Regulation of estrogen responsive genes by human Estrogen Receptor alpha

In women, breast cancer is the most common cancer and accounts for most cancer deaths. The molecular mechanisms underlying this pathology are diverse and contribute to the complexity of the disease. Early diagnosis and detailed molecular characterization of tumours significantly increase the prognosis for the patients. A limited number of breast cancer markers are already used for diagnosis, characterization, and determination of the most promising therapy of breast cancer tumours. Although new therapeutic approaches such as herceptin antibodies are now available on the market, new markers that are specific for a subset of breast cancer patients are urgently needed. A major criterion in breast cancer diagnosis is the presence of the estrogen receptor alpha (ERα) which is associated with better prognosis and often sensitivity to anti-estrogen therapy. ERα is a ligand-inducible transcription factor that modulates expression of estrogen responsive target genes involved in both, physiological and pathological conditions such as breast cancer. Despite the complexity of the regulatory mechanisms, a variety of mechanisms resulting in transcriptional activation of target genes have been characterised. Although about 50 % of estrogen-responsive genes are repressed in response to estrogen treatment, the mechanisms underlying this regulation are just beginning to be discovered. This thesis aimed to study expression and regulation of the breast cancer and salivary gland expression gene (BASE). The evaluation of this gene was interesting for two reasons: firstly, its expression is strongly repressed by estrogen suggesting involvement of ERα, and secondly, previous studies indicate that the expression of this putative secreted protein is restricted to breast cancer cells and salivary gland. Therefore, BASE has the potential to function as a new breast cancer marker. One major finding of this study is the strong separation of expression and regulation of BASE. Expression of the gene is depending on the transcription factor FoxA1, which binds in a regulatory region about 2 kb upstream of the transcription start site. Although essential for expression, FoxA1 has no function in BASE regulation. Furthermore, this study shows that the BASE gene is rapidly repressed after estrogen-treatment and that ERα is required for this regulation. ERα can bind the BASE promoter in the same regulatory region as FoxA1, however, direct binding seems not to be a critical prerequisite. Based on the data obtained in this study, two molecular models for the mechanism of repression are proposed. Furthermore, analysis of normal and primary breast tumour samples in collaboration with M. Kerins group in Galway confirmed BASE expression in about 50 % of the samples. Therefore, BASE remains an interesting candidate as breast cancer marker. In a side project investigating the mechanism of estrogen mediated activation of target genes, the CTSD gene has been further characterized. Besides the well characterized proximal promoter, the functionality of two further ERα binding sites, located 9 kb and 33 kb upstream of the transcription start site, have been reported. This study confirmed binding of ERα and PolII to the 9 kb upstream enhancer and moreover, the ability of this site to convey estrogen-stimulation was confirmed. Whether the enhancer requires physical interaction with the proximal promoter to enable transcriptional activation remains to be further examined

SFTA2 - a novel secretory peptide highly expressed in the lung - is modulated by lipopolysaccharide but not hyperoxia

Tissue-specific transcripts are likely to be of importance for the corresponding organ. While attempting to define the specific transcriptome of the human lung, we identified the transcript of a yet uncharacterized protein, SFTA2. In silico analyses, biochemical methods, fluorescence imaging and animal challenge experiments were employed to characterize SFTA2. Human SFTA2 is located on Chr. 6p21.33, a disease-susceptibility locus for diffuse panbronchiolitis. RT-PCR verified the abundance of SFTA2-specific transcripts in human and mouse lung. SFTA2 is synthesized as a hydrophilic precursor releasing a 59 amino acid mature peptide after cleavage of an N-terminal secretory signal. SFTA2 has no recognizable homology to other proteins while orthologues are present in all mammals. SFTA2 is a glycosylated protein and specifically expressed in nonciliated bronchiolar epithelium and type II pneumocytes. In accordance with other hydrophilic surfactant proteins, SFTA2 did not colocalize with lamellar bodies but colocalized with golgin97 and clathrin-labelled vesicles, suggesting a classical secretory pathway for its expression and secretion. In the mouse lung, Sfta2 was significantly downregulated after induction of an inflammatory reaction by intratracheal lipopolysaccharides paralleling surfactant proteins B and C but not D. Hyperoxia, however, did not alter SFTA2 mRNA levels. We have characterized SFTA2 and present it as a novel unique secretory peptide highly expressed in the lung

Genetic Characterization of Listeria from Food of Non-Animal Origin Products and from Producing and Processing Companies in Bavaria, Germany

Reported cases of listeriosis from food of non-animal origin (FNAO) are increasing. In order to assess the risk of exposure to Listeria monocytogenes from FNAO, the genetic characterization of the pathogen in FNAO products and in primary production and processing plants needs to be investigated. For this, 123 samples of fresh and frozen soft fruit and 407 samples of 39 plants in Bavaria, Germany that produce and process FNAO were investigated for Listeria contamination. As a result, 64 Listeria spp. isolates were detected using ISO 11290-1:2017. Environmental swabs and water and food samples were investigated. L. seeligeri (36/64, 56.25%) was the most frequently identified species, followed by L. monocytogenes (8/64, 12.50%), L. innocua (8/64, 12.50%), L. ivanovii (6/64, 9.38%), L. newyorkensis (5/64, 7.81%), and L. grayi (1/64, 1.56%). Those isolates were subsequently sequenced by whole-genome sequencing and subjected to pangenome analysis to retrieve data on the genotype, serotype, antimicrobial resistance (AMR), and virulence markers. Eight out of sixty-four Listeria spp. isolates were identified as L. monocytogenes. The serogroup analysis detected that 62.5% of the L. monocytogenes isolates belonged to serogroup IIa (1/2a and 3a) and 37.5% to serogroup IVb (4b, 4d, and 4e). Furthermore, the MLST (multilocus sequence typing) analysis of the eight detected L. monocytogenes isolates identified seven different sequence types (STs) and clonal complexes (CCs), i.e., ST1/CC1, ST2/CC2, ST6/CC6, ST7/CC7, ST21/CC21, ST504/CC475, and ST1413/CC739. The core genome MLST analysis also showed high allelic differences and suggests plant-specific isolates. Regarding the AMR, we detected phenotypic resistance against benzylpenicillin, fosfomycin, and moxifloxacin in all eight L. monocytogenes isolates. Moreover, virulence factors, such as prfA, hly, plcA, plcB, hpt, actA, inlA, inlB, and mpl, were identified in pathogenic and nonpathogenic Listeria species. The significance of L. monocytogenes in FNAO is growing and should receive increasing levels of attention

Genome-wide identification of highaffinity estrogen response elements in human and mouse. Mol Endocrinol

caspase 7, apoptosis-related cysteine protease; CDS, coding sequence; ChIP; chromatin immunoprecipitation; COX7A2L, cytochrome c oxidase subunit VIIa polypeptide 2 like; CTSD, cathepsin D; EBAG9, estrogen receptor binding site associated, antigen 9; EFP, estrogen-responsive finger protein; ER, estrogen receptor; ERE, estrogen response element; FGF9, fibroblast growth factor 9; GAD2, glutamate decarboxylase 2; GAPD, glyceraldehyde-3-phosphate dehydrogenase; GREB1, gene regulated by estrogen in breast cancer protein; IGFBP4, insulin-like growth factor binding protein 4; KHDRBS3 (T-STAR/ETOILE) KH domain containing, RNA binding, signal transduction associated 3; LY6E, lymphocyte antigen 6 complex, locus E; NRIP1 (RIP140), nuclear receptor interacting protein 1; OVGP1, oviductal glycoprotein 1; P-PolII, phosphorylated RNA polymerase II; RBM, RNA-binding motif; RNF14, ring finger protein 14; SCNN1A, sodium channel, nonvoltage-gated 1 alpha; TBP, TATA bo

Whole-Genome Sequence Comparisons of Listeria monocytogenes Isolated from Meat and Fish Reveal High Inter- and Intra-Sample Diversity

Interpretation of whole-genome sequencing (WGS) data for foodborne outbreak investigations is complex, as the genetic diversity within processing plants and transmission events need to be considered. In this study, we analyzed 92 food-associated Listeria monocytogenes isolates by WGS-based methods. We aimed to examine the genetic diversity within meat and fish production chains and to assess the applicability of suggested thresholds for clustering of potentially related isolates. Therefore, meat-associated isolates originating from the same samples or processing plants as well as fish-associated isolates were analyzed as distinct sets. In silico serogrouping, multilocus sequence typing (MLST), core genome MLST (cgMLST), and pangenome analysis were combined with screenings for prophages and genetic traits. Isolates of the same subtypes (cgMLST types (CTs) or MLST sequence types (STs)) were additionally compared by SNP calling. This revealed the occurrence of more than one CT within all three investigated plants and within two samples. Analysis of the fish set resulted in predominant assignment of isolates from pangasius catfish and salmon to ST2 and ST121, respectively, potentially indicating persistence within the respective production chains. The approach not only allowed the detection of distinct subtypes but also the determination of differences between closely related isolates, which need to be considered when interpreting WGS data for surveillance

Identification of knowledge gaps in whole-genome sequence analysis of multi-resistant thermotolerant Campylobacter spp.

Abstract Background Campylobacter spp. is the most frequent cause of bacterial food-borne gastroenteritis and a high priority antibiotic resistant bacterium according to the World Health Organization (WHO). European monitoring of thermotolerant Campylobacter spp. does not reflect the global burden of resistances already circulating within the bacterial population worldwide. Methods We systematically compared whole genome sequencing with comprehensive phenotypic antimicrobial susceptibility, analyzing 494 thermotolerant Campylobacter poultry isolates from Vietnam and Germany. Any discrepancy was checked by repeating the wet lab and improving the dry lab part. Selected isolates were additionally analyzed via long-read Oxford Nanopore technology, leading to closed chromosomes and plasmids. Results Overall, 22 different resistance genes and gene variants (e. g. erm(B), aph(3’)-IIIa, aph(2’’)-If, catA, lnu(C), bla OXA, sat4) and point mutations in three distinct genes (gyrA, 23S rRNA, rpsL) associated with AMR were present in the Campylobacter isolates. Two AMR genes were missing in the database and one falsely associated with resistance. Bioinformatic analysis based on short-read data partly failed to identify tet(O) and aadE, when the genes were present as duplicate or homologous gene variants. Intriguingly, isolates also contained different determinants, redundantly conferring resistance to chloramphenicol, gentamicin, kanamycin, lincomycin and streptomycin. We found a novel tet(W) in tetracycline sensitive strains, harboring point mutations. Furthermore, analysis based on assemblies from short-read data was impaired to identify full length phase variable aad9, due to variations of the poly-C tract within the gene. The genetic determinant responsible for gentamicin resistance of one isolate from Germany could not be identified. GyrT86I, presenting the main determinant for (fluoro-)quinolone resistance led to a rare atypical phenotype of ciprofloxacin resistance but nalidixic acid sensitivity. Long-read sequencing predicted AMR genes were mainly located on the chromosome, and rarely on plasmids. Predictions from long- and short-read sequencing, respectively, often differed. AMR genes were often organized in multidrug resistance islands (MDRI) and partially located in proximity to transposase genes, suggesting main mobilization of resistance determinants is via natural transformation and transposition in Campylobacter. Conclusions The results of this study suggest that there is frequent resistance gene duplication, mosaicism, and mutation leading to gene variation and truncation in Campylobacter strains that have not been reported in previous studies and are missing from databases. Furthermore, there is a need for deciphering yet unknown resistance mechanisms and resistance spread in thermotolerant Campylobacter spp. that may pose a challenge to global food safety

RNA-Sequencing as Useful Screening Tool in the Combat against the Misuse of Anabolic Agents

The abuse of anabolic substances in animal husbandry is forbidden within the EU and well controlled by detecting substance residues in different matrices. The application of newly designed drugs or substance cocktails represents big problems. Therefore developing sensitive test methods is important. The analysis of physiological changes caused by the use of anabolic agents on the molecular level, for example, by quantifying gene expression response, is a new approach to develop such screening methods. A novel technology for holistic gene expression analysis is RNA sequencing. In this study, the potential of this high-throughput method for the identification of biomarkers was evaluated. The effect of trenbolone acetate plus estradiol on gene expression in liver from Nguni heifers was analyzed with RNA sequencing. The expression of 40 selected candidate genes was verified via RT-qPCR, whereby 20 of these genes were significantly regulated. To extract the intended information from these regulated genes, biostatistical tools for pattern recognition were applied and resulted in a clear separation of the treatment groups. Those candidate genes could be verified in boars and in calves treated with anabolic substances. These results show the potential of RNA sequencing to screen for biomarker candidates to detect the abuse of anabolics. The verification of these biomarkers in boars and calves leads to the assumption that gene expression biomarkers are independent of breed or even species and that biomarkers, identified in farm animals could also act as potential biomarker candidates to detect the abuse of anabolic substances in human sports

RNA-Sequencing as Useful Screening Tool in the Combat against the Misuse of Anabolic Agents

The abuse of anabolic substances in animal husbandry is forbidden within the EU and well controlled by detecting substance residues in different matrices. The application of newly designed drugs or substance cocktails represents big problems. Therefore developing sensitive test methods is important. The analysis of physiological changes caused by the use of anabolic agents on the molecular level, for example, by quantifying gene expression response, is a new approach to develop such screening methods. A novel technology for holistic gene expression analysis is RNA sequencing. In this study, the potential of this high-throughput method for the identification of biomarkers was evaluated. The effect of trenbolone acetate plus estradiol on gene expression in liver from Nguni heifers was analyzed with RNA sequencing. The expression of 40 selected candidate genes was verified via RT-qPCR, whereby 20 of these genes were significantly regulated. To extract the intended information from these regulated genes, biostatistical tools for pattern recognition were applied and resulted in a clear separation of the treatment groups. Those candidate genes could be verified in boars and in calves treated with anabolic substances. These results show the potential of RNA sequencing to screen for biomarker candidates to detect the abuse of anabolics. The verification of these biomarkers in boars and calves leads to the assumption that gene expression biomarkers are independent of breed or even species and that biomarkers, identified in farm animals could also act as potential biomarker candidates to detect the abuse of anabolic substances in human sports