Egr-1 Activation by Cancer-Derived Extracellular Vesicles Promotes Endothelial Cell Migration via ERK1/2 and JNK Signaling Pathways

Various mammalian cells, including cancer cells, shed extracellular vesicles (EVs), also known as exosomes and microvesicles, into surrounding tissues. These EVs play roles in tumor growth and metastasis by promoting angiogenesis. However, the detailed mechanism of how cancer-derived EVs elicit endothelial cell activation remains unknown. Here, we provide evidence that early growth response-1 (Egr-1) activation in endothelial cells is involved in the angiogenic activity of colorectal cancer cell-derived EVs. Both RNA interference-mediated downregulation of Egr-1 and ERK1/2 or JNK inhibitor significantly blocked EV-mediated Egr-1 activation and endothelial cell migration. Furthermore, lipid raft-mediated endocytosis inhibitor effectively blocked endothelial Egr-1 activation and migration induced by cancer-derived EVs. Our results suggest that Egr-1 activation in endothelial cells may be a key mechanism involved in the angiogenic activity of cancer-derived EVs. These findings will improve our understanding regarding the proangiogenic activities of EVs in diverse pathological conditions including cancer, cardiovascular diseases, and neurodegenerative diseases.open11617sciescopu

miR-93/miR-106b/miR-375-CIC-CRABP1: a novel regulatory axis in prostate cancer progression

Capicua (CIC) has been implicated in pathogenesis of spinocerebellar ataxia type-1 (SCA1) neurodegenerative disease and some types of cancer; however, the role of CIC in prostate cancer remains unknown. Here we show that CIC suppresses prostate cancer progression. CIC expression was markedly decreased in human prostatic carcinoma. CIC overexpression suppressed prostate cancer cell proliferation, invasion, and migration, whereas CIC RNAi exerted opposite effects. We found that knock-down of CIC derepresses expression of ETV5 and CRABP1 in LNCaP and PC-3 cells, respectively, thereby promoting cell proliferation and invasion. We also discovered that miR-93, miR-106b, and miR-375, which are known to be frequently overexpressed in prostate cancer patients, cooperatively down-regulate CIC levels to promote cancer progression. Altogether, we suggest miR-93/miR-106b/miR-375-CIC-CRABP1 as a novel key regulatory axis in prostate cancer progression.113324Ysciescopu

Easy detection of chromatin binding proteins by the histone association assay

The Histone Association Assay provides an easy approach for detecting proteins that bind chromatin in vivo. This technique is based on a chromatin immunoprecipitation protocol using histone H3-specific antibodies to precipitate bulk chromatin from crosslinked whole cell extracts. Proteins that co-precipitate with chromatin are subsequently detected by conventional SDS-PAGE and Western blot analysis. Unlike techniques that separate chromatin and non-chromatin interacting proteins by centrifugation, this method can be used to delineate whether a protein is chromatin associated regardless of its innate solubility. Moreover, the relative amount of protein bound to DNA can be ascertained under quantitative conditions. Therefore, this technique may be utilized for analyzing the chromatin association of proteins involved in diverse cellular processes

The roles of tumor necrosis factor-alpha in colon tight junction protein expression and intestinal mucosa structure in a mouse model of acute liver failure

<p>Abstract</p> <p>Background</p> <p>Spontaneous bacterial peritonitis (SBP) is a common clinical disease and one of the most severe complications of acute liver failure (ALF). Although the mechanism responsible for SBP is unclear, cytokines play an important role. The aim of this study was to investigate the effects of tumor necrosis factor-alpha (TNF-α) on the structure of the intestinal mucosa and the expression of tight junction (Zona Occludens 1; ZO-1) protein in a mouse model of ALF.</p> <p>Methods</p> <p>We induced ALF using D-galactosamine/lipopolysaccharide (GalN/LPS) or GalN/TNF-α and assessed the results using transmission electron microscopy, immunohistochemistry, Western blotting, ELISA and real-time quantitative PCR. The effects of administration of anti-TNF-α IgG antibody or anti-TNF-α R1 antibody before administration of GalN/LPS or GalN/TNF-α, respectively, on TNF-α were also assessed.</p> <p>Results</p> <p>Morphological abnormalities in the intestinal mucosa of ALF mice were positively correlated with serum TNF-α level. Electron microscopic analysis revealed tight junction (TJ) disruptions, epithelial cell swelling, and atrophy of intestinal villi. Gut bacteria invaded the body at sites where TJ disruptions occurred. Expression of ZO-1 mRNA was significantly decreased in both ALF models, as was the level of ZO-1 protein. Prophylactic treatment with either anti-TNF-α IgG antibody or anti-tumor necrosis factor-a receptor1 (anti-TNF-α R1) antibody prevented changes in intestinal tissue ultrastructure and ZO-1 expression.</p> <p>Conclusion</p> <p>TNF-α affects the structure of the intestinal mucosa, decreases expression of ZO-1, and affects the morphology of the colon in a mouse model of ALF. It also may participate in the pathophysiological mechanism of SBP complicated to ALF.</p

Mapping Dynamic Histone Acetylation Patterns to Gene Expression in Nanog-depleted Murine Embryonic Stem Cells

Embryonic stem cells (ESC) have the potential to self-renew indefinitely and to differentiate into any of the three germ layers. The molecular mechanisms for self-renewal, maintenance of pluripotency and lineage specification are poorly understood, but recent results point to a key role for epigenetic mechanisms. In this study, we focus on quantifying the impact of histone 3 acetylation (H3K9,14ac) on gene expression in murine embryonic stem cells. We analyze genome-wide histone acetylation patterns and gene expression profiles measured over the first five days of cell differentiation triggered by silencing Nanog, a key transcription factor in ESC regulation. We explore the temporal and spatial dynamics of histone acetylation data and its correlation with gene expression using supervised and unsupervised statistical models. On a genome-wide scale, changes in acetylation are significantly correlated to changes in mRNA expression and, surprisingly, this coherence increases over time. We quantify the predictive power of histone acetylation for gene expression changes in a balanced cross-validation procedure. In an in-depth study we focus on genes central to the regulatory network of Mouse ESC, including those identified in a recent genome-wide RNAi screen and in the PluriNet, a computationally derived stem cell signature. We find that compared to the rest of the genome, ESC-specific genes show significantly more acetylation signal and a much stronger decrease in acetylation over time, which is often not reflected in an concordant expression change. These results shed light on the complexity of the relationship between histone acetylation and gene expression and are a step forward to dissect the multilayer regulatory mechanisms that determine stem cell fate.Comment: accepted at PLoS Computational Biolog

A Histone Map of Human Chromosome 20q13.12

We present a systematic search for regulatory elements in a 3.5 Mb region on human chromosome 20q13.12, a region associated with a number of medical conditions such as type II diabetes and obesity.We profiled six histone modifications alongside RNA polymerase II (PolII) and CTCF in two cell lines, HeLa S3 and NTERA-2 clone D1 (NT2/D1), by chromatin immunoprecipitation using an in-house spotted DNA array, constructed with 1.8 kb overlapping plasmid clones. In both cells, more than 90% of transcription start sites (TSSs) of expressed genes showed enrichments with PolII, di-methylated lysine 4 of histone H3 (H3K4me2), tri-methylated lysine 4 of histone H3 (H3K4me3) or acetylated H3 (H3Ac), whereas mono-methylated lysine 4 of histone H3 (H3K4me1) signals did not correlate with expression. No TSSs were enriched with tri-methylated lysine 27 of histone H3 (H3K27me3) in HeLa S3, while eight TSSs (4 expressed) showed enrichments in NT2/D1. We have also located several CTCF binding sites that are potential insulator elements.In summary, we annotated a number of putative regulatory elements in 20q13.12 and went on to verify experimentally a subset of them using dual luciferase reporter assays. Correlating this data to sequence variation can aid identification of disease causing variants

An Integrated Approach to Identifying Cis-Regulatory Modules in the Human Genome

In eukaryotic genomes, it is challenging to accurately determine target sites of transcription factors (TFs) by only using sequence information. Previous efforts were made to tackle this task by considering the fact that TF binding sites tend to be more conserved than other functional sites and the binding sites of several TFs are often clustered. Recently, ChIP-chip and ChIP-sequencing experiments have been accumulated to identify TF binding sites as well as survey the chromatin modification patterns at the regulatory elements such as promoters and enhancers. We propose here a hidden Markov model (HMM) to incorporate sequence motif information, TF-DNA interaction data and chromatin modification patterns to precisely identify cis-regulatory modules (CRMs). We conducted ChIP-chip experiments on four TFs, CREB, E2F1, MAX, and YY1 in 1% of the human genome. We then trained a hidden Markov model (HMM) to identify the labels of the CRMs by incorporating the sequence motifs recognized by these TFs and the ChIP-chip ratio. Chromatin modification data was used to predict the functional sites and to further remove false positives. Cross-validation showed that our integrated HMM had a performance superior to other existing methods on predicting CRMs. Incorporating histone signature information successfully penalized false prediction and improved the whole performance. The dataset we used and the software are available at http://nash.ucsd.edu/CIS/

An integrated ChIP-seq analysis platform with customizable workflows

<p>Abstract</p> <p>Background</p> <p>Chromatin immunoprecipitation followed by next generation sequencing (ChIP-seq), enables unbiased and genome-wide mapping of protein-DNA interactions and epigenetic marks. The first step in ChIP-seq data analysis involves the identification of peaks (i.e., genomic locations with high density of mapped sequence reads). The next step consists of interpreting the biological meaning of the peaks through their association with known genes, pathways, regulatory elements, and integration with other experiments. Although several programs have been published for the analysis of ChIP-seq data, they often focus on the peak detection step and are usually not well suited for thorough, integrative analysis of the detected peaks.</p> <p>Results</p> <p>To address the peak interpretation challenge, we have developed ChIPseeqer, an integrative, comprehensive, fast and user-friendly computational framework for in-depth analysis of ChIP-seq datasets. The novelty of our approach is the capability to combine several computational tools in order to create easily customized workflows that can be adapted to the user's needs and objectives. In this paper, we describe the main components of the ChIPseeqer framework, and also demonstrate the utility and diversity of the analyses offered, by analyzing a published ChIP-seq dataset.</p> <p>Conclusions</p> <p>ChIPseeqer facilitates ChIP-seq data analysis by offering a flexible and powerful set of computational tools that can be used in combination with one another. The framework is freely available as a user-friendly GUI application, but all programs are also executable from the command line, thus providing flexibility and automatability for advanced users.</p

ChromaSig: A Probabilistic Approach to Finding Common Chromatin Signatures in the Human Genome

Computational methods to identify functional genomic elements using genetic information have been very successful in determining gene structure and in identifying a handful of cis-regulatory elements. But the vast majority of regulatory elements have yet to be discovered, and it has become increasingly apparent that their discovery will not come from using genetic information alone. Recently, high-throughput technologies have enabled the creation of information-rich epigenetic maps, most notably for histone modifications. However, tools that search for functional elements using this epigenetic information have been lacking. Here, we describe an unsupervised learning method called ChromaSig to find, in an unbiased fashion, commonly occurring chromatin signatures in both tiling microarray and sequencing data. Applying this algorithm to nine chromatin marks across a 1% sampling of the human genome in HeLa cells, we recover eight clusters of distinct chromatin signatures, five of which correspond to known patterns associated with transcriptional promoters and enhancers. Interestingly, we observe that the distinct chromatin signatures found at enhancers mark distinct functional classes of enhancers in terms of transcription factor and coactivator binding. In addition, we identify three clusters of novel chromatin signatures that contain evolutionarily conserved sequences and potential cis-regulatory elements. Applying ChromaSig to a panel of 21 chromatin marks mapped genomewide by ChIP-Seq reveals 16 classes of genomic elements marked by distinct chromatin signatures. Interestingly, four classes containing enrichment for repressive histone modifications appear to be locally heterochromatic sites and are enriched in quickly evolving regions of the genome. The utility of this approach in uncovering novel, functionally significant genomic elements will aid future efforts of genome annotation via chromatin modifications

Genome-wide promoter analysis of histone modifications in human monocyte-derived antigen presenting cells

<p>Abstract</p> <p>Background</p> <p>Monocyte-derived macrophages and dendritic cells (DCs) are important in inflammatory processes and are often used for immunotherapeutic approaches. Blood monocytes can be differentiated into macrophages and DCs, which is accompanied with transcriptional changes in many genes, including chemokines and cell surface markers.</p> <p>Results</p> <p>To study the chromatin modifications associated with this differentiation, we performed a genome wide analysis of histone H3 trimethylation on lysine 4 (H3K4me3) and 27 (H3K27me3) as well as acetylation of H3 lysines (AcH3) in promoter regions. We report that both H3K4me3 and AcH3 marks significantly correlate with transcriptionally active genes whereas H3K27me3 mark is associated with inactive gene promoters. During differentiation, the H3K4me3 levels decreased on monocyte-specific CD14, CCR2 and CX3CR1 but increased on DC-specific TM7SF4/DC-STAMP, TREM2 and CD209/DC-SIGN genes. Genes associated with phagocytosis and antigen presentation were marked by H3K4me3 modifications. We also report that H3K4me3 levels on clustered chemokine and surface marker genes often correlate with transcriptional activity.</p> <p>Conclusion</p> <p>Our results provide a basis for further functional correlations between gene expression and histone modifications in monocyte-derived macrophages and DCs.</p