PRMT1 promotes the tumor suppressor function of p14ARF and is indicative for pancreatic cancer prognosis.

The p14ARF protein is a well-known regulator of p53-dependent and p53-independent tumor-suppressive activities. In unstressed cells, p14ARF is predominantly sequestered in the nucleoli, bound to its nucleolar interaction partner NPM. Upon genotoxic stress, p14ARF undergoes an immediate redistribution to the nucleo- and cytoplasm, where it promotes activation of cell cycle arrest and apoptosis. Here, we identify p14ARF as a novel interaction partner and substrate of PRMT1 (protein arginine methyltransferase 1). PRMT1 methylates several arginine residues in the C-terminal nuclear/nucleolar localization sequence (NLS/NoLS) of p14ARF . In the absence of cellular stress, these arginines are crucial for nucleolar localization of p14ARF . Genotoxic stress causes augmented interaction between PRMT1 and p14ARF , accompanied by arginine methylation of p14ARF . PRMT1-dependent NLS/NoLS methylation promotes the release of p14ARF from NPM and nucleolar sequestration, subsequently leading to p53-independent apoptosis. This PRMT1-p14ARF cooperation is cancer-relevant and indicative for PDAC (pancreatic ductal adenocarcinoma) prognosis and chemotherapy response of pancreatic tumor cells. Our data reveal that PRMT1-mediated arginine methylation is an important trigger for p14ARF 's stress-induced tumor-suppressive function.

Antigen receptor-mediated depletion of FOXP3 in induced regulatory T-lymphocytes via PTPN2 and FOXO1.

Regulatory T-cells induced via IL-2 and TGFß in vitro (iTreg) suppress immune cells and are potential therapeutics during autoimmunity. However, several reports described their re-differentiation into pathogenic cells in vivo and loss of their key functional transcription factor (TF) FOXP3 after T-cell antigen receptor (TCR)-signalling in vitro. Here, we show that TCR-activation antagonizes two necessary TFs for foxp3 gene transcription, which are themselves regulated by phosphorylation. Although the tyrosine phosphatase PTPN2 is induced to restrain IL-2-mediated phosphorylation of the TF STAT5, expression of the TF FOXO1 is downregulated and miR-182, a suppressor of FOXO1 expression, is upregulated. TGFß counteracts the FOXP3-depleting TCR-signal by reassuring FOXO1 expression and by re-licensing STAT5 phosphorylation. Overexpressed phosphorylation-independent active versions of FOXO1 and STAT5 or knockdown of PTPN2 restores FOXP3 expression despite TCR-signal and absence of TGFß. This study suggests novel targets for stabilisation and less dangerous application of iTreg during devastating inflammation.

Complementation of non-tumorigenicity of HPV18-positive cervical carcinoma cells involves differential mRNA expression of cellular genes including potential tumor suppressor genes on chromosome 11q13.

The fusion between human tumorigenic cells and normal human diploid fibroblasts results in non-tumorigenic hybrid cells, suggesting a dominant role for tumor suppressor genes in the generated hybrid cells. After long-term cultivation in vitro, tumorigenic segregants may arise. The loss of tumor suppressor genes on chromosome 11q13 has been postulated to be involved in the induction of the tumorigenic phenotype of human papillomavirus (HPV)18-positive cervical carcinoma cells and their derived tumorigenic hybrid cells after subcutaneous injection in immunocompromised mice. The aim of this study was the identification of novel cellular genes that may contribute to the suppression of the tumorigenic phenotype of non-tumorigenic hybrid cells in vivo. We used cDNA microarray technology to identify differentially expressed cellular genes in tumorigenic HPV18-positive hybrid and parental HeLa cells compared to non-tumorigenic HPV18-positive hybrid cells. We detected several as yet unknown cellular genes that play a role in cell differentiation, cell cycle progression, cell-cell communication, metastasis formation, angiogenesis, antigen presentation, and immune response. Apart from the known differentially expressed genes on 11q13 (e.g., phosphofurin acidic cluster sorting protein 1 (PACS1) and FOS ligand 1 (FOSL1 or Fra-1)), we detected novel differentially expressed cellular genes located within the tumor suppressor gene region (e.g., EGF-containing fibulin-like extracellular matrix protein 2 (EFEMP2) and leucine rich repeat containing 32 (LRRC32) (also known as glycoprotein-A repetitions predominant (GARP)) that may have potential tumor suppressor functions in this model system of non-tumorigenic and tumorigenic HeLa x fibroblast hybrid cells.

Screening assays for epigenetic targets using native histones as substrates.

In the past years, a lot of attention has been given to the identification and characterization of selective and potent inhibitors of chromatin-modifying enzymes to better understand their specific role in transcriptional regulation. As aberrant histone methylation is involved in different pathological processes, the search for methyltransferase and demethylase inhibitors has emerged as a crucial issue in current medicinal chemistry research. High-throughput in vitro assays are important tools for the identification of new methyltransferase or demethylase inhibitors. These usually use oligopeptide substrates derived from histone sequences, although in many cases, they are not good substrates for these enzymes. Here, the authors report about the setup and establishment of in vitro assays that use native core histones as substrates, enabling an assay environment that better resembles native conditions. They have applied these substrates for the known formaldehyde dehydrogenase assay for the histone demethylase LSD1 and have established two new antibody-based assays. For LSD1, a heterogeneous assay format was set up, and a homogeneous assay was used for the characterization of the arginine methyltransferase PRMT1. Validation of the system was achieved with reference inhibitors in each case.

DXS6797 contains two STRs which can be easily haplotyped in both sexes.

DXS6797 is a complex X-chromosomal locus which contains two variable short tandem repeats (STRs) (motif ATCT) separated by 128 non-polymorphic nucleotides. The two STRs can be cleaved apart by Taq I digestion. Conventionally, DXS6797 is typed by measuring the overall amplicon length, providing only eight alleles [polymorphism information content (PIC) 0.733, mean exclusion chance (MEC) 0.712]. Separate amplification would increase the discrimination but obscure the haplotype constellation in females. Therefore, we proceed by amplifying the whole sequence containing both repeats (DXS6797 I and DXS6797 II) using a Fam-labelled forward primer and a Tamra-labelled reverse primer. We then measure the length of the entire double-labelled amplicon and a Taq-I-digested aliquot to infer, for both males and females, compound haplotypes consisting of DXS6797 I and DXS6797 II repeat length. This procedure has the potential to provide 42 DXS6797 haplotypes. If the crossover rate between both STRs is assumed to be <1.5x10(-6), DXS6797 haplotypes could be used for kinship testing like STR alleles. In our German sample (780 X chromosomes), we determined 27 haplotypes (PIC 0.842, MEC 0,834) and in 220 meioses, we found no new mutations.

The problem of single parent/child paternity analysis--practical results involving 336 children and 348 unrelated men.

In a certain amount of paternity investigations, only DNA from child and alleged father is analyzed, thus increasing the possibility of false paternity inclusions. The aim of this study was to determine how many wrong paternity inclusions could be detected in a rather small geographical area comparing empirical results from 336 children and 348 men (13-15 STRs were investigated per person). This comparison between each child and all unrelated men (i.e. all putative fathers from the other cases) with an especially designed computer program resulted in 116,004 man/child pairs. Less than three excluding STRs were found in 1666 child/unrelated man pairs (1.44% of the comparisons). At least one unrelated man with only two or less STR mismatches could be determined for 322 children (95.8% of all investigated children). In 26 comparisons no STR mismatches between a child and an unrelated man were detected, thus at least one and up to three "second father(s)" under 350 men could be found for 23 children, if the mother is excluded. Paternity probabilities between 95.475% and 99.996% were calculated. Our results underline the difficulties in motherless paternity cases using only STR analysis and advise great precaution in assigning verbal predicates such as "paternity proven" in those investigations.

Development of two pentaplex systems with X-chromosomal STR loci and their allele frequencies in a northeast German population.

In this study we present two new pentaplex systems for the coamplification of X-chromosomal short tandem repeats (STRs). X-penta-1 comprises DXS9898, DXS6807, HPRTB, DXS101, and androgen receptor (ARA); X-penta-2 consists of DXS7133, DXS10011, DXS7424, DXS8377, and DXS8378. In addition, allele frequencies for these loci in a northeast German population comprising 100 females and 105 males were shown. The applicability and usefulness of our two PCR pentaplex approaches in paternity deficiency cases is demonstrated by a combined power of discrimination (PD(c)) for both females and males with PD(c)>0.999999.

Comparative analysis of promoter regions containing binding sites of the heterodimeric transcription factor Ino2/Ino4 involved in yeast phospholipid biosynthesis.

The inositol/choline responsive element (ICRE) functions as a UAS element mediating coordinate expression of structural genes required for yeast phospholipid biosynthesis. However, ICRE motifs could be detected upstream of various genes apparently not involved in lipid metabolism. In this work we investigated the expression pattern of selected genes containing ICRE promoter motifs, as identified by in silico analysis (ARG4, ERG20, FAR8, GPD2, RSF1, URA8, VHT1 and YEL073C). It turned out that the presence of an ICRE upstream of a gene of unknown function indeed allows to conclude for regulation by phospholipid precursors, which is mediated by activators Ino2/Ino4 and the repressor Opi1. We also demonstrated in vitro binding of Ino2/Ino4 heterodimers to promoter regions. Thus, our analysis supports the view that identification of regulatory elements by a database search provides evidence for a specific pattern of gene expression. Activation by pathway-specific regulators may suggest a physiological function for as yet uncharacterized genes.

Constitutive expression of yeast phospholipid biosynthetic genes by variants of Ino2 activator defective for interaction with Opi1 repressor.

Regulated expression of structural genes involved in yeast phospholipid biosynthesis is mediated by inositol/choline-responsive element (ICRE) upstream motifs, bound by the heterodimeric activator complex Ino2 + Ino4. Gene repression occurs in the presence of sufficient inositol and choline, requiring an intact Opi1 repressor which binds to Ino2. For a better understanding of interactions among regulators, we mapped an 18 aa repressor interaction domain (RID, aa 118-135) within Ino2 necessary and sufficient for binding by Opi1. By alanine scanning mutagenesis of the entire RID we were able to identify nine residues critical for Opi1-dependent repression of Ino2 function. Consequently, the corresponding dominant Ino2 variants conferred constitutive expression of an ICRE-dependent reporter gene and were no longer inhibited even by overproduction of Opi1. Interestingly, Ino2 RID partially overlaps with transcriptional activation domain TAD2. As certain mutations exclusively affect repression while others affect both repression and activation, both functions of Ino2 can be functionally uncoupled. Correspondingly, we mapped the RID-binding activator interaction domain (AID, aa 321-380) at the C-terminus of Opi1 and introduced missense mutations at selected positions. An Opi1 variant simultaneously mutated at three highly conserved positions showed complete loss of repressor function, confirming RID-AID interaction as the crucial step of regulated expression of ICRE-dependent genes.

[Paternity analysis in deficiency cases with related putative fathers: simulation of a deficiency analysis in 27 families]. / Abstammungsbegutachtungen in Defizienzfällen mit verwandten Putativvätern: eine simulierte Analyse in 27 Familien.

During the last few years, the number of privately ordered paternity investigations has increased considerably. Probably due to financial reasons in more and more cases only the putative father and the child are investigated. Additionally, very often only one method, such as STR analysis, is employed. This raises the question whether such a reduced analysis leads to reliable and clear results when investigating cases with related putative fathers. We investigated 165 individuals from 27 families using the AmpFlSTRIdentifiler multiplex PCR and calculated the paternity probabilities of the children to their biological fathers, uncles, grand fathers and other relatives. In more than 30% less than three exclusions between child and relative were detected. In five cases no exclusions were found between child and uncle, always leading to paternity probabilities >99.9%. These results show that the calculation of high probabilities (>99.9%) does not necessarily lead to the accurate conclusion of fatherhood. In many of our cases misleadingly the brother of the real father or another close relative would have been declared to be the biological father.