Project description:KRAS mutation is a negative predictive factor for treatment with anti-epidermal growth factor receptor (EGFR) antibodies in metastatic colorectal cancer (mCRC). Novel predictive markers are required to further improve the selection of patients for this treatment. Here, we assessed the influence of modification of KRAS by gene copy number aberration (CNA) and microRNAs (miRNAs) in correlation to clinical outcome in mCRC patients treated with cetuximab in combination with chemotherapy and bevacizumab. Formalin-fixed paraffin-embedded primary tumour tissue was used from 34 mCRC patients in a phase III trial, who were selected based upon their good (n=17) or poor (n=17) progression-free survival (PFS) upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab. Gene copy number at the KRAS locus was assessed using high resolution genome-wide array CGH and the expression levels of 17 miRNAs targeting KRAS were determined by real-time PCR. Good response was associated with 12p12.1 copy number loss, even in patients with a KRAS mutation, while copy number gain in wild-type KRAS patients was correlated with a poor response. In KRAS mutated tumours increased miR-200b and decreased miR-143 expression were associated with a good response. In wild-type KRAS patients, miRNA expression did not predict response in a multivariate model. Thus, assessment of KRAS CNA and miRNAs targeting KRAS might further optimize the selection of patients eligible for anti-EGFR therapy. Copy number detection was performed using NimbleScan and Nexus software Formalin-fixed paraffin-embedded primary tumour tissue was used from 34 metastisized colorectal cancer patients in a phase III trial (CKTO 2005-02; ClinTrials.gov NCT00208546) of the Dutch Colorectal Cancer Group (DCCG), who were selected based upon their good (n=17) or poor (n=17) progression-free survival (PFS) upon treatment with cetuximab in combination with capecitabine, oxaliplatin, and bevacizumab.

Project description:Background: Lignocellulosic biomass is a promising renewable feedstock for the microbial production of fuels. To release the major fermentable sugars such as glucose and xylose, pretreatment and enzymatic hydrolysis of biomass feedstock are needed. During this process, many toxic compounds are produced or introduced which subsequently inhibit microbial growth and eventually the production rate and yield. Acetate is one of the major inhibitors liberated from hemicelluloses during dilute acid pretreatment. An understanding of the toxic effects of acetate on the fermentation microorganism is critical to improving biofuel yields in the process. In addition, the efficient utilization of mixed sugars of glucose and xylose in the presence of hydrolysate inhibitors is crucial for economic biofuel production. Results: We have observed previously that some pretreatment inhibitors affect growth and performance in Zymomonas mobilis 8b differently when different sugars (e.g. glucose or xylose) are used as substrate. To investigate this phenomenon at the cellular level, microarray technology was used to investigate the acetate stress responses of Z. mobilis 8b when using single carbon sources of glucose or xylose, and mixed sugars of glucose and xylose. We designed a microarray based on the most up-to-date genome annotation for both coding sequences and intergenic regions. In the presence of acetate, 8b still can utilize all the glucose (though xylose utilization was inhibited) with similar ethanol yield although the growth, final biomass, and ethanol production rate were reduced. The presence of acetate caused genes related to biosynthesis, flagellar system, and glycolysis to be downregulated, and genes related to stress responses and energy metabolism to be upregulated. Our result indicates that Z. mobilis utilized different mechanism for xylose utilization compared to that of glucose, with even more dramatic results than those caused by treatment of the culture with the inhibitor acetate. Our study also suggests that redox imbalance caused by stressful conditions may trigger a metabolic reaction that leads to the accumulation of toxic intermediates such as xylitol, but Z. mobilis appears to be capable of managing its carbon and energy metabolism through the control of individual reactions to overcome the inhibition caused by stressful conditions. Several target gene candidates based on transcriptomic result have been selected for genetic manipulation and a TonB-dependent receptor knockout mutant was confirmed to have advantage on acetate tolerance. Conclusions: We have gained insights into the molecular responses of the model ethanologenic bacterium Z. mobilis to the inhibitor acetate when grown in different sugar sources. These insights will facilitate future metabolic modeling studies and help further strain metabolic engineering efforts for better xylose utilization and acetate tolerance. Two series of microarray studies using total RNA extracted from Zymomonas mobilis subsp mobilis 8b (an xylose-utilizing recombinant) were carried out to investigate the effect of carbon source and acetate on Z. mobilis. One study compared the acetate effect in either glucose or xylose at exponential phase and another study investigated the acetate effect in mixed sugar of glucose and xylose at three growth phases of exponential, transition, and stationary. Tthree biological replicates were used for each condition.

Project description:Recent genome-scale ChIP-chip studies of transcription factors have shown that a low percentage of experimentally determined binding sites contain the consensus motif for the immunoprecipitated factor. In most cases, differences between in vivo target sites that contain or lack a consensus motif have not been explored. We have previously shown that most sites to which E2F family members are bound in vivo do not contain E2F consensus motifs. The main purpose of this study was to develop an understanding of how E2F binding specificity is achieved in vivo. In particular, we have addressed how E2F family members are recruited to core promoter regions that lack a consensus motif and are excluded from other regions that contain a consensus motif. Using promoter and ENCODE arrays, we have shown that the predominant factors specifying whether E2F is recruited to an in vivo binding site are a) the site must be in a core promoter and b) the promoter region must be utilized as a promoter by the transcriptional machinery in that particular cell type. We have tested three models for recruitment of E2F to core promoters lacking a consensus site, including a) indirect recruitment, b) looping to the core promoter mediated by an E2F bound to a distal consensus motif, and c) assisted binding of E2F to a site that weakly resembles an E2F consensus motif within the core promoter. To test these models, we developed a new in vivo assay, termed eChIP, which allows analysis of transcription factor binding to isolated promoter fragments. Our findings suggest that in vivo a) the presence of a consensus motif is not sufficient to recruit E2Fs, b) E2Fs can bind to isolated regions that lack a consensus motif, and c) binding can require regions other than the best match to the E2F PWM in the core promoter. Keywords: E2F, ChIP-chip, transcription factor binding, consensus motifs 37 ChIP-chip arrays (of these, 14 array sets are biological duplicates). 22 samples are included in this series, the rest can be found in supplementary info to the following papers: Xu 2007, Jin 2006, Komashko 2008

Project description:We performed a genome-scale ChIP-chip comparison of two modifications (trimethylation of lysine 9 and trimethylation of lysine 27) of histone H3 in Ntera2 testicular carcinoma cells and in three different anatomical sources of primary human fibroblasts. We found that in each of the cell types the two modifications were differentially enriched at the promoters of the two largest classes of transcription factors. Specifically, zinc finger genes were bound by H3me3K9 and homeobox genes were bound by H3me3K27. We have previously shown that the PRC2 complex is responsible for mediating trimethylation of lysine 27 of histone H3 in human cancer cells. In contrast, there is little overlap between H3me3K9 targets and components of the PRC2 complex, suggesting that a different histone methyltransferase is responsible for the H3me3K9 modification. Previous studies have shown that SETDB1 can trimethylate H3 on lysine 9, using in vitro or artificial tethering assays. SETDB1 is thought to be recruited to chromatin by complexes containing the KAP1 co-repressor. To determine if a KAP1-containing complex mediates trimethylation of the identified H3me3K9 targets, we performed ChIP-chip assays and identified KAP1 target genes using human 5kb promoter arrays. We found that a large number of promoters of zinc finger transcription factors were bound by both KAP1 and H3me3K9 in normal and cancer cells. To expand our studies of KAP1, we next performed a complete genomic analysis of KAP1 binding using a 38 array tiling set, identifying ~7000 KAP1 binding sites. The identified KAP1 targets were highly enriched for C2H2 zinc fingers, especially those containing KRAB domains. Interestingly, although most KAP1 binding sites were within core promoter regions, the binding sites near ZNF genes were greatly enriched within transcribed regions of the target genes. Because KAP1 is recruited to the DNA via interaction with KRAB-ZNF proteins, we suggest that expression of KRAB-ZNF genes may be controlled via an auto-regulatory mechanism involving KAP1. Keywords: Human whole genome tiling array Amplicons were applied either to ENCODE arrays, 5 kb promoter arrays, or to the human genome tiling array set consisting of 38 arrays (see www.nimblegen.com for details). The labeling and hybridization of DNA samples for ChIP-chip analysis was performed by NimbleGen Systems, Inc, except that ENCODE arrays were hybridized at UC Davis. Briefly, each DNA sample (1 μg) was denatured in the presence of 5'-Cy3- or Cy5-labeled random nonamers (TriLink Biotechnologies) and incubated with 100 units (exo-) Klenow fragment (NEB) and dNTP mix [6 mM each in TE buffer (10 mM Tris/1 mM EDTA, pH 7.4; Invitrogen)] for 2 h at 37°C. Reactions were terminated by addition of 0.5 M EDTA (pH 8.0), precipitated with isopropanol, and resuspended in water. Then, 13ug of the Cy5-labeled ChIP sample and 13ug of the Cy3- labeled total sample were mixed, dried down, and resuspended in 40 μl of NimbleGen Hybridization Buffer (NimbleGen Systems) plus 1.5 ug of human COT1 DNA. After denaturation, hybridization was carried out in a MAUI Hybridization System (BioMicro Systems) for 18 h at 42°C. The arrays were washed using NimbleGen Wash Buffer System (NimbleGen Systems), dried by centrifugation, and scanned at 5-μm resolution using the GenePix 4000B scanner (Axon Instruments). Fluorescence intensity raw data were obtained from scanned images of the oligonucleotide tiling arrays using NIMBLESCAN 2.0 extraction software (NimbleGen Systems). For each spot on the array, log2-ratios of the Cy5-labeled test sample versus the Cy3-labeled reference sample were calculated. Then, the biweight mean of this log2 ratio was subtracted from each point; this procedure is approximately equivalent to mean-normalization of each channel. Total RNA was prepared from 5x106 Ntera cells using RNAeasy Kit (Qiagen) following the manufacturer’s instructions. RNA quality was ensured using the Agilent Systems Bioanalyzer. The RNA was hybridized to human whole genome expression microarrays from Nimblegen, which contain probes for every human gene based on genome build HG18 from the UCSC database (more details at http://www.nimblegen.com). 10 ug total RNA were used to synthesize cDNA using the SuperScript Double-Stranded cDNA synthesis kit (Invitrogen). The labeling of RNA samples, array hybridization and preliminary RNA expression analysis (data normalization) was performed by NimbleGen Systems, Inc.

Project description:Transcriptional profiling of the M-NM-^T835np mutant vs the wild-type strain grown in BHI to OD600=0.5. An intimate linkage between the regulation of biofilm formation, stress tolerance and genetic competence exists in the dental caries pathogen Streptococcus mutans. We previously reported that the rcrRPQ genes, which encode ABC exporters (RcrPQ) and a MarR-family transcriptional regulator (RcrR) that represses the expression of the rcr operon, play a dominant role in regulation of development of genetic competence and connect competence with stress tolerance and (p)ppGpp production in S. mutans. Here we identify the region required for efficient RcrR binding to the rcr promoter region using purified recombinant RcrR (rRcrR) protein in electrophoretic mobility shift and fluorescence polarization assays. DNA fragments carrying mutations in the binding region resulted in decreased binding of rRcrR. Direct binding of RcrR to the comX gene promoter in vitro revealed that RcrR has the potential to directly regulate competence development. Likewise, direct interactions of rRcrR with the promoter region of relP, encoding a (p)ppGpp synthase, show the potential for RcrR-dependent modulation of (p)ppGpp pools. Introduction of mutations into the RcrR binding sites in the rcrR promoter region in vivo demonstrated that optimal RcrR binding is essential for proper expression of competence genes and optimal transformation efficiency. Transcription profiling by RNA-seq and mircroarrays of strains with mutations in rcrR showed that genes in the competence pathway were those most differentially expressed. Thus, here and in a accompanying paper, we establish mechanisms by which the products of the rcrRPQ operon integrate com gene expression, genetic transformation, ppGpp metabolism and stress tolerance in S. mutans Two-condition experiment, M-NM-^T835np mutant vs the wild-type strain. Biological replicates: 4 per strain, independently grown and harvested. One replicate per array

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Chip-chip for H4K16ac in HEK293 cells

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