Bovine spongiform encephalopathy infection alters endogenous retrovirus expression in distinct brain regions of cynomolgus macaques (Macaca fascicularis)

<p>Abstract</p> <p>Background</p> <p>Prion diseases such as bovine spongiform encephalopathies (BSE) are transmissible neurodegenerative diseases which are presumably caused by an infectious conformational isoform of the cellular prion protein. Previous work has provided evidence that in murine prion disease the endogenous retrovirus (ERV) expression is altered in the brain. To determine if prion-induced changes in ERV expression are a general phenomenon we used a non-human primate model for prion disease.</p> <p>Results</p> <p>Cynomolgus macaques (<it>Macaca fasicularis</it>) were infected intracerebrally with BSE-positive brain stem material from cattle and allowed to develop prion disease. Brain tissue from the <it>basis pontis </it>and <it>vermis cerebelli </it>of the six animals and the same regions from four healthy controls were subjected to ERV expression profiling using a retrovirus-specific microarray and quantitative real-time PCR. We could show that Class I gammaretroviruses HERV-E4-1, ERV-9, and MacERV-4 increase expression in BSE-infected macaques. In a second approach, we analysed ERV-K-(HML-2) RNA and protein expression in extracts from the same cynomolgus macaques. Here we found a significant downregulation of both, the macaque ERV-K-(HML-2) Gag protein and RNA in the frontal/parietal cortex of BSE-infected macaques.</p> <p>Conclusions</p> <p>We provide evidence that dysregulation of ERVs in response to BSE-infection can be detected on both, the RNA and the protein level. To our knowledge, this is the first report on the differential expression of ERV-derived structural proteins in prion disorders. Our findings suggest that endogenous retroviruses may induce or exacerbate the pathological consequences of prion-associated neurodegeneration.</p

c-MYB is a transcriptional regulator of ESPL1/Separase in BCR-ABL-positive chronic myeloid leukemia

Background: Genomic instability and clonal evolution are hallmarks of progressing chronic myeloid leukemia (CML). Recently, we have shown that clonal evolution and blast crisis correlate with altered expression and activity of Separase, a cysteine endopeptidase that is a mitotic key player in chromosomal segregation and centriole duplication. Hyperactivation of Separase in human hematopoietic cells has been linked to a feedback mechanism that posttranslationally stimulates Separase proteolytic activity after imatinib therapy-induced reduction of Separase protein levels. Methods and Results: In search for potential therapy-responsive transcriptional mechanisms we have investigated the role of the transcription factor c-MYB for Separase expression in CML cell lines (LAMA-84, K562, BV-173) and in clinical samples. Quantitative RT-PCR and Western blot immunostaining experiments revealed that c-MYB expression levels are decreased in an imatinib-dependent manner and positively correlate with Separase expression levels in cell lines and in clinical CML samples. RNA silencing of c-MYB expression in CML cell lines resulted in reduced Separase protein levels. Gelshift and ChIP assays confirmed that c-MYB binds to a putative c-MYB binding sequence located within the ESPL1 promoter. Conclusions: Our data suggest that ESPL1/Separase is a regulatory target of c-MYB. Therefore, c-MYB, known to be required for BCR-ABL-dependent transformation of hematopoietic progenitors and leukemogenesis, may also control the Separase-dependent fidelity of mitotic chromosomal segregation and centriole duplication essential for maintenance of genomic stability

Expression pattern analysis of transcribed HERV sequences is complicated by ex vivo recombination

<p>Abstract</p> <p>Background</p> <p>The human genome comprises numerous human endogenous retroviruses (HERVs) that formed millions of years ago in ancestral species. A number of loci of the HERV-K(HML-2) family are evolutionarily much younger. A recent study suggested an infectious HERV-K(HML-2) variant in humans and other primates. Isolating such a variant from human individuals would be a significant finding for human biology.</p> <p>Results</p> <p>When investigating expression patterns of specific HML-2 proviruses we encountered HERV-K(HML-2) cDNA sequences without proviral homologues in the human genome, named HERV-KX, that could very well support recently suggested infectious HML-2 variants. However, detailed sequence analysis, using the software RECCO, suggested that HERV-KX sequences were produced by recombination, possibly arising <it>ex vivo</it>, between transcripts from different HML-2 proviral loci.</p> <p>Conclusion</p> <p>As RT-PCR probably will be instrumental for isolating an infectious HERV-K(HML-2) variant, generation of "new" HERV-K(HML-2) sequences by <it>ex vivo </it>recombination seems inevitable. Further complicated by an unknown amount of allelic sequence variation in HERV-K(HML-2) proviruses, newly identified HERV-K(HML-2) variants should be interpreted very cautiously.</p

Expression patterns of transcribed human endogenous retrovirus HERV-K(HML-2) loci in human tissues and the need for a HERV Transcriptome Project

Background: A significant proportion of the human genome is comprised of human endogenous retroviruses (HERVs). HERV transcripts are found in every human tissue. Expression of proviruses of the HERV-K(HML-2) family has been associated with development of human tumors, in particular germ cell tumors (GCT). Very little is known about transcriptional activity of individual HML-2 loci in human tissues, though. Results: By employing private nucleotide differences between loci, we assigned ~1500 HML-2 cDNAs to individual HML-2 loci, identifying, in total, 23 transcriptionally active HML-2 proviruses. Several loci are active in various human tissue types. Transcription levels of some HML-2 loci appear higher than those of other loci. Several HML-2 Rec-encoding loci are expressed in GCT and non-GCT tissues. A provirus on chromosome 22q11.21 appears strongly upregulated in pathologic GCT tissues and may explain high HML-2 Gag protein levels in GCTs. Presence of Gag and Env antibodies in GCT patients is not correlated with activation of individual loci. HML-2 proviruses previously reported capable of forming an infectious HML-2 variant are transcriptionally active in germ cell tissue. Our study furthermore shows that Expressed Sequence Tag (EST) data are insufficient to describe transcriptional activity of HML-2 and other HERV loci in tissues of interest. Conclusion: Our, to date, largest-scale study reveals in greater detail expression patterns of individual HML-2 loci in human tissues of clinical interest. Moreover, large-scale, specialized studies are indicated to better comprehend transcriptional activity and regulation of HERVs. We thus emphasize the need for a specialized HERV Transcriptome Project

Modulation of human endogenous retrovirus (HERV) transcription during persistent and de novo HIV-1 infection

Background: The human genome contains multiple LTR elements including human endogenous retroviruses (HERVs) that together account for approximately 8–9% of the genomic DNA. At least 40 different HERV groups have been assigned to three major HERV classes on the basis of their homologies to exogenous retroviruses. Although most HERVs are silenced by a variety of genetic and epigenetic mechanisms, they may be reactivated by environmental stimuli such as exogenous viruses and thus may contribute to pathogenic conditions. The objective of this study was to perform an in-depth analysis of the influence of HIV-1 infection on HERV activity in different cell types. Results: A retrovirus-specific microarray that covers major HERV groups from all three classes was used to analyze HERV transcription patterns in three persistently HIV-1 infected cell lines of different cellular origins and in their uninfected counterparts. All three persistently infected cell lines showed increased transcription of multiple class I and II HERV groups. Up-regulated transcription of five HERV taxa (HERV-E, HERV-T, HERV-K (HML-10) and two ERV9 subgroups) was confirmed by quantitative reverse transcriptase PCR analysis and could be reversed by knock-down of HIV-1 expression with HIV-1-specific siRNAs. Cells infected de novo by HIV-1 showed stronger transcriptional up-regulation of the HERV-K (HML-2) group than persistently infected cells of the same origin. Analysis of transcripts from individual members of this group revealed up-regulation of predominantly two proviral loci (ERVK-7 and ERVK-15) on chromosomes 1q22 and 7q34 in persistently infected KE37.1 cells, as well as in de novo HIV-1 infected LC5 cells, while only one single HML-2 locus (ERV-K6) on chromosome 7p22.1 was activated in persistently infected LC5 cells. Conclusions: Our results demonstrate that HIV-1 can alter HERV transcription patterns of infected cells and indicate a correlation between activation of HERV elements and the level of HIV-1 production. Moreover, our results suggest that the effects of HIV-1 on HERV activity may be far more extensive and complex than anticipated from initial studies with clinical material

Development of a LightCycler PCR assay for detection and quantification of Aspergillus fumigatus DNA in clinical samples from neutropenic patients

The increasing incidence of invasive aspergillosis, a life-threatening infection in immunocompromised patients, emphasizes the need to improve the diagnostic tools for this disease. We established a LightCyclerbased real-time PCR assay to detect and quantify rapidly, specifically, and sensitively Aspergillus fumigatus DNA in both bronchoalveolar lavage (BAL) and blood samples from high-risk patients. The primers and hybridization probes were derived from an A. fumigatus-specific sequence of the mitochondrial cytochrome b gene. The assay is linear in the range between 13.2 fg and 1.3 ng of A. fumigatus DNA, corresponding to 3 to 300,000 CFU per ml of BAL fluid or blood. No cross-amplification was observed with human DNA or with the DNA of fungal or bacterial pathogens. For clinical evaluation we investigated 10 BAL samples from nine neutropenic patients with malignant hematological diseases and 12 blood samples from seven neutropenic patients with malignant hematological diseases. Additionally, we tested one blood sample and one BAL sample from each of two neutropenic patients. In order to characterize the validity of the novel PCR assay, only samples that had shown positive results by a previously described sensitive and specific nested PCR assay were tested. Twelve of 12 BAL samples and 6 of 14 blood samples gave positive results by the LightCycler PCR assay. Eight of 14 blood samples gave negative results by the novel method. The LightCycler PCR-mediated quantification of the fungal burden showed 15 to 269,018 CFU per ml of BAL sample and 298 to 104,114 CFU per ml of blood sample. Twenty of 20 BAL samples and 50 of 50 blood samples from subjects without evidence of invasive pulmonary aspergillosis (IPA) were PCR negative. Compared to a previously described nested PCR assay, these preliminary data for the novel real-time PCR assay indicate a less sensitive rate of detection of IPA in high-risk patients, but the assay may be valuable for quantification of the fungal burden in individual clinical samples

Expression of Human Endogenous Retrovirus-W Including Syncytin-1 in Cutaneous T-Cell Lymphoma

Peer reviewe

Effect of ABCG2, OCT1, and ABCB1(MDR1) Gene Expression on Treatment-Free Remission in a EURO-SKI Subtrial

Introduction Tyrosine kinase inhibitors (TKIs) can safely be discontinued in chronic myeloid leukemia (CML) patients with sustained deep molecular response. ABCG2 (breast cancer resistance protein), OCT1 (organic cation transporter 1), and ABCB1 (multidrug resistance protein 1) gene products are known to play a crucial role in acquired pharmacogenetic TKI resistance. Their influence on treatment-free remission (TFR) has not yet been investigated. Materials and Methods RNA was isolated on the last day of TKI intake from peripheral blood leukocytes of 132 chronic phase CML patients who discontinued TKI treatment within the European Stop Tyrosine Kinase Inhibitor Study trial. Plasmid standards were designed including subgenic inserts of OCT1, ABCG2, and ABCB1 together with GUSB as reference gene. For expression analyses, quantitative real-time polymerase chain reaction was used. Multiple Cox regression analysis was performed. In addition, gene expression cutoffs for patient risk stratification were investigated. Results The TFR rate of 132 patients, 12 months after TKI discontinuation, was 54% (95% confidence interval [CI], 46%-62%). ABCG2 expression (‰) was retained as the only significant variable (P = .02; hazard ratio, 1.04; 95% CI, 1.01-1.07) in multiple Cox regression analysis. Only for the ABCG2 efflux transporter, a significant cutoff was found (P = .04). Patients with an ABCG2/GUSB transcript level >4.5‰ (n = 93) showed a 12-month TFR rate of 47% (95% CI, 37%-57%), whereas patients with low ABCG2 expression (≤4.5‰; n = 39) had a 12-month TFR rate of 72% (95% CI, 55%-82%). Conclusion In this study, we investigated the effect of pharmacogenetics in the context of a CML treatment discontinuation trial. The transcript levels of the efflux transporter ABCG2 predicted TFR after TKI discontinuation

Epigenetic Regulation of a Murine Retrotransposon by a Dual Histone Modification Mark

Large fractions of eukaryotic genomes contain repetitive sequences of which the vast majority is derived from transposable elements (TEs). In order to inactivate those potentially harmful elements, host organisms silence TEs via methylation of transposon DNA and packaging into chromatin associated with repressive histone marks. The contribution of individual histone modifications in this process is not completely resolved. Therefore, we aimed to define the role of reversible histone acetylation, a modification commonly associated with transcriptional activity, in transcriptional regulation of murine TEs. We surveyed histone acetylation patterns and expression levels of ten different murine TEs in mouse fibroblasts with altered histone acetylation levels, which was achieved via chemical HDAC inhibition with trichostatin A (TSA), or genetic inactivation of the major deacetylase HDAC1. We found that one LTR retrotransposon family encompassing virus-like 30S elements (VL30) showed significant histone H3 hyperacetylation and strong transcriptional activation in response to TSA treatment. Analysis of VL30 transcripts revealed that increased VL30 transcription is due to enhanced expression of a limited number of genomic elements, with one locus being particularly responsive to HDAC inhibition. Importantly, transcriptional induction of VL30 was entirely dependent on the activation of MAP kinase pathways, resulting in serine 10 phosphorylation at histone H3. Stimulation of MAP kinase cascades together with HDAC inhibition led to simultaneous phosphorylation and acetylation (phosphoacetylation) of histone H3 at the VL30 regulatory region. The presence of the phosphoacetylation mark at VL30 LTRs was linked with full transcriptional activation of the mobile element. Our data indicate that the activity of different TEs is controlled by distinct chromatin modifications. We show that activation of a specific mobile element is linked to a dual epigenetic mark and propose a model whereby phosphoacetylation of histone H3 is crucial for full transcriptional activation of VL30 elements