Project description:The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-M-NM-:B subunit p65 in relation to active enhancers and promoters of transcribed genes by ChIP-seq experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin (IL)-1-induced H3K27ac and p65 binding. Inhibition of TAK1, IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-M-NM-:B p50 and of AP-1 transcription factors to both, promoters and enhancers. This study provides a high resolution view of epigenetic changes occurring during the IL-1 response and allows the first genome-wide identification of a novel class of inducible p65 NF-M-NM-:B-dependent enhancers in epithelial cells. Two sets of experiments were performed as biological replicate series (rep1 and rep2) each comprising of the following 4 conditions: Human epithelial KB cells were 1) left untreated or were 2) treated with Interleukin-1-alpha (10ng/M-BM-5l) for 1 hour, 3) treated for 1.5h with the TAK1 inhibitor 5Z-7-oxozeaenol (1M-BM-5M) or were 4) treated with 5Z-7-oxozeaenol for 30 minutes followed by Interleukin-1-alpha for 1 hour.
Project description:The inflammatory gene response requires activation of the protein kinase TAK1, but it is currently unknown how TAK1-derived signals coordinate transcriptional programs in the genome. We determined the genome-wide binding of the TAK1-controlled NF-M-NM-:B subunit p65 in relation to active enhancers and promoters of transcribed genes by ChIP-seq experiments. Out of 35,000 active enhancer regions, 410 H3K4me1-positive enhancers show interleukin (IL)-1-induced H3K27ac and p65 binding. Inhibition of TAK1, IKK2 or depletion of p65 blocked inducible enhancer activation and gene expression. As exemplified by the CXC chemokine cluster located on chromosome 4, the TAK1-p65 pathway also regulates the recruitment kinetics of the histone acetyltransferase CBP, of NF-M-NM-:B p50 and of AP-1 transcription factors to both, promoters and enhancers. This study provides a high resolution view of epigenetic changes occurring during the IL-1 response and allows the first genome-wide identification of a novel class of inducible p65 NF-M-NM-:B-dependent enhancers in epithelial cells. Two sets of experiments were performed as biological replicate series (rep1 and rep2) each comprising 5 dual-color microarray hybridizations. The two series were performed with inverted Cy-Dye allocation (Dye-swap). Human epithelial KB cells were treated with Interleukin-1-alpha (10ng/M-BM-5l) for 0.5h, 1h, 3h or 24h, or, were left untreated. An individual set of samples was treated identically with IL1a but after an initial preincubation for 1h with PD98059 (50 micromol/l). Each dual-color microarray represents a direct comparison of samples preincubated or not with PD98059.
Project description:Maize (Zea mays L.) and other monocot cereals develop extensive shoot-borne root systems from nodal structures of the shoot to secure water and nutrient uptake and provide anchorage in the soil. In the present study, the early stages of coleoptilar node (first shoot-node) development of maize were subjected to a detailed morphological and histological analysis followed by a comprehensive microarray profiling via 105 k oligonucleotide microarrays. Expression profiling identified 1,179 microarray features corresponding to 974 unique transcripts which were differentially expressed between three stages of wild-type B73 coleoptilar node development within the first four days after the initiation of this structure. Significantly, all but one of these microarray features were preferentially expressed at day four compared to day zero of coleoptilar node formation. Moreover, comparative microarray profiling of wild-type versus mutant rtcs coleoptilar nodes which do not initiate shoot-borne roots revealed 958 microarray features corresponding to 828 unique transcripts that were expressed in a RTCS dependent fashion. Differential gene expression during wild-type coleoptilar node development and between wild-type and rtcs coleoptilar nodes was confirmed for a subset of 24 genes by qRT-PCR. In order to identify direct downstream targets of RTCS in silico promoter analyses revealed 191 differentially expressed genes that contained at least one LBD motif in a 1 kb promoter region upstream of the ATG start codon. For six promoter sequences containing LBD motives EMSA experiments demonstrated RTCS binding, hence supporting the notion that differentially accumulated genes containing LBD motifs are direct downstream targets of RTCS. The complex RTCS dependent regulation of coleoptilar node development and crown root formation was illustrated by a wide variety of cellular functions represented by the differentially accumulated genes and in particular by the differentially accumulated genes containing LBD promoter motifs. 24 samples in total: 3 developmental stages (0, 2, 4) * 2 genotypes (WT, rtcs) * 4 biological replicates
Project description:1-day-old C57BL/6 mice were left untreated (co) or orally infected with 10E2 CFU wildtype (wt) or delta invC SPI1 mutant Salmonella Typhimurium (ATCC14028). Four biological replicates obtained from individual animals were exmained; each group contained animals from at least 2 different litters. On day 4 p.i., animals were sacrificed and intestinal epithelial cells were isolated from total small intestine (protocol according to: Lotz et al., J. Exp. Med. 2006). Total RNA was isolated by TriZol and its purity was examined using a Bioanalyzer. We used microarrays to detail the global gene expression in primary total isolated intestinal epithelial cells. Four biological replicates of isolated intestinal epithelial cells obtained 4 daysp.i. from neonate mice infected at the age of 1 day (each group with animals from at least two litteres). Single color array.
Project description:Intestinal epithelial cells of 28-day-old female mice were isolated according to a recently published protocol (Lotz et al., J Exp. Med. 2006). Isolated intestinal epithelial cells of four individual animals (four biological replicates) were examined. Single color array.
Project description:Inflammation is a hallmark of multiple disease processes, including inflammatory bowel disease (IBD). The transcription factor NF-kappaB plays a critical role in this response through its effects on pro-inflammatory gene expression. Consequently, homeostatic mechanisms that control NF-kappaB activity have been found to play important roles in controlling inflammatory responses in physiologic and pathologic states. Copper Metabolism Murr1 Domain containing 1 (COMMD1), a regulator of copper excretion and other transport pathways, has been shown to play a role in limiting NF-kappaB activation. However, it remains unclear if this factor plays a necessary role in the control of inflammation or in the pathogenesis of inflammatory disorders in vivo. Using a myeloid-cell specific model of Commd1 deficiency in mice we evaluated the contribution of this factor to inflammatory responses. We found that this gene plays an important role as a negative regulator of the LPS transcriptional response, and deficiency in this factor led to sensitization to in vivo models of sepsis. In addition, we identified single nucleotide variants located near COMMD1 that are associated with decreased expression of this gene in humans. Interestingly, these polymorphisms also show a suggestive association signal with ulcerative colitis risk. Consistent with the possibility that genetic variation in COMMD1 expression may predispose to colitis, we find that myeloid deletion of Commd1 leads to more severe colonic inflammation and cancer progression in experimental colitis models. Altogether, the data support the notion that COMMD1 expression in myeloid cells plays an important anti-inflammatory role in physiologic states and human disease. Bone marrow derived macrophages (BMDM) were isolated from two individual wildtype - and two Commd1 knockout mice. Cells from individual mice were splitted and were treated with LPS for 3h, 24h, or, were left untreated. The corresponding 12 cell samples were subjected to total RNA preparation and subsequently used for Single-Color-based Microarray Analysis (Agilent Technologies).
Project description:Intestinal epithelial cells were isolated from total small intestine of each four 3- and 21-day-old C57BL/6 mice (protocol according to: Lotz et al., J. Exp. Med. 2006). Each two 3- and 21-day-old animals were obtained from one litter. Total RNA was isolated by TriZol and ist purity was examined using a Bioanalyszer. We used microarrays to detail the global gene expression in primary total isolated intestinal epithelial cells of 3- versus 21-day-old mice. Four biological replicates from isolated intestinal epithelial cells obtained from 3- and 21-day-old mice (each two animals from one litter). Colour change.
Project description:Intestinal epithelial cells were isolated from total small intestine of each four 28-day old conventionally raised (conv) and germ-free (GF) bred C57BL/6 mice (protocol according to: Lotz et al., J. Exp Med. 2006). Total RNA was isolated by TriZol and ist purity was examined using a Bioanalyszer. We used microarrays to comparatively detail the global gene expression in primary total isolated intestinal epithelial cells. Four biological replicates from isolated intestinal epithelial cells obtained from each 4 germ-free bred and conventionally raised mice. Colour change.
Project description:Exercise leads to a rapid change in the profile of gene expression in circulating neutrophils. We hypothesized that miRNA expression in circulating neutrophils would also be affected by brief exercise. Eleven healthy men (19-30 yr old) performed 10 2-min bouts of cycle ergometer exercise interspersed with 1-min rest at a constant work equivalent to about 76% of VO2max. A baseline blood sample was taken before the and immediately after the exercise. Neutrophils were isolated using OptiPrep Density Gradient Medium (SIGMA). Total RNA was extracted using TRIzolM-BM-.. For this study we used Agilent Human miRNA microarrays V2 (total of 22 chips).