Two non-homologous brain diseases-related genes, SERPINI1 and PDCD10, are tightly linked by an asymmetric bidirectional promoter in an evolutionarily conserved manner

BACKGROUND: Despite of the fact that mammalian genomes are far more spacious than prokaryotic genomes, recent nucleotide sequencing data have revealed that many mammalian genes are arranged in a head-to-head orientation and separated by a small intergenic sequence. Extensive studies on some of these neighboring genes, in particular homologous gene pairs, have shown that these genes are often co-expressed in a symmetric manner and regulated by a shared promoter region. Here we report the identification of two non-homologous brain disease-related genes, with one coding for a serine protease inhibitor (SERPINI1) and the other for a programmed cell death-related gene (PDCD10), being tightly linked together by an asymmetric bidirectional promoter in an evolutionarily conserved fashion. This asymmetric bidirectional promoter, in cooperation with some cis-acting elements, is responsible for the co-regulation of the gene expression pattern as well as the tissue specificity of SERPINI1 and PDCD10. RESULTS: While SERPINI1 is predominantly expressed in normal brain and down-regulated in brain tumors, PDCD10 is ubiquitously expressed in all normal tissues but its gene transcription becomes aberrant in different types of cancers. By measuring the luciferase activity in various cell lysates, their 851-bp intergenic sequence was shown to be capable of driving the reporter gene expression in either direction. A 175-bp fragment from nt 1 to 175 in the vicinity of PDCD10 was further determined to function as a minimal bidirectional promoter. A critical regulatory fragment, from nt 176-473 outside the minimal promoter in the intergenic region, was identified to contain a strong repressive element for SERPINI1 and an enhancer for PDCD10. These cis-acting elements may exist to help coordinate the expression and regulation of the two flanking genes. CONCLUSION: For all non-homologous genes that have been described to be closely adjacent in the mammalian genomes, the intergenic region of the head-to-head PDCD10-SERPINI1 gene pair provides an interesting and informative example of a complex regulatory system that governs the expression of both genes not only through an asymmetric bidirectional promoter, but also through fine-tuned regulations with some cis-acting elements

Bi-directional and shared epigenomic signatures following proton and 56Fe irradiation.

The brain's response to radiation exposure is an important concern for patients undergoing cancer therapy and astronauts on long missions in deep space. We assessed whether this response is specific and prolonged and is linked to epigenetic mechanisms. We focused on the response of the hippocampus at early (2-weeks) and late (20-week) time points following whole body proton irradiation. We examined two forms of DNA methylation, cytosine methylation (5mC) and hydroxymethylation (5hmC). Impairments in object recognition, spatial memory retention, and network stability following proton irradiation were observed at the two-week time point and correlated with altered gene expression and 5hmC profiles that mapped to specific gene ontology pathways. Significant overlap was observed between DNA methylation changes at the 2 and 20-week time points demonstrating specificity and retention of changes in response to radiation. Moreover, a novel class of DNA methylation change was observed following an environmental challenge (i.e. space irradiation), characterized by both increased and decreased 5hmC levels along the entire gene body. These changes were mapped to genes encoding neuronal functions including postsynaptic gene ontology categories. Thus, the brain's response to proton irradiation is both specific and prolonged and involves novel remodeling of non-random regions of the epigenome

How human papillomavirus replication and immune evasion strategies take advantage of the host DNA damage repair machinery

The DNA damage response (DDR) is a complex signalling network activated when DNA is altered by intrinsic or extrinsic agents. DDR plays important roles in genome stability and cell cycle regulation, as well as in tumour transformation. Viruses have evolved successful life cycle strategies in order to ensure a chronic persistence in the host, virtually avoiding systemic sequelae and death. This process promotes the periodic shedding of large amounts of infectious particles to maintain a virus reservoir in individual hosts, while allowing virus spreading within the community. To achieve such a successful lifestyle, the human papilloma virus (HPV) needs to escape the host defence systems. The key to understanding how this is achieved is in the virus replication process that provides by itself an evasion mechanism by inhibiting and delaying the host immune response against the viral infection. Numerous studies have demonstrated that HPV exploits both the ataxia-telangiectasia mutated (ATM) and ataxia-telangiectasia and rad3-related (ATR) DDR pathways to replicate its genome and maintain a persistent infection by downregulating the innate and cell-mediated immunity. This review outlines how HPV interacts with the ATM-and ATR-dependent DDR machinery during the viral life cycle to create an environment favourable to viral replication, and how the interaction with the signal transducers and activators of transcription (STAT) protein family and the deregulation of the Janus kinase (JAK)-STAT pathways may impact the expression of interferon-inducible genes and the innate immune responses

Ultra-high throughput sequencing-based small RNA discovery and discrete statistical biomarker analysis in a collection of cervical tumours and matched controls

Background: Ultra-high throughput sequencing technologies provide opportunities both for discovery of novel molecular species and for detailed comparisons of gene expression patterns. Small RNA populations are particularly well suited to this analysis, as many different small RNAs can be completely sequenced in a single instrument run.Results: We prepared small RNA libraries from 29 tumour/normal pairs of human cervical tissue samples. Analysis of the resulting sequences (42 million in total) defined 64 new human microRNA (miRNA) genes. Both arms of the hairpin precursor were observed in twenty-three of the newly identified miRNA candidates. We tested several computational approaches for the analysis of class differences between high throughput sequencing datasets and describe a novel application of a log linear model that has provided the most effective analysis for this data. This method resulted in the identification of 67 miRNAs that were differentially-expressed between the tumour and normal samples at a false discovery rate less than 0.001.Conclusions: This approach can potentially be applied to any kind of RNA sequencing data for analysing differential sequence representation between biological sample sets. ?? 2010 Witten et al; licensee BioMed Central Ltd

Small Molecule Inhibitors of Human Papillomavirus Protein - Protein Interactions

Human papillomaviruses (HPV) have now been identified as a necessary cause of benign and malignant lesions of the differentiating epithelium, particularly cervical cancer, the second most prevalent cancer in women worldwide. While two prophylactic HPV vaccines and screening programs are available, there is currently no antiviral drug for the treatment of HPV infections and associated diseases. The recent progress toward the identification and characterization of specific molecular targets for small molecule-based approaches provides prospect for the development of effective HPV antiviral compounds. Traditionally, antiviral therapies target viral enzymes. HPV encode for few proteins, however, and rely extensively on the infected cell for completion of their life cycle. This article will review the functions of the viral E1 helicase, which encodes the only enzymatic function of the virus, of the E2 regulatory protein, and of the viral E6 and E7 oncogenes in viral replication and pathogenesis. Particular emphasis will be placed on the recent progress made towards the development of novel small molecule inhibitors that specifically target and inhibit the functions of these viral proteins, as well as their interactions with other viral and/or cellular proteins

Papilloomiviiruse genoomi replikatsiooni ja säilumise uurimine

Väitekirja elektrooniline versioon ei sisalda publikatsioone.Papilloomiviirused on ümbriseta ja kaheahelalise DNA genoomiga viirused, mis on rangelt liigispetsiifilised. Siiski on inimest võimelised nakatama enam kui sada erinevat HPV tüüpi. Kõige levinumateks kasvajaid tekitavateks viirusteks on HPV16 ja HPV18, millede nakkuse vältimiseks on arendatud ka vaktsiinid. HPV vastane vaktsineerimine vähendab haigestumise tõenäosust vähemalt 70%, kuid täielikku kaitset see paraku pakkuda ei suuda. Lisaks on olemasolevad vaktsiinid mõeldud ainult haiguste ennetamiseks ja on kasutud viirusega juba nakatunud inimestele. Paraku ei ole veel välja töötatud ravimeid, mis oleksid võimelised papilloomiviiruse infektsiooni inimese organismist kõrvaldama. HPV on kõrge koespetsiifilisusega viirus ja nakatab epiteeli peamist rakutüüpi – keratinotsüüte. Nakatamiseks peab viirus sisenema naha epiteeli basaalkihi rakkudesse või genitaalide ja teiste organite limaskestade vastavatesse rakkudesse. Kuna HPV elustükkel sõltub täielikult keratinotsüütide diferentseerumisest, siis selle viiruse uurimist raskendab asjaolu, et uurimistöös kasutatavates rakulistes mudelsüsteemides on keratinotsüütide diferentseerumise käivitamine väga keerukas. Antud uurimistöös kirjeldatakse inimese sääreluu kasvajast algatatud U2OS rakuliinil põhinevat rakulist mudelsüsteemi, mida saab kasutada HPV genoomide replikatsiooni uurimiseks. Loodud mudelsüsteem on võimeline toetama erinevat tüüpi HPVde DNA replikatsiooni nii nagu see toimub viiruse loomuliku elutsükli eri etappidel. Samuti võimaldab mudelsüsteem võrdlevalt uurida nii limaskesta spetesiifilisi kõrgeriski (HPV16, HPV18), kui ka madalariski (HPV6b, HPV11) ning nahka nakatavaid (HPV5, HPV8) papilloomiviirusi, mille uurimine teistes rakulistes mudelsüsteemides on seni olnud üsna keerukas. Samuti näitame, et HPV DNA replikatsiooni on kaasatud vähemalt kaks erinevat mehhanismi. Ühte neist on varasemalt juba kirjeldatud kui võimalikku papilloomiviiruste päriliku materjali paljundamise meetodit – selleks on kahesuunaline theta-tüüpi replikatsioon. U2OS rakuliinil põhineva mudelsüsteemi abil tuvastasime aga veel teise, ühesuunalise replikatsioonimehhanismi, mis oletatavalt põhineb molekulidevahelisel homoloogsel rekombinatsioonil ja mille roll HPV elutsüklis on veel ebaselge. Lisaks ülaltoodule on käesolevas töös näidatud, et kõikide vajalike viiruseliste trans-faktorite juuresolekul on heteroloogsed originid, mis sisaldavad veise papilloomiviirusest (BPV1) või ka Epstein-Barr viirusest (EBV) pärinevaid segregatsioonielemente, võimelised tagama neid elemente sisaldavate originiplasmiidide stabiilse säilumise.Papillomavirus infections are common in both people and animals. Generally, these viruses may result in asymptomatic infections and can cause benign tumors as condylomas and papillomas in mucous membranes or warts and skin papillomas. However, in the case of persistent infections, these viruses may induce malignant tumors at various body sites. Currently, there are two commonly used HPV vaccines on the market. The Gardasil (against HPV6/ HPV11/ HPV16/ HPV18) and Cervarix (HPV16 / HPV18) vaccines are preventive, they do not eliminate existing infection and have no therapeutic effect in diseases caused by HPV. A multivalent HPV therapeutic vaccine candidate against six HPVs was reported; however, this vaccine is only in preclinical studies, so there is an urgent medical need for drugs that suppress HPV DNA replication. HPV DNA replicates in the epithelial tissue, which is mostly composed of differentiating keratinocytes. HPV genomic DNA replication occurs in three clearly distinguishable phases, making the study and control of the virus complicated, because it is difficult to mimic all three stages of the virus infection cycle in the regular tissue culture cells under laboratory conditions. We demonstrate that HPV genomes replicate efficiently in the human osteosarcoma cell line U2OS, which is capable of supporting the genome replication of many different subtypes (α-HPVs of high-risk HPV18 and HPV16; low-risk HPV6b and HPV11 and β-HPVs of HPV5 and HPV8). U2OS cells capable of supporting the maintenance of the HPV genomes provide a useful model system to study the mechanisms and regulation of viral DNA replication during various phases of the viral life cycle, including initial amplification, stable maintenance and late amplification. We have demonstrated that two different mechanisms are involved in the HPV genome replication, shedding new light on the events that occur during the different phases of papillomavirus DNA replication. Finally, an analysis of the papillomavirus and the Epstein-Barr virus genomes segregation functions are included. Our data demonstrated that BPV1 E2 protein and minichromosome maintenance element (MME), or EBNA1 and FR-element dependent stable maintenance functions are necessary for the stable episomal multicopy nuclear replication of the plasmids with hybrid replication origin in different types of dividing cells

Novel principles of gamma-retroviral insertional transcription activation in murine leukemia virus-induced end-stage tumors

BACKGROUND: Insertional mutagenesis screens of retrovirus-induced mouse tumors have proven valuable in human cancer research and for understanding adverse effects of retroviral-based gene therapies. In previous studies, the assignment of mouse genes to individual retroviral integration sites has been based on close proximity and expression patterns of annotated genes at target positions in the genome. We here employed next-generation RNA sequencing to map retroviral-mouse chimeric junctions genome-wide, and to identify local patterns of transcription activation in T-lymphomas induced by the murine leukemia gamma-retrovirus SL3-3. Moreover, to determine epigenetic integration preferences underlying long-range gene activation by retroviruses, the colocalization propensity with common epigenetic enhancer markers (H3K4Me1 and H3K27Ac) of 6,117 integrations derived from end-stage tumors of more than 2,000 mice was examined. RESULTS: We detected several novel mechanisms of retroviral insertional mutagenesis: bidirectional activation of mouse transcripts on opposite sides of a provirus including transcription of unannotated mouse sequence; sense/antisense-type activation of genes located on opposite DNA strands; tandem-type activation of distal genes that are positioned adjacently on the same DNA strand; activation of genes that are not the direct integration targets; combination-type insertional mutagenesis, in which enhancer activation, alternative chimeric splicing and retroviral promoter insertion are induced by a single retrovirus. We also show that irrespective of the distance to transcription start sites, the far majority of retroviruses in end-stage tumors colocalize with H3K4Me1 and H3K27Ac-enriched regions in murine lymphoid tissues. CONCLUSIONS: We expose novel retrovirus-induced host transcription activation patterns that reach beyond a single and nearest annotated gene target. Awareness of this previously undescribed layer of complexity may prove important for elucidation of adverse effects in retroviral-based gene therapies. We also show that wild-type gamma-retroviruses are frequently positioned at enhancers, suggesting that integration into regulatory regions is specific and also subject to positive selection for sustaining long-range gene activation in end-stage tumors. Altogether, this study should prove useful for extrapolating adverse outcomes of retroviral vector therapies, and for understanding fundamental cellular regulatory principles and retroviral biology