Transcriptome profiling of human hepatocytes treated with Aroclor 1254 reveals transcription factor regulatory networks and clusters of regulated genes

BACKGROUND: Aroclor 1254 is a well-known hepatotoxin and consists of a complex mixture of polychlorinated biphenyls (PCBs), some of which have the ability to activate the aryl hydrocarbon receptor (AhR) and other transcription factors (TFs). Altered transcription factor expression enables activation of promoters of many genes, thereby inducing a regulatory gene network. In the past, computational approaches were not applied to understand the combinatorial interplay of TFs acting in concert after treatment of human hepatocyte cultures with Aroclor 1254. We were particularly interested in interrogating promoters for transcription factor binding sites of regulated genes. RESULTS: Here, we present a framework for studying a gene regulatory network and the large-scale regulation of transcription on the level of chromatin structure. For that purpose, we employed cDNA and oligomicroarrays to investigate transcript signatures in human hepatocyte cultures treated with Aroclor 1254 and found 910 genes to be regulated, 52 of which code for TFs and 47 of which are involved in cell cycle and apoptosis. We identified regulatory elements proximal to AhR binding sites, and this included recognition sites for the transcription factors ETS, SP1, CREB, EGR, NF-kB, NKXH, and ZBP. Notably, ECAT and TBP binding sites were identified for Aroclor 1254-induced and E2F, MAZ, HOX, and WHZ for Aroclor 1254-repressed genes. We further examined the chromosomal distribution of regulated genes and observed a statistically significant high number of gene pairs within a distance of 200 kb. Genes regulated by Aroclor 1254, are much closer located to each other than genes distributed randomly all over the genome. 37 regulated gene pairs are even found to be directly neighbored. Within these directly neighbored gene pairs, not all genes were bona fide targets for AhR (primary effect). Upon further analyses many were targets for other transcription factors whose expression was regulated by Aroclor 1254 (secondary effect). CONCLUSION: We observed coordinate events in transcript regulation upon treatment of human hepatocytes with Aroclor 1254 and identified a regulatory gene network of different TFs acting in concert. We determined molecular rules for transcriptional regulation to explain, in part, the pleiotropic effect seen in animals and humans upon exposure to Aroclor 1254

Topoisomerase II inhibition involves characteristic chromosomal expression patterns

<p>Abstract</p> <p>Background</p> <p>The phenomenon of co-localization of transcriptionally upregulated genes showing similar expression levels is known across all eukaryotic genomes. We recently mapped the Aroclor 1254-regulated transcriptome back onto the genome and provided evidence for the statistically significant co-localization of regulated genes. They did, however, not always show similar expression levels, and many of the regulated genes were, in fact, repressed.</p> <p>Results</p> <p>In this study, we were able to reproduce this observation with microarray data stemming from 1) human hepatocytes treated with the gyrase and potential topoisomerase II inhibitor trovafloxacin, 2) human hepatocytes treated with the topoisomerase II inhibitor doxorubicin and 3) mouse lymphoma cells treated with the topoisomerase II inhibitor etoposide. We found statistically significant co-localization of regulated gene pairs – induced and repressed – within the window size of 0–100 kbp. Notably, by using microarray data stemming from lung tissue of a mouse transgenic line overexpressing the transcription factor c-myc, which served as a negative control, we found regulated genes to be located with regard to each other nearly in the same way as genes distributed randomly all over the genome (0–100 kbp).</p> <p>Conclusion</p> <p>We suggest topoisomerase II inhibition by Aroclor 1254, trovafloxacin, doxorubicin, and etoposide to be responsible for significant co-localization of regulated genes through the inability of the stabilized enzyme complexes to religate DNA. Within the permanently opened chromatin domains, neighbored genes might be allowed to be regulated. Overexpression of c-myc, however, does not inhibit topoisomerase II activity. Consequently, the enzyme is able to perform its normal function of transiently breaking and rejoining the DNA double strand. As a result, exclusively target genes are regulated.</p

A 6-months assessment of the alcohol-related clinical burden at emergency rooms (ERs) in 11 acute care hospitals of an urban area in Germany

BACKGROUND: The purpose of the study was to identify and to profile alcohol-related attendances to emergency rooms (ERs) of 11 hospitals of various medical specialties covering a large urban population, to assess risk factors associated with short-stay cases, repeat attendances and higher degree of alcohol consumption and to estimate their impact on the alcohol-related burden at ERs. METHODS: A 6-months study was carried out to obtain clinical and administrative data on single and multiple attendances at ERs in 11 governmental acute hospitals in a large city in Germany. All alcohol-related attendances at ERs of study hospitals were eligible. A broad definition of alcohol-related attendances independently from alcohol diagnosis and various demographic, clinical and administrative measures were used. Odds ratios for the associations of these measures with duration of stay, repeat attendances and higher degrees of alcohol consumption were derived from multivariate binomial and multinomial logistic regression models. RESULTS: 1,748 patients with symptoms of alcohol consumption or withdrawal (inclusion rate 83.8%) yielded 2,372 attendances (3% of all medical admissions), and resulted in 12,629 inpatient-days. These patients accounted for 10.7 cases per 1,000 inhabitants. The average duration of inpatient stay was 10 days. 1,451 of all patients (83%) presented once, whereas the median of repeat attendances was three for the remaining 297 patients. Short-stay cases (<24 hours) were significantly linked with male gender, alcohol misuse, trauma (or suspicion of a trauma) and medical specialties. Increased levels of alcohol consumption at first attendance were significantly associated with repeat attendances in due course. In a multinomial logistic regression model higher degrees of alcohol consumption were significantly associated with male gender, trauma, short-stays, attendance outside regular working time, and with repeat attendances and self-discharge. CONCLUSION: Apart from demographic factors, the alcohol-related clinical burden is largely determined by short-stay cases, repeat attendances and cases with higher levels of alcohol consumption at first attendance varying across medical specialties. These findings could be relevant for the planning of anti-alcoholic interventions at ERs

Transcription profiling of lung adenocarcinomas of c-myc-transgenic mice: Identification of the c-myc regulatory gene network

<p>Abstract</p> <p>Background</p> <p>The transcriptional regulator c-Myc is the most frequently deregulated oncogene in human tumors. Targeted overexpression of this gene in mice results in distinct types of lung adenocarcinomas. By using microarray technology, alterations in the expression of genes were captured based on a female transgenic mouse model in which, indeed, c-Myc overexpression in alveolar epithelium results in the development of bronchiolo-alveolar carcinoma (BAC) and papillary adenocarcinoma (PLAC). In this study, we analyzed exclusively the promoters of induced genes by different in silico methods in order to elucidate the c-Myc transcriptional regulatory network.</p> <p>Results</p> <p>We analyzed the promoters of 361 transcriptionally induced genes with respect to c-Myc binding sites and found 110 putative binding sites in 94 promoters. Furthermore, we analyzed the flanking sequences (+/- 100 bp) around the 110 c-Myc binding sites and found Ap2, Zf5, Zic3, and E2f binding sites to be overrepresented in these regions. Then, we analyzed the promoters of 361 induced genes with respect to binding sites of other transcription factors (TFs) which were upregulated by c-Myc overexpression. We identified at least one binding site of at least one of these TFs in 220 promoters, thus elucidating a potential transcription factor network. The analysis correlated well with the significant overexpression of the TFs Atf2, Foxf1a, Smad4, Sox4, Sp3 and Stat5a. Finally, we analyzed promoters of regulated genes which where apparently not regulated by c-Myc or other c-Myc targeted TFs and identified overrepresented Oct1, Mzf1, Ppargamma, Plzf, Ets, and HmgIY binding sites when compared against control promoter background.</p> <p>Conclusion</p> <p>Our in silico data suggest a model of a transcriptional regulatory network in which different TFs act in concert upon c-Myc overexpression. We determined molecular rules for transcriptional regulation to explain, in part, the carcinogenic effect seen in mice overexpressing the c-Myc oncogene.</p

Transcription profiling of lung adenocarcinomas of c-myc-transgenic mice: Identification of the c-myc regulatory gene network-0

C: LightCycler). Fold changes are shown on the y-axis. Significant changes of gene expression are indicated either with a diamond for array analysis or with an asterisk for real time PCR.<p><b>Copyright information:</b></p><p>Taken from "Transcription profiling of lung adenocarcinomas of c-myc-transgenic mice: Identification of the c-myc regulatory gene network"</p><p>http://www.biomedcentral.com/1752-0509/2/46</p><p>BMC Systems Biology 2008;2():46-46.</p><p>Published online 22 May 2008</p><p>PMCID:PMC2430022.</p><p></p

Beware the intruder: Real time observation of infiltrated neutrophils and neutrophil-Microglia interaction during stroke in vivo.

Inflammation plays an important role in the pathogenesis of ischemic stroke including an acute and prolonged inflammatory process. The role of neutrophil granulocytes as first driver of the immune reaction from the blood site is under debate due to controversial findings. In bone marrow chimeric mice we were able to study the dynamics of tdTomato-expressing neutrophils and GFP-expressing microglia after photothrombosis using intravital two-photon microscopy. We demonstrate the infiltration of neutrophils into the brain parenchyma and confirm a long-lasting contact between neutrophils and microglia as well as an uptake of neutrophils by microglia clearing the brain from peripheral immune cells

The automatically analysis of the different neutrophil granulocytes conditions.

<p><b>A</b> Quantification of the sphericity of neutrophil granulocytes inside the capillaries (15 cells), in the tissue (28 cells) and in contact with microglia (6 cells) (**p<0.01 capillaries vs. tissue and interaction, n = 4 mice). <b><i>B</i></b> Quantification of neutrophil granulocytes velocity in the tissue (22 cells) and in contact with microglia (6 cells) (**p<0.01 tissue vs. interaction, n = 4 mice).</p

Neutrophils infiltrate the ischemic affected brain parenchyma after photothrombosis.

<p><b>A</b> C57BL/6 animals were lethally irradiated and reconstituted with bone marrow from Catchup^IVM mice. 8 weeks after recovery mice were subjected to photothrombosis and the affected brain areas investigated by intravital 2-photon microscopy. Shown is a 3D reconstruction (x-y orientation) of infiltrated tdTomato⁺ neutrophils (red, white arrows) within the infarct zone as well as FITC-dextran⁺ capillaries (green). <b>B</b> 3D reconstruction of <b>A</b> showing the depth (z orientation) of neutrophil infiltration. <b>B</b>_<b>1</b> and <b>B</b>_<b>2</b> displaying 2D transversal sections in x-y orientation in different depth of <b><i>B</i></b>. The arrowheads in <b>B</b>_<b>1</b> and <b>B</b>_<b>2</b> showing infiltrated neutrophils in the parenchyma apart the capillaries. <b>A, B, B</b>_<b>1</b> <b>and B</b>_<b>2</b> The black rhombus and the white asterisk show two cortex penetrating vessels with the capillary bed in between indicating an appropriate depth of imaging. <b>C</b> Staining of a 50 μm thick section displaying infiltrated tdTomato⁺ neutrophils (red, arrowheads) within the parenchyma and a neutrophil in a capillary (asterisk) as well as showing neutrophils, capillaries and a microglia (arrow) in green by using FITC-labeled <i>solanum tuberosum</i> (Potato) lectin. Scale bars: A, B, B₁, B₂: 10 μm and C 5 μm.</p