Project description:We have studied CH1 cells that undergo G1 arrest upon anti-IgM treatment after 16 hrs of stimulation. First we studied the differential gene expression under anti-IgM and IL-4 stimulation condition individually and in combination, and this revealed the affected genes to be directly or indirectly playing a role for arresting the cells in the G1 phase of the cell cycle. We then performed Western blotting experiments for the selected signaling molecule candidates from various pathways, and the phosphorylation kinetic profiles were used to study their role in regulating the gene expression under anti-IgM and/or IL-4 stimulus. Finally, we profiled how the signaling pathways are regulating the activation and deactivation of 345 transcription factors, which are responsible for regulating the anti-IgM and/or IL-4 responsive genes, which in turn leads to the functional output.
Project description:To unravel genes and molecular pathways involved in the pathogenesis of type 1 diabetes (T1D), we performed genome-wide gene expression profiling of prospective venous blood samples from children developing T1D-associated autoantibodies or progressing towards clinical diagnosis. 247 peripheral blood RNA samples from 18 prediabetic children and their matched controls were analyzed with Illumina Human HT-12 Expression BeadChips version 3 arrays, in order to study the gene expression changes occuring during the pathogenesis of Type 1 diabetes (T1D). Each case child (with T1D-specific autoantibodies) was matched with a persistently autoantibody-negative control child, with the same HLA-DQB1 risk category, gender, and place and date of birth. Two control children were selected for T1D cases 3, 5, 13 and 17. Seroconversion is determined as the first detection of T1D-specific autoantibody/autoantibodies (ICA titre >4 JDFU, IAA >3.47 RU, GADA >5.4 RU, IA-2A >0.43 RU, ZnT8A >0.61 RU).
Project description:To unravel genes and molecular pathways involved in the pathogenesis of type 1 diabetes (T1D), we performed genome-wide gene expression profiling of prospective venous blood samples from children developing T1D-associated autoantibodies or progressing towards clinical diagnosis. 58 peripheral blood RNA samples from 4 autoantibody-positive children and their matched controls were analyzed with Illumina Sentrix WG-6 v2 genome-wide arrays, in order to study the gene expression changes occuring during the pathogenesis of Type 1 diabetes (T1D). Each case child (positive for T1D-specific autoantibodies) was matched with a persistently autoantibody-negative control child, with the same HLA-DQB1 risk category, gender, and place and date of birth. Seroconversion is determined as the first detection of T1D-specific autoantibody/autoantibodies (ICA titre >4 JDFU, IAA >3.47 RU, GADA >5.4 RU, IA-2A >0.43 RU, ZnT8A >0.61 RU).
Project description:30 patients were randomized equally into three groups: no treatment, bicalutamide (antiandrogen) 150 mg daily, and goserelin (GnRH agonist) 3.6 mg every 4 weeks. Following the neoadjuvant treatment for 12 weeks, patients underwent radical prostatectomy. Freshly frozen specimens were collected for expression profiling using Illumina microarray (probes for >25 000 mRNAs) <br><br>A file containing processed data is available on the FTP site for this experiment.
Project description:Fusion of the EWS gene to FLI1 produces a fusion oncoprotein that drives an aberrant gene expression program responsible for the development of Ewing sarcoma. We used a homogenous proximity assay to screen for compounds that disrupt the binding of EWS-FLI1 to its cognate DNA targets. A number of DNA-binding chemotherapeutic agents were found to non-specifically disrupt protein binding to DNA. In contrast, actinomycin D was found to preferentially disrupt EWS-FLI1 binding by comparison to p53 binding to their respective cognate DNA targets in vitro. In cell-based assays, low concentrations of actinomycin preferentially blocked EWS-FLI1 binding to chromatin, and disrupted EWS-FLI1-mediated gene expression. Higher concentrations of actinomycin globally repressed transcription. These results demonstrate that actinomycin preferentially disrupts EWS-FLI1 binding to DNA at selected concentrations. Although the window between this preferential effect and global suppression is too narrow to exploit in a therapeutic manner, these results suggest that base-preferences may be exploited to find DNA-binding compounds that preferentially disrupt subclasses of transcription factors. Using proximity assays in A673 Ewing Sarcoma cells, we screened 7 bioactive-enriched small molecule libraries, totaling 5,200 compounds to identify compounds that could disrupt the binding of EWS-FLI1 to its cognate DNA binding sequence. We defined a set of EWS-FLI1-regulated genes by shRNA depletion of EWS-FLI1in the same cell line. Duplicate knock down experiments were carried out and compared to duplicate scrambled shRNA controls. This signature was used to interrogate the effects in duplicate experiments of low- and high-dose actinomycin D treatment in A673 cells as compared to DMSO and untreated controls (2 each).
Project description:Lysine Specific Demethylase 1 (LSD1, KDM1A) functions as a transcriptional corepressor through demethylation of histone 3 lysine 4 (H3K4), but has coactivator function on some genes through unclear mechanisms. We show that LSD1, interacting with CoREST, associates with and coactivates androgen receptor (AR) on a large fraction of androgen-stimulated genes. A subset of these AR/LSD1-associated enhancer sites have histone 3 threonine 6 phosphorylation (H3T6ph), and these sites are further enriched for androgen-stimulated genes. Significantly, despite its coactivator activity, LSD1 still mediates H3K4me2 demethylation at these androgen-stimulated enhancers. FOXA1 is also associated with LSD1 at AR regulated enhancer sites, and a FOXA1 interaction with LSD1 enhances binding of both proteins at these sites. These findings show LSD1 functions broadly as a regulator of AR function, that it maintains a transcriptional repression function at AR-regulated enhancers through H3K4 demethylation, and has a distinct AR-linked coactivator function mediated by demethylation of other substrates. Determine the role of LSD1 in androgen signaling.