RNA non-codificanti indotti da danno al DNA regolano il fattore di trascrizione HIF-1α

Recenti analisi sull’intero trascrittoma hanno rivelato una estensiva trascrizione di RNA non codificanti (ncRNA), le quali funzioni sono tuttavia in gran parte sconosciute. In questo lavoro è stato dimostrato che alte dosi di camptotecina (CPT), un farmaco antitumorale inibitore della Top1, aumentano la trascrizione di due ncRNA antisenso in 5’ e 3’ (5'aHIF-1α e 3'aHIF-1α rispettivamente) al locus genico di HIF-1α e diminuiscono i livelli dell’mRNA di HIF-1α stesso. Gli effetti del trattamento sono Top1-dipendenti, mentre non dipendono dal danno al DNA alla forca di replicazione o dai checkpoint attivati dal danno al DNA. I ncRNA vengono attivati in risposta a diversi tipi di stress, il 5'aHIF-1α è lungo circa 10 kb e possiede sia il CAP in 5’ sia poliadenilazione in 3’ (in letteratura è noto che il 3'aHIF-1α è un trascritto di 1,7 kb, senza 5’CAP né poliadenilazione). Analisi di localizzazione intracellulare hanno dimostrato che entrambi sono trascritti nucleari. In particolare 5'aHIF-1α co-localizza con proteine del complesso del poro nucleare, suggerendo un suo possibile ruolo come mediatore degli scambi della membrana nucleare. È stata dimostrata inoltre la trascrizione dei due ncRNA in tessuti di tumore umano del rene, evidenziandone possibili ruoli nello sviluppo del cancro. È anche noto in letteratura che basse dosi di CPT in condizioni di ipossia diminuiscono i livelli di proteina di HIF-1α. Dopo aver dimostrato su diverse linee cellulari che i due ncRNA sopracitati non potessero essere implicati in tale effetto, abbiamo studiato le variazioni dell’intero miRnoma alle nuove condizioni sperimentali. In tal modo abbiamo scoperto che il miR-X sembra essere il mediatore molecolare dell’abbattimento di HIF-1α dopo trattamento con basse dosi di CPT in ipossia. Complessivamente, questi risultati suggeriscono che il fattore di trascrizione HIF-1α venga finemente regolato da RNA non-codificanti indotti da danno al DNA.

Regulation von "Silent mating type information regulation 2 homolog Type" 1 (SIRT1) durch die nach DNA-Schaden induzierte Kinase "Homeodomain-interaction kinase" (HIPK2)

"Silent mating type information regulation 2 Type" 1 (SIRT1), das humane Homolog der NAD+-abhängigen Histondeacetylase Sir2 aus Hefe, besitzt Schlüsselfunktionen in der Regulation des Metabolismus, der Zellalterung und Apoptose. Letztere wird vor allem durch die Deacetylierung von p53 an Lys382 und der dadurch verringerten Transkription proapoptotischer Zielgene vermittelt. Im Rahmen der vorliegenden Arbeit wurde die SIRT1 Regulation im Zusammenhang mit der DNA-Schadensantwort untersucht.rnIn der Apoptoseregulation übernimmt die Serin/Threonin-Kinase "Homeodomain interacting protein kinase" 2 (HIPK2) eine zentrale Rolle und daher wurde die SIRT1 Modifikation und Regulation durch HIPK2 betrachtet. Durch Phosphorylierung des Tumorsuppressorproteins p53 an Ser46 aktiviert HIPK2 das Zielprotein und induziert die Transkription proapoptotischer Zielgene von p53. Es wurde beschrieben, dass HIPK2 nach DNA-Schädigung über einen bisher unbekannten Mechnismus die Acetylierung von p53 potenzieren kann.rnIn der vorliegenden Arbeit konnte gezeigt werden, dass SIRT1 von HIPK2 in vitro und in Zellen an Serin 27 und 682 phosphoryliert wird. Weiterhin ist die Interaktion von SIRT1 mit HIPK2 sowie die SIRT1 Phosphorylierung an Serin 682 durch DNA-schädigende Adriamycinbehandlung erhöht. Es gibt Hinweise, dass HIPK2 die Expression von SIRT1 reguliert, da HIPK2 RNA-Interferenz zur Erniedrigung der SIRT1 Protein- und mRNA-Mengen führt.rnEin weiterer interessanter Aspekt liegt in der Beobachtung, dass Ko-Expression von PML-IV, welches SIRT1 sowie HIPK2 in PML-Kernkörper rekrutiert, die SIRT1 Phosphorylierung an Serin 682 verstärkt. Phosphorylierung von SIRT1 an Serin 682 interferiert wiederum mit der SUMO-1 Modifikation, welche für die Lokalisation in PML-Kernkörpen wichtig ist.rnBemerkenswerterweise reduziert die DNA-schadendsinduzierte SIRT1 Phosphorylierung die Bindung des SIRT1 Ko-Aktivators AROS, beeinflusst aber nicht diejenige des Inhibitors DBC1. Dies führt zur Reduktion der enzymatischen Aktivität von SIRT1 und der darausfolgenden weniger effizienten Deacetylierung des Zielproteins p53.rnDurch die von mir in der vorliegenden Promotionsarbeit erzielten Ergebnisse konnte ein neuer molekularer Mechanismus entschlüsselt werden, welcher die durch HIPK2 modulierte Acetylierung von p53 und die daran anschließende Induktion der Apoptose beschreibt.rnHIPK2-vermittelte SIRT1 Phosphorylierung resultiert in einer verminderten Deacetylasefunktion von SIRT1 und führt so zu einer verstärkten acetylierungsinduzierten Expression proapoptotischer p53 Zielgene.

Quantitative 1-Step DNA Methylation Analysis with Native Genomic DNA as Template

DNA methylation analysis currently requires complex multistep procedures based on bisulfite conversion of unmethylated cytosines or on methylation-sensitive endonucleases. To facilitate DNA methylation analysis, we have developed a quantitative 1-step assay for DNA methylation analysis.

Loss of sphingosine kinase-1 in carcinoma cells increases formation of reactive oxygen species and sensitivity to doxorubicin-induced DNA damage

Sphingosine kinases (SK) catalyse the formation of sphingosine 1-phosphate, which is a key lipid mediator regulating cell responses such as proliferation, survival and migration. Here we have investigated the effect of targeted inhibition of SK-1 on cell damage and elucidated the mechanisms involved.

Effect of early antiretroviral therapy during primary HIV-1 infection on cell-associated HIV-1 DNA and plasma HIV-1 RNA

Early initiation of combination antiretroviral therapy (ART) during primary HIV-1 infection may prevent the establishment of large viral reservoirs, possibly resulting in improved control of plasma viraemia rebound after ART cessation.

Relationship of porcine IGF2 imprinting status to DNA methylation at the H19 DMD and the IGF2 DMRs 1 and 2

BACKGROUND: Porcine IGF2 and the H19 genes are imprinted. The IGF2 is paternally expressed, while the H19 gene is maternally expressed. Extensive studies in mice established a boundary model indicating that the H19 differentially methylated domain (DMD) controls, upon binding with the CTCF protein, reciprocal imprinting of the IGF2 and the H19 genes. IGF2 transcription is tissue and development specific involving the use of 4 promoters. In the liver of adult Large White boars IGF2 is expressed from both parental alleles, whereas in skeletal muscle and kidney tissues we observed variable relaxation of IGF2 imprinting. We hypothesized that IGF2 expression from both paternal alleles and relaxation of IGF2 imprinting is reflected in differences in DNA methylation patterns at the H19 DMD and IGF2 differentially methylated regions 1 and 2 (DMR1 and DMR2). RESULTS: Bisulfite sequencing analysis did not show any differences in DNA methylation at the three porcine CTCF binding sites in the H19 DMD between liver, muscle and kidney tissues of adult pigs. A DNA methylation analysis using methyl-sensitive restriction endonuclease SacII and 'hot-stop' PCR gave consistent results with those from the bisulfite sequencing analysis. We found that porcine H19 DMD is distinctly differentially methylated, at least for the region formally confirmed by two SNPs, in liver, skeletal muscle and kidney of foetal, newborn and adult pigs, independent of the combined imprinting status of all IGF2 expressed transcripts. DNA methylation at CpG sites in DMR1 of foetal liver was significantly lower than in the adult liver due to the presence of hypomethylated molecules. An allele specific analysis was performed for IGF2 DMR2 using a SNP in the IGF2 3'-UTR. The maternal IGF2 DMR2 of foetal and newborn liver revealed a higher DNA methylation content compared to the respective paternal allele. CONCLUSIONS: Our results indicate that the IGF2 imprinting status is transcript-specific. Biallelic IGF2 expression in adult porcine liver and relaxation of IGF2 imprinting in porcine muscle were a common feature. These results were consistent with the IGF2 promoter P1 usage in adult liver and IGF2 promoter P2, P3 and P4 usages in muscle. The results showed further that bialellic IGF2 expression in liver and relaxation of imprinting in muscle and kidney were not associated with DNA methylation variation at and around at least one CTCF binding site in H19 DMD. The imprinting status in adult liver, muscle and kidney tissues were also not reflected in the methylation patterns of IGF2 DMRs 1 and 2.

A subset of equine sarcoids harbours BPV-1 DNA in a complex with L1 major capsid protein

Bovine papillomavirus type 1 or 2 (BPV-1, BPV-2) are accepted causal factors in equine sarcoid pathogenesis. Whereas viral genomes are consistently found and expressed within lesions, intact virions have never been detected, thus permissiveness of sarcoids for BPV-1 replication remains unclear. To reassess this issue, an immunocapture PCR (IC/PCR) was established using L1-specific antibodies to capture L1-DNA complexes followed by amplification of the viral genome. Following validation of the assay, 13 sarcoid-bearing horses were evaluated by IC/PCR. Samples were derived from 21 tumours, 4 perilesional/intact skin biopsies, and 1 serum. Tissue extracts from sarcoid-free equines served as controls. IC/PCR scored positive in 14/24 (58.3%) specimens obtained from sarcoid-patients, but negative for controls. Quantitative IC/PCR demonstrated <125 immunoprecipitable viral genomes/50 microl extract for the majority of specimens. Moreover, full-length BPV-1 genomes were detected in a complex with L1 proteins. These complexes may correspond to virion precursors or intact virions.

Role of DNA methylation in the tissue-specific expression of the CYP17A1 gene for steroidogenesis in rodents

The CYP17A1 gene is the qualitative regulator of steroidogenesis. Depending on the presence or absence of CYP17 activities mineralocorticoids, glucocorticoids or adrenal androgens are produced. The expression of the CYP17A1 gene is tissue as well as species-specific. In contrast to humans, adrenals of rodents do not express the CYP17A1 gene and have therefore no P450c17 enzyme for cortisol production, but produce corticosterone. DNA methylation is involved in the tissue-specific silencing of the CYP17A1 gene in human placental JEG-3 cells. We investigated the role of DNA methylation for the tissue-specific expression of the CYP17A1 gene in rodents. Rats treated with the methyltransferase inhibitor 5-aza-deoxycytidine excreted the cortisol metabolite tetrahydrocortisol in their urine suggesting that treatment induced CYP17 expression and 17alpha-hydroxylase activity through demethylation. Accordingly, bisulfite modification experiments identified a methylated CpG island in the CYP17 promoter in DNA extracted from rat adrenals but not from testes. Both methyltransferase and histone deacetylase inhibitors induced the expression of the CYP17A1 gene in mouse adrenocortical Y1 cells which normally do not express CYP17, indicating that the expression of the mouse CYP17A1 gene is epigenetically controlled. The role of DNA methylation for CYP17 expression was further underlined by the finding that a reporter construct driven by the mouse -1041 bp CYP17 promoter was active in Y1 cells, thus excluding the lack of essential transcription factors for CYP17 expression in these adrenal cells.

Local gene transfer and expression following intramuscular administration of FGF-1 plasmid DNA in patients with critical limb ischemia

NV1FGF is an expression plasmid encoding sp.FGF-1(21-154) currently under investigation for therapeutic angiogenesis in clinical trials. NV1FGF plasmid distribution and transgene expression following intramuscular (IM) injection in patients is unknown. The study involved six patients with chronic critical limb ischemia (CLI) planned to undergo amputation. A total dose of 0.5, 2, or 4 mg NV1FGF was administered as eight IM injections (0.006, 0.25, or 0.5 mg per injection) 3-5 days before amputation. Injected sites (30 cm(3)) were divided into equally sized smaller pieces to assess spatial distribution of NV1FGF sequences (PCR), NV1FGF mRNA (reverse transcriptase-PCR), and fibroblast growth factor-1 (FGF-1)-expressing cells (immunohistochemistry). Data indicated gene expression at all doses. The distribution area was within 5-12 cm for NV1FGF sequences containing the expression cassette, up to 5 cm for NV1FGF mRNA, and up to 3 cm for FGF-1-expressing myofibers. All FGF receptors were detected indicating robust potential for bioactivity after NV1FGF gene transfer. Circulating levels of NV1FGF sequences were shown to decrease within days after injection. Data support demonstration of plasmid-mediated gene transfer and expression in muscles from patients with CLI. FGF-1 expression was shown to be limited to injection sites, which supports the concept of multiple-site injection for therapeutic use.

CREB trans-activation of disruptor of telomeric silencing-1 mediates forskolin inhibition of CTGF transcription in mesangial cells.

Connective tissue growth factor (CTGF) participates in diverse fibrotic processes including glomerulosclerosis. The adenylyl cyclase agonist forskolin inhibits CTGF expression in mesangial cells by unclear mechanisms. We recently reported that the histone H3K79 methyltransferase disruptor of telomeric silencing-1 (Dot1) suppresses CTGF gene expression in collecting duct cells (J Clin Invest 117: 773-783, 2007) and HEK 293 cells (J Biol Chem In press). In the present study, we characterized the involvement of Dot1 in mediating the inhibitory effect of forskolin on CTGF transcription in mouse mesangial cells. Overexpression of Dot1 or treatment with forskolin dramatically suppressed basal CTGF mRNA levels and CTGF promoter-luciferase activity, while hypermethylating H3K79 in chromatin associated with the CTGF promoter. siRNA knockdown of Dot1 abrogated the inhibitory effect of forskolin on CTGF mRNA expression. Analysis of the Dot1 promoter sequence identified a CREB response element (CRE) at -384/-380. Overexpression of CREB enhanced forskolin-stimulated Dot1 promoter activity. A constitutively active CREB mutant (CREB-VP16) strongly induced Dot1 promoter-luciferase activity, whereas overexpression of CREBdLZ-VP16, which lacks the CREB DNA-binding domain, abolished this activation. Mutation of the -384/-380 CRE resulted in 70% lower levels of Dot1 promoter activity. ChIP assays confirmed CREB binding to the Dot1 promoter in chromatin. We conclude that forskolin stimulates CREB-mediated trans-activation of the Dot1 gene, which leads to hypermethylation of histone H3K79 at the CTGF promoter, and inhibition of CTGF transcription. These data are the first to describe regulation of the Dot1 gene, and disclose a complex network of genetic and epigenetic controls on CTGF transcription.