongly SV40 T-antigen-expressing WAP-T mouse line. In the bitransgenic mice, T-antigen-induced chromatin remodeling led to re-expression of epigenetically silenced mutp53 transgene(s). In these mice, mutp53 expression was much more variable compared to SV40 T-antigen expression, and seemed to depend on the coexpression of SV40 T-antigen. Mutp53 expression in this system thus resembles the situation in many human tumors, where one can observe a heterogeneous expression of mutp53, despite a homogeneous distribution of the p53 mutation in the tumor cells.

Inhibition of LINE-1 expression in the heart decreases ischemic damage by activation of Akt/PKB signaling.

Microarray analyses indicate that ischemic and pharmacological preconditioning suppress overexpression of the non-long terminal repeat retrotransposon long interspersed nuclear element 1 (LINE-1, L1) after ischemia-reperfusion in the rat heart. We tested whether L1 overexpression is mechanistically involved in postischemic myocardial damage. Isolated, perfused rat hearts were treated with antisense or scrambled oligonucleotides (ODNs) against L1 for 60 min and exposed to 40 min of ischemia followed by 60 min of reperfusion. Functional recovery and infarct size were measured. Effective nuclear uptake was determined by FITC-labeled ODNs, and downregulation of L1 transcription was confirmed by RT-PCR. Immunoblot analysis was used to assess changes in expression levels of the L1-encoded proteins ORF1p and ORF2p. Immunohistochemistry was performed to localize ORF1/2 proteins in cardiac tissue. Effects of ODNs on prosurvival protein kinase B (Akt/PKB) expression and activity were also determined. Antisense ODNs against L1 prevented L1 burst after ischemia-reperfusion. Inhibition of L1 increased Akt/PKBbeta expression, enhanced phosphorylation of PKB at serine 473, and markedly improved postischemic functional recovery and decreased infarct size. Antisense ODN-mediated protection was abolished by LY-294002, confirming the involvement of the Akt/PKB survival pathway. ORF1p and ORF2p were found to be expressed in rat heart. ORF1p showed a predominantly nuclear localization in cardiomyocytes, whereas ORF2p was exclusively present in endothelial cells. ORF1p levels increased in response to ischemia, which was reversed by antisense ODN treatment. No significant changes in ORF2p were noted. Our results demonstrate that L1 suppression favorably affects postischemic outcome in the heart. Modifying transcriptional activity of L1 may represent a novel anti-ischemic therapeutic strategy.

Interaction in vitro of type III intermediate filament proteins with higher order structures of single-stranded DNA, particularly with G-quadruplex DNA.

Cytoplasmic intermediate filament (cIF) proteins interact strongly with single-stranded (ss) DNAs and RNAs, particularly with G-rich sequences. To test the hypothesis that this interaction depends on special nucleotide sequences and, possibly, higher order structures of ssDNA, a random mixture of mouse genomic ssDNA fragments generated by a novel "whole ssDNA genome PCR" technique via RNA intermediates was subjected to three rounds of affinity binding to in vitro reconstituted vimentin IFs at physiological ionic strength with intermediate PCR amplification of the bound ssDNA segments. Nucleotide sequence and computer folding analysis of the vimentin-selected fragments revealed an enrichment in microsatellites, predominantly of the (GT)n type, telomere DNA, and C/T-rich sequences, most of which, however, were incapable of folding into stable stem-loop structures. Because G-rich sequences were underrepresented in the vimentin-bound fraction, it had to be assumed that such sequences require intramolecular folding or lateral assembly into multistrand structures to be able to stably interact with vimentin, but that this requirement was inadequately fulfilled under the conditions of the selection experiment. For that reason, the few vimentin-selected G-rich ssDNA fragments and a number of telomere models were analyzed for their capacity to form inter- and intramolecular Gquadruplexes (G4 DNAs) under optimized conditions and to interact as such with vimentin and its type III relatives, glial fibrillary acidic protein, and desmin. Band shift assays indeed demonstrated differential binding of the cIF proteins to parallel four-stranded G4 DNAs and, with lower affinity, to bimolecular G'2 and unimolecular G'4 DNA configurations, whereby the transition regions from four- to single-strandedness played an additional role in the binding reaction. In this respect, the binding activity of cIF proteins was comparable with that toward other noncanonical DNA structures, like ds/ss DNA forks, triplex DNA, four-way junction DNA and Z-DNA, which also involve configurational transitions in their interaction with the filament proteins. Association of the cIF proteins with the corresponding nonfolded G-rich ssDNAs was negligible. Considering the almost universal involvement of ssDNA regions and G-quadruplexes in nuclear processes, including DNA transcription and recombination as well as telomere maintenance and dynamics, it is plausible to presume that cIF proteins as complementary constituents of the nuclear matrix participate in the cell- and tissue-specific regulation of these processes.