Chromatin Immunoprecipitation Data Research Articles

The role of Sp1 and EZH2 in the regulation of LMX1A in cervical cancer cells

We have reported previously that LIM homeobox transcription factor 1α (LMX1A) is hypermethylated and functions as a metastasis suppressor in cervical cancer cells. However, the regulation of LMX1A in carcinogenesis has not been reported. We aim to clarify whether specificity protein 1 (Sp1) and enhancer of zeste homolog 2 (EZH2) are involved in the regulation of LMX1A in cervical cancer. First we characterized the LMX1A promoter and used overexpression, knockdown, and reporter assays to show that Sp1 increased LMX1A promoter activity. Next, we used site-directed mutagenesis and electrophoresis mobility shift assays (EMSAs) to demonstrate that Sp1-binding sites were important for Sp1-mediated activation of the LMX1A promoter. Chromatin immunoprecipitation data demonstrated that Sp1 could bind directly to the LMX1A promoter and activate endogenous LMX1A expression in cells pretreated with 5-aza-2′-deoxycytidine (5-aza-dC). Knockdown of EZH2 decreased H3K27me3 histone modification but was insufficient to restore LMX1A expression. To explore the effect of EZH2 on the endogenous LMX1A promoter, we treated EZH2-knockdown cells with 5-aza-dC and trichostatin A (TSA) and then depleted the cells of drugs for 3days. H3K14ac was enriched at the LMX1A promoter in EZH2-knockdown cells and LMX1A mRNA was still expressed. Taken together, these data imply that Sp1 may activate LMX1A expression upon oncogenic stress during cervical cancer development. Moreover, suppression of EZH2 may delay resilencing of LMX1A after the removal of 5-aza-dC and TSA.

CTW: A Software System for ChIP Data Analysis Based on Workflow

In recent research, to analyze the ChIP (chromatin immunoprecipitation) data, many algorithms and computer tools have been developed. Since these programs are developed by academic institutes independently, the lack of common standard makes the talks between them difficult. In this paper, we developed a software system based on workflow for ChIP data analysis, namely CTW (ChIP-Tool-Workflow). CTW is a java-based software platform, which provides a common development interface to integrate data and programs. At the moment, many data types and tools abstracted from CisGenome, MAT and QuEST are integrated. CTW is designed to meet all basic needs of ChIP data analyses, including visualization, data normalization, peak detection, false discovery rate computation, gene-peak association, and sequence and motif analysis. Because the interface developed is very general and flexible, new analysis tools can be integrated effectively as required. CTW implies an ideal environment for integrative Biological research.

15: An intron of IRF-8 harbors myeloid lineage specific regulatory element that is regulated by dynamic chromatin architecture

Interferon Regulatory Factor-8 (IRF-8) is an IFN-gamma responsive gene that serves as a key factor in the hierarchical differentiation of bone marrow derived cells towards monocyte/macrophage lineage. In attempt to identify the molecular mechanisms leading to this lineage restricted expression of IRF-8, we are using IRF-8 Bacterial Artificial Chromosome (BAC) reporter constructs transfected either to permissive macrophage cell line or to restrictive non-hematopoietic fibroblast cell line. We present data showing that the control of lineage specific expression of IRF-8 is confined to an intronic segment. Deletion of this intron leads to loss of lineage specific expression of BAC IRF-8 reporter. Interestingly, this IRF-8 intron exhibits evolutionary conserved regions, which are not typical to “benign” intronic sequences. To gain molecular insight to this restricted expression, we compared histone modifications and nucleosome occupancy in this intron between expression permissive and restrictive cell lines. For that purpose, we employed Chromatin Immuno-Precipitation (ChIP) and Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) analyses. The ChIP data revealed subtle differences in the overall histone modification profile (”histone code”). The FAIRE analysis pointed to a significant nucleosome density difference between permissive and restrictive cell lines. Accordingly, IRF-8 repression in a restrictive myeloid progenitor cell line was partially alleviated upon treatment with histone methyltransferase inhibitors. Altogether, our data clearly suggests that an intron of IRF-8 harbors myeloid lineage specific regulatory element that is regulated by dynamic chromatin architecture. Taken together, it is most probable that this intron serves as nucleation core for chromatin condensation in restrictive cells.

Abstract 238: Class I HDAC inhibition prevents Ncx1 upregulation by stabilizing an HDAC5-HDAC1/Sin3a Repressor Complex during cardiac hypertrophy.

Cardiac hypertrophy is often associated with the activation of signaling pathways that perpetuate altered calcium efflux and influx. One gene that is upregulated and contributes to altered intracellular calcium concentrations and worsening contractility during cardiac hypertrophy is the Sodium Calcium Exchanger ( Ncx1 ). Molecular studies implicate histone deacetylases (HDACs) in possibly regulating the expression of this gene. Our recent work reveals that HDAC1, HDAC5 and Sin3a interact and are recruited to the Ncx1 promoter through the Nkx2.5 transcription factor. Interestingly, we observed greater associated/interaction of the HDAC1-HDAC5/Sin3a repressor complex upon broad HDAC inhibition. Taken together, we hypothesized that HDAC inhibition, stabilizes an HDAC1-HDAC5/Sin3a repressor complex during cardiac hypertrophy. We addressed this hypothesis by treating isolated adult cardiomyocytes with class specific HDAC inhibitors since HDAC1 is a Class I HDAC and HDAC5 is a Class IIa HDAC. Co-Immunoprecipitation (Co-IP) revealed a greater association of repressor complex molecules in the presence of Entinostat, a Class I HDAC inhibitor compared to both non-treated control and TSA, a broad HDAC inhibitor (n=3). These works show enhanced recruitment Sin3a (co-repressor) at the proximal promoter of NCX1 as demonstrated by Chromatin-Immunoprecipitation (ChIP) (n=3). To test whether these observations translated into in vivo models, we subjected mice to transaortic constriction (TAC) to induce hypertrophy. In this model, Co-IP revealed results that similar to our in vitro studies with greater immuno- detection of repressor complex component, Sin3a after immune-precipitation with HDAC1. Furthermore, our ChIP data showed a greater PCR product amplification of proximal Ncx1 promoter, from experimental groups that were subjected to Entinostat (n=3). Our cumulative data suggests that Class I HDAC inhibition stabilizes a repressor complex on the Ncx1 promoter that hinders hypertrophy- mediated Ncx1 upregulation. Class specific HDAC inhibition may be useful in the stabilization and repression of aberrantly expressed genes that contribute to poor clinical outcomes in cardiac hypertrophy.

Abstract A23: Repression of Id4 (inhibitor of DNA binding 4) by the polycomb group protein EZH2 in prostate cancer

Abstract Introduction: Inhibitor of DNA binding/differentiation protein 4 (Id4) is dominant negative regulator of basic helix loop helix (bHLH) family of transcription factors. Id4 shares the homology of HLH domain with other Ids and lack the basic DNA binding region. Unlike Id1, Id2 and Id3, Id4 acts as a tumor suppressor in prostate cancer by attenuating cell proliferation and promote apoptosis. We have previously shown that decreased Id4 expression in prostate cancer is due to its promoter hypermethylation. In this study we investigated the molecular mechanism by which Id4 promoter is hypermethylated. Here, we demonstrate that Id4 promoter methylation is initiated by EZH2 dependent tri-methylation of histone 3 at lysine 27 (H3K27me3). Experimental procedures: Id4 protein expression was analyzed on human prostate adenocarcinoma samples by Immunohistochemistry and prostate cancer cell lines (LNCaP, C81 and DU145) by Western blot. EZH2 was silenced in DU145 cells and confirmed through western blot and Immunocytochemistry. Chromatin Immunoprecipitation (ChIP) was performed with IgG, RNA polymerase, EZH2 and H3K27Me3 antibodies in both prostate cancer cell lines and prostate cancer tissue samples. Results: IHC demonstrated decreased Id4 protein expression in human prostate tissue samples whereas higher nuclear Id4 expression was found in normal prostate tissues. Re-expression of Id4 in EZH2 silenced Du145 demonstrated that Id4 is regulated in an EZH2 dependent manner. ChIP data on prostate cancer samples and cell lines suggested EZH2 occupancy and H3K27Me3 marks in the Id4 promoter region in cell lines and prostate cancer tissue specimens. Conclusions: Id4 appears as a potential tumor suppressor gene that is epigenetically silenced during prostate cancer development. H3K27Me3 marks and EZH2 occupancy suggest a PRC2 dependent mechanism in Id4 promoter silencing in prostate cancer. Acknowledgement: The research was supported by NIH/NCI-RO1CA128914 and in part by NIH/NCRR/RCMI G12RR03062 Citation Format: Swathi Chinaranagari, Pankaj Sharma, Jaideep Chaudhary. Repression of Id4 (inhibitor of DNA binding 4) by the polycomb group protein EZH2 in prostate cancer. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A23.

Abstract B58: Implementation of a synthetic reagent to mimic chromatin provides a chromatin immunoprecipitation quantitative control

Abstract Chromatin immunoprecipitation (ChIP) is an analytical method used to investigate interaction of proteins with specific genomic DNA regions in vivo to provide a better understanding of mechanisms of gene regulation, DNA replication and DNA repair. However, variations in the efficiency of immunoprecipitation, background signals from carryover in immunoprecipitation, variability between chromatin preps, and loss of material during immunoprecipitation and purification of the ChIP DNA are sources of variability that restrict the use of ChIP as a quantitative tool. To address these limitations, we have developed a simple spike-in control that enables labs to generate results that are more consistent and more quantitative. The approach we have developed uses a synthetic peptide-DNA reagent that is spiked into chromatin immunoprecipitation reactions. Because this reagent mimics the behavior of cross-linked chromatin and can be quantitatively recovered by immunoprecipitation, it can be readily incorporated in to virtually any ChIP protocol. To utilize this internal control, a fixed amount of the synthetic peptide-DNA complex is spiked into the chromatin and co-ChIPed simultaneously with the antibody of interest. The recovered synthetic DNA is then used to normalize the results obtained with the antibody of interest. This spike-in control can be designed using a peptide specific to the ChIP antibody of interest and recovered using a single antibody. Alternatively, a universal spike-in control can be designed using a peptide that is recognized by an epitope tag antibody. This universal spike-in control and epitope tagged antibody are used along with the antibody of interest in a standard ChIP reaction. Our data show that the inclusion of our internal control significantly reduces variability between ChIP assays, thus increasing the accuracy of the ChIP data. Because of the universal feature, this method can also be used to compare ChIP data between different antibodies or to normalize data variations that often result between users and multiple experiments. Furthermore, experiments are underway to confirm that such a spike in control can be incorporated upstream of chromatin preparation, and is capable of being adapted for NGS library construction to aid in normalization between chromatin NGS experiments. Citation Format: John M. Rosenfeld, Zirong Li, Konstantin Taganov, Tracy Cooke, Bhaskar Thyagarajan, Alejandra Solache. Implementation of a synthetic reagent to mimic chromatin provides a chromatin immunoprecipitation quantitative control. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr B58.

Abstract A58: Systematic optimization of key parameters involved in the preparation of chromatin and the chromatin immunoprecipitation (ChIP) workflow

Abstract The ChIP protocol is a fundamental tool that has been used to gain insight into the impact of epigenetic phenomenon and their effect on cancer development and progression. Despite the abundance of research tools that support ChIP, very little innovation has occurred around the ChIP method itself to make the process more reproducible and reliable. Issues surrounding antibody specificity and performance, lack of reliable high-throughput options for limited amounts of chromatin, and accepted criteria to make determinations on the quality of chromatin suitable for ChIP are just some of the challenges faced when performing this method. Improvements in the ChIP process would enable more reliable high resolution genomic mapping of chromatin events that are critical to unambiguously dissect epigenetic regulatory mechanisms and their influence on gene expression. To address these issues, we have engaged in an iterative process of examining multiple parameters that influence ChIP data. For chromatin fragmentation, we prepared chromatin on a variety of devices and utilized various buffer systems to develop an approach to optimize this process. To allow rapid and evaluation of histone antibody specificity, we have produced tools that allow researchers to evaluate specificity utilizing a simple dot blot western blotting approach. In addition, we have developed low-cell protocol that simplifies the ChIP process, reduces variability and significantly improve signal-to-noise ratios. This protocol has been demonstrated to work in both a low throughput version as well as a high-throughput version that is amenable to automation. Our approach is well suited to those with limited experience in ChIP, but is also sufficiently robust and flexible to enable ChIP from either abundant or limited amounts of chromatin derived from cells or tissues. Using this systematic approach, we present data comparing the impact of different sonication protocols on ChIP, analysis of user-to-user variation, as well as approaches to optimize and control for factors that introduce variability in ChIP. Citation Format: Tracy Cooke, Zirong Li, Kan Saito, Michael R. Sturges, Konstantin Taganov, Bhaskar Thyagarajan, John M. Rosenfeld, Alejandra Solache. Systematic optimization of key parameters involved in the preparation of chromatin and the chromatin immunoprecipitation (ChIP) workflow. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Jun 19-22, 2013; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2013;73(13 Suppl):Abstract nr A58.

Selaginella tamariscina Attenuates Metastasis via Akt Pathways in Oral Cancer Cells

BackgroundCrude extracts of Selaginella tamariscina , an oriental medicinal herb, have been evidenced to treat several human diseases. This study investigated the mechanisms by which Selaginella tamariscina inhibits the invasiveness of human oral squamous-cell carcinoma (OSCC) HSC-3 cells.Methodology/Principal FindingsHerein, we demonstrate that Selaginella tamariscina attenuated HSC-3 cell migration and invasion in a dose-dependent manner. The anti-metastatic activities of Selaginella tamariscina occurred at least partially because of the down-regulation of matrix metalloproteinases (MMP)-2 and MMP-9 gelatinase activity and the down-regulation of protein expression. The expression and function of both MMP-2 and MMP-9 were regulated by Selaginella tamariscina at a transcriptional level, as shown by quantitative real-time PCR and reporter assays. Chromatin immunoprecipitation (ChIP) data further indicated that binding of the cAMP response element-binding (CREB) protein and activating protein-1 (AP-1) to the MMP-2 promoter diminished at the highest dosage level of Selaginella tamariscina . The DNA-binding activity of specificity protein 1 (SP-1) to the MMP-9 promoter was also suppressed at the same concentration. Selaginella tamariscina did not affect the mitogen-activated protein kinase signaling pathway, but did inhibit the effects of gelatinase by reducing the activation of serine–threonine kinase Akt.ConclusionsThese results demonstrate that Selaginella tamariscina may be a potent adjuvant therapeutic agent in the prevention of oral cancer.

Genome-wide inference of natural selection on human transcription factor binding sites

For decades, it has been hypothesized that gene regulation has had a central role in human evolution, yet much remains unknown about the genome-wide impact of regulatory mutations. Here we use whole-genome sequences and genome-wide chromatin immunoprecipitation and sequencing data to demonstrate that natural selection has profoundly influenced human transcription factor binding sites since the divergence of humans from chimpanzees 4-6 million years ago. Our analysis uses a new probabilistic method, called INSIGHT, for measuring the influence of selection on collections of short, interspersed noncoding elements. We find that, on average, transcription factor binding sites have experienced somewhat weaker selection than protein-coding genes. However, the binding sites of several transcription factors show clear evidence of adaptation. Several measures of selection are strongly correlated with predicted binding affinity. Overall, regulatory elements seem to contribute substantially to both adaptive substitutions and deleterious polymorphisms with key implications for human evolution and disease.

PiDNA: predicting protein–DNA interactions with structural models

Predicting binding sites of a transcription factor in the genome is an important, but challenging, issue in studying gene regulation. In the past decade, a large number of protein–DNA co-crystallized structures available in the Protein Data Bank have facilitated the understanding of interacting mechanisms between transcription factors and their binding sites. Recent studies have shown that both physics-based and knowledge-based potential functions can be applied to protein–DNA complex structures to deliver position weight matrices (PWMs) that are consistent with the experimental data. To further use the available structural models, the proposed Web server, PiDNA, aims at first constructing reliable PWMs by applying an atomic-level knowledge-based scoring function on numerous in silico mutated complex structures, and then using the PWM constructed by the structure models with small energy changes to predict the interaction between proteins and DNA sequences. With PiDNA, the users can easily predict the relative preference of all the DNA sequences with limited mutations from the native sequence co-crystallized in the model in a single run. More predictions on sequences with unlimited mutations can be realized by additional requests or file uploading. Three types of information can be downloaded after prediction: (i) the ranked list of mutated sequences, (ii) the PWM constructed by the favourable mutated structures, and (iii) any mutated protein–DNA complex structure models specified by the user. This study first shows that the constructed PWMs are similar to the annotated PWMs collected from databases or literature. Second, the prediction accuracy of PiDNA in detecting relatively high-specificity sites is evaluated by comparing the ranked lists against in vitro experiments from protein-binding microarrays. Finally, PiDNA is shown to be able to select the experimentally validated binding sites from 10 000 random sites with high accuracy. With PiDNA, the users can design biological experiments based on the predicted sequence specificity and/or request mutated structure models for further protein design. As well, it is expected that PiDNA can be incorporated with chromatin immunoprecipitation data to refine large-scale inference of in vivo protein–DNA interactions. PiDNA is available at: http://dna.bime.ntu.edu.tw/pidna.

Models incorporating chromatin modification data identify functionally important p53 binding sites

Genome-wide prediction of transcription factor binding sites is notoriously difficult. We have developed and applied a logistic regression approach for prediction of binding sites for the p53 transcription factor that incorporates sequence information and chromatin modification data. We tested this by comparison of predicted sites with known binding sites defined by chromatin immunoprecipitation (ChIP), by the location of predictions relative to genes, by the function of nearby genes and by analysis of gene expression data after p53 activation. We compared the predictions made by our novel model with predictions based only on matches to a sequence position weight matrix (PWM). In whole genome assays, the fraction of known sites identified by the two models was similar, suggesting that there was little to be gained from including chromatin modification data. In contrast, there were highly significant and biologically relevant differences between the two models in the location of the predicted binding sites relative to genes, in the function of nearby genes and in the responsiveness of nearby genes to p53 activation. We propose that these contradictory results can be explained by PWM and ChIP data reflecting primarily biophysical properties of protein–DNA interactions, whereas chromatin modification data capture biologically important functional information.

Abstract 2861: A novel negative regulation principle between DAPK and STAT3 in inflammation-associated carcinogenesis.

Abstract It has been shown that the TNF-IL-6-STAT3 pathway plays a crucial role in promoting ulcerative colitis associated carcinoma (UCC). As inflammation compromises gut homeostasis and is associated with cancer progression, it is important to understand the role of key signalling molecules that are involved in the activation of the inflammatory cascade. Interestingly, we found that intestinal epithelial cells (IEC) of UCC in comparison to ulcerative colitis (UC) show high expression level of the anti-inflammatory Death associated protein kinase (DAPK) but only low levels of pSTAT3. Accordingly, epithelial DAPK expression was enhanced in STAT3IEC-KO mice. To unravel a possible regulatory mechanism we used an in vitro model of TNF-treated IEC. We identified a new function of DAPK in suppressing TNF-induced STAT3 activation as DAPK si knockdown and treatment with a DAPK inhibitor potentiated STAT3 activation and IL-6 secretion. DAPK attenuated STAT3 activity directly by physical interaction shown in a 3D structural model. This model suggests that DAPK induced conformational changes in the STAT3 dimer masked its nuclear localization signal. Vice versa, pharmacologic inactivation of STAT3 led to an increase in DAPK mRNA and protein levels. Chromatin immunoprecipitation data showed that STAT3 restricted DAPK expression by promoter binding, thereby reinforcing its own activation by inducing IL-6. This novel negative regulation principle was confirmed in an AOM-DSS-colon carcinogenesis mouse model. It might balance TNF-induced inflammation and seems to play an important role in the inflammation-associated transformation process. DAPK as a negative regulator of STAT3 emerges as therapeutic option in the treatment of UC and UCC. Citation Format: Saritha Chakilam, Muktheswar Gandesiri, Tilman Rau, Clemens Neufert, Abbas Agaimy, Vijayalakshmi Mahadevan, Jelena Ivanovska, Ralph Wirtz, Natalya Benderska, Jan Schulze-Lührmann, Nadine Wittkopf, Ajithavalli Chellappan, Petra Ruemmele, Michael Vieth, Margret Rave-Fränk, Hans Christiansen, Arndt Hartmann, Raja Atreya, Christoph Becker, Pablo Steinberg, Regine Schneider-Stock. A novel negative regulation principle between DAPK and STAT3 in inflammation-associated carcinogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2861. doi:10.1158/1538-7445.AM2013-2861 Note: This abstract was not presented at the AACR Annual Meeting 2013 because the presenter was unable to attend.

CREB-regulated transcription co-activator family stimulates promoter II-driven aromatase expression in preadipocytes.

The dramatically increased prevalence of breast cancer after menopause is of great concern and is correlated with elevated local levels of estrogens. This is mainly due to an increase in aromatase expression driven by its proximal promoter II (PII). We have previously demonstrated that the CREB co-activator CRTC2 binds directly to PII and stimulates its activity via mechanisms involving LKB1-AMPK in response to prostaglandin E(2) (PGE(2)). There are three members of the CRTC family (CRTC1-3) and this study aimed to characterize the role of other CRTCs in the activation of aromatase PII. The expression and subcellular localization of CRTCs were examined in preadipocytes using qPCR and immunofluorescence. Under basal conditions, CRTC1 expression was the lowest, whereas CRTC3 transcripts were present at higher levels. Basally, CRTC2 and CRTC3 were mainly cytoplasmic and PGE(2) caused their nuclear translocation. Reporter assays and chromatin immunoprecipitation (ChIP) were performed to assess the effect of CRTCs on PII activity and binding. Basal PII activity was significantly increased with all CRTCs. Forskolin (FSK)/phorbol 12-myristate 13-acetate (PMA), to mimic PGE(2), resulted in a further significant increase in PII activity with all CRTCs, with CRTC2 and CRTC3 having greater effects. This was consistent with ChIP data showing an increased binding of CRTCs to PII with FSK/PMA. Moreover, gene silencing of CRTC2 and CRTC3 significantly reduced the FSK/PMA-mediated stimulation of aromatase activity. Interestingly, CRTCs acted cooperatively with CREB1 to increase PII activity, and both CREs were found to be essential for the maximal induction of PII activity by CRTCs. Phosphorylation of CRTC2 at its AMPK target site, Ser 171, dictated its subcellular localization, and the activation of aromatase PII in preadipocytes. In conclusion, this study demonstrates that aromatase regulation in primary human breast preadipocytes involves more than one CRTC.

Set7/9 represses SirT1 to induce collagen type II expression in 3D cultured human chodrocytes

Set7/9 represses SirT1 to induce collagen type II expression in 3D cultured human chodrocytes

Liver receptor homologue-1 expression in ovarian epithelial and granulosa cell tumours

Granulosa cell tumours of the ovary (GCT) express aromatase and produce oestrogens. The ovarian-specific aromatase promoter (pII) is regulated by members of the group 5A nuclear receptor family, SF-1 and LRH-1. Since both SF-1 and LRH-1 are implicated in proliferation and cancer, we hypothesised that alteration in the expression of either or both receptors may be associated with GCT. We therefore determined the expression of LRH-1, SF-1 and aromatase in a cohort of GCT, mucinous and serous cystadenocarcinomas, and normal ovaries. LRH-1 mRNA was present at low level in normal ovary and serous cystadenocarcinoma, but was elevated approximately 30-fold in GCT, and 8-fold in mucinous cystadenocarcinoma, compared to normal ovary. LRH-1 protein expression was confirmed in GCT by immunohistochemistry. SF-1 mRNA was significantly lower that of LRH-1 in all samples and not significantly altered in GCT, compared to normal ovary. Aromatase mRNA was present at low level in normal ovary and serous and mucinous cystadenocarcinoma, and significantly elevated (18-fold) in GCT compared to normal ovary. Despite the coordinate over-expression of both LRH-1 and aromatase in GCT versus normal ovary, their levels did not correlate in individual patients; rather, aromatase expression correlated with that of SF-1. Finally, although both LRH-1 and SF-1 activated aromatase promoter activity in transient transfection studies, gel-shift and chromatin immunoprecipitation data indicated that SF-1, but not LRH-1, bound to the aromatase promoter. We conclude that SF-1 regulates aromatase expression in GCT; over-expression of LRH-1 suggests that this receptor may be involved in the pathogenesis of GCT by mechanisms other than the regulation of aromatase. Its role in this disease therefore warrants further investigation.

Comparative study of artificial chromosome centromeres in human and murine cells

Human artificial chromosomes (HAC) are a valuable tool in the analysis of complex chromatin structures such as the human centromere because of their small size and relative simplicity compared with normal human chromosomes. This report includes a comprehensive study of the centromere and chromatin composition of HAC, expressing human genes, generated in human cells and transferred to murine cells. The analysis involved chromatin immuno-precipitation and immuno-FISH on metaphase chromosomes and chromatin fibres. In both the cell types, the HAC consisted of alphoid and non-alphoid DNA and were mainly euchromatic in composition, although a pericentromeric heterochromatic region was present on all the HAC. Fibre-FISH and chromatin immuno-precipitation data indicated that the position of the centromere differed between HAC in human cells and in murine cells. Our work highlights the importance and utilisation of HAC for understanding the epigenetic aspects of chromosome biology.

Inferring functional transcription factor-gene binding pairs by integrating transcription factor binding data with transcription factor knockout data

BackgroundChromatin immunoprecipitation (ChIP) experiments are now the most comprehensive experimental approaches for mapping the binding of transcription factors (TFs) to their target genes. However, ChIP data alone is insufficient for identifying functional binding target genes of TFs for two reasons. First, there is an inherent high false positive/negative rate in ChIP-chip or ChIP-seq experiments. Second, binding signals in the ChIP data do not necessarily imply functionality.MethodsIt is known that ChIP-chip data and TF knockout (TFKO) data reveal complementary information on gene regulation. While ChIP-chip data can provide TF-gene binding pairs, TFKO data can provide TF-gene regulation pairs. Therefore, we propose a novel network approach for identifying functional TF-gene binding pairs by integrating the ChIP-chip data with the TFKO data. In our method, a TF-gene binding pair from the ChIP-chip data is regarded to be functional if it also has high confident curated TFKO TF-gene regulatory relation or deduced hypostatic TF-gene regulatory relation.Results and conclusionsWe first validated our method on a gathered ground truth set. Then we applied our method to the ChIP-chip data to identify functional TF-gene binding pairs. The biological significance of our identified functional TF-gene binding pairs was shown by assessing their functional enrichment, the prevalence of protein-protein interaction, and expression coherence. Our results outperformed the results of three existing methods across all measures. And our identified functional targets of TFs also showed statistical significance over the randomly assigned TF-gene pairs. We also showed that our method is dataset independent and can apply to ChIP-seq data and the E. coli genome. Finally, we provided an example showing the biological applicability of our notion.

Evidence classification of high-throughput protocols and confidence integration in RegulonDB

RegulonDB provides curated information on the transcriptional regulatory network of Escherichia coli and contains both experimental data and computationally predicted objects. To account for the heterogeneity of these data, we introduced in version 6.0, a two-tier rating system for the strength of evidence, classifying evidence as either ‘weak’ or ‘strong’ (Gama-Castro,S., Jimenez-Jacinto,V., Peralta-Gil,M. et al. RegulonDB (Version 6.0): gene regulation model of Escherichia Coli K-12 beyond transcription, active (experimental) annotated promoters and textpresso navigation. Nucleic Acids Res., 2008;36:D120–D124.). We now add to our classification scheme the classification of high-throughput evidence, including chromatin immunoprecipitation (ChIP) and RNA-seq technologies. To integrate these data into RegulonDB, we present two strategies for the evaluation of confidence, statistical validation and independent cross-validation. Statistical validation involves verification of ChIP data for transcription factor-binding sites, using tools for motif discovery and quality assessment of the discovered matrices. Independent cross-validation combines independent evidence with the intention to mutually exclude false positives. Both statistical validation and cross-validation allow to upgrade subsets of data that are supported by weak evidence to a higher confidence level. Likewise, cross-validation of strong confidence data extends our two-tier rating system to a three-tier system by introducing a third confidence score ‘confirmed’.Database URL: http://regulondb.ccg.unam.mx/

Role of the repressor Oaf3p in the recruitment of transcription factors and chromatin dynamics during the oleate response

Cellular responses to environmental stimuli are mediated by the co-ordinated activity of multiple control mechanisms, which result in the dynamics of cell function. Communication between different levels of regulation is central for this adaptability. The present study focuses on the interplay between transcriptional regulators and chromatin modifiers to co-operatively regulate transcription in response to a fatty acid stimulus. The genes involved in the β-oxidation of fatty acids are highly induced in response to fatty acid exposure by four gene-specific transcriptional regulators, Oaf (oleate-activated transcription factor) 1p, Pip2p (peroxisome induction pathway 2), Oaf3p and Adr1p (alcohol dehydrogenase regulator 1). In the present study, we examine the interplay of these factors with Htz1p (histone variant H2A.Z) in regulating POT1 (peroxisomal oxoacyl thiolase 1) encoding peroxisomal thiolase and PIP2 encoding the autoregulatory oleate-specific transcriptional activator. Temporal resolution of ChIP (chromatin immunoprecipitation) data indicates that Htz1p is required for the timely removal of the transcriptional repressor Oaf3p during oleate induction. Adr1p plays an important role in the assembly of Htz1p-containing nucleosomes on the POT1 and PIP2 promoters. We also investigated the function of the uncharacterized transcriptional inhibitor Oaf3p. Deletion of OAF3 led to faster POT1 mRNA accumulation than in the wild-type. Most impressively, a highly protected nucleosome structure on the POT1 promoter during activation was observed in the OAF3 mutant cells in response to oleate induction.

A Monte Carlo-based framework enhances the discovery and interpretation of regulatory sequence motifs

BackgroundDiscovery of functionally significant short, statistically overrepresented subsequence patterns (motifs) in a set of sequences is a challenging problem in bioinformatics. Oftentimes, not all sequences in the set contain a motif. These non-motif-containing sequences complicate the algorithmic discovery of motifs. Filtering the non-motif-containing sequences from the larger set of sequences while simultaneously determining the identity of the motif is, therefore, desirable and a non-trivial problem in motif discovery research.ResultsWe describe MotifCatcher, a framework that extends the sensitivity of existing motif-finding tools by employing random sampling to effectively remove non-motif-containing sequences from the motif search. We developed two implementations of our algorithm; each built around a commonly used motif-finding tool, and applied our algorithm to three diverse chromatin immunoprecipitation (ChIP) data sets. In each case, the motif finder with the MotifCatcher extension demonstrated improved sensitivity over the motif finder alone. Our approach organizes candidate functionally significant discovered motifs into a tree, which allowed us to make additional insights. In all cases, we were able to support our findings with experimental work from the literature.ConclusionsOur framework demonstrates that additional processing at the sequence entry level can significantly improve the performance of existing motif-finding tools. For each biological data set tested, we were able to propose novel biological hypotheses supported by experimental work from the literature. Specifically, in Escherichia coli, we suggested binding site motifs for 6 non-traditional LexA protein binding sites; in Saccharomyces cerevisiae, we hypothesize 2 disparate mechanisms for novel binding sites of the Cse4p protein; and in Halobacterium sp. NRC-1, we discoverd subtle differences in a general transcription factor (GTF) binding site motif across several data sets. We suggest that small differences in our discovered motif could confer specificity for one or more homologous GTF proteins. We offer a free implementation of the MotifCatcher software package at http://www.bme.ucdavis.edu/facciotti/resources_data/software/.