ABSTRACT: We report that WT1 transcriptional repressor protein BASP1 interacts with oestrogen receptor alpha (Erα), and interaction which in enhanced in the presence of Tamoxifen. We utilised RNASeq to identify common BASP1 and ERα target genes as well as Tamoxifen responsive genes that are altered in the absence of BASP1. Total mRNA sequencing analysis of MCF7 cells treated with either siRNA against BASP1 or negative control siRNA, with and without Tamoxifen treatment. Each experiment was performed in triplicate.
Project description:We identified protein-protein interactions and chromatin binding sites for two isforms of BRD1 (BRD1-S and BRD1-L) using Co-IP MS/MS and ChIP-seq. BRD1 isoforms were cloned, epitope-tagged (pcDNA 6 V5-His6, Lifetechnologies) and stably expressed in HEK293T cells. Chromatin immunoprecipitations were performed using anti V5-antibody conjugated agarose beads (Sigma-Aldrich) and anti HA antibody conjugated agarose beads (Sigma-Aldrich) as controls
Project description:We analyze the globel gene expression changes in the tumor initiating cells of regressing miR-125b addicted tumors after oncomiR withdrawal For Histone-H2BGFP expression in DTG tumor cells, the U6 promoter was deleted in the PLKO-PGK-H2B-GFP vector throughNdeI-AgeI digestion, Quick Blunting Kit treatment (NEB), and self-ligation. The resulting PLNA-PGK-H2BGFP plasmid was packaged into lentivirus and used to infect ~1x106freshly sorted α6hiβ1hi DTG tumor cells. After 30 min, cells were then extensively washed and immediately engrafted onto backskins of Nude mice by intradermal transplantation. GFP+α6hiβ1hicells were FACS-isolated from resulting tumors and then serially transplanted as above. For RNA seq analysis of the miR-125b addicted tumor regression process, H2BGFP labeled DTG tumor cells were intradermally engrafted onto backskins of Nude mice (1x104cells/site). Tumors were allowed to grow to ~1cm diameter. Mice were then taken Off Dox by transferring them to regular food for 0, 4, 7 days. GFP+α6hiβ1hiwere then FACS-isolated from the tumors. Two or three independent replicates were collected for each time point. Total RNAswere extracted from the FACS-sorted cells using the miRNeasy Mini Kit (Qiagen) according to the vender’s protocol. Expression of miR-125b in each sample was quantified by RT-PCR using TaqMan MicroRNA Assays (Applied Biosystems). For RNA seq, RNA samples were submitted to the Genomics Resources Core Facility of the Weill Cornell Medical College for library construction using IlluminaTruSeq Stranded mRNA Sample Prep Kit and then sequencing using Illumina HiSeq2000. Resultswere analyzed via the Galaxy web platform using TopHat for initiate mapping and Cufflinks for transcripts assembling and expression level estimation (Computing FPKM: fragments per kilobase of exon per million fragments mapped). The MM9 genome assembly (UCSC Genome Browser) was used as reference genome for all analyses. Low expression genes
Project description:Estrogen signaling pathway is critical for breast cancer development and has remained the major adjuvant therapeutic target for this disease. Tamoxifen has been used in clinic for many years to treat ER-positive breast cancer. However a great many (30%) suffer relapse due to drug resistance. In this study, the bromodomain inhibitor JQ1 was found to down-regulate ERalpha gene expression and have anti-tumor effect in cultured tamoxifen-resisant breast cancer cells. We used microarrays to detail the global programme of gene expression in tamoxifen-resistant MCF7 cells treated with the bromodomain inhibitor JQ1. Tamoxifen-resistant breast cancer MCF7 cells were treated with DMSO (vehicle) or JQ1 (0.2 uM) for 24 hours before total RNA was purified for microarray. Each sample was triplicated.
Project description:RNA-Seq of Arabidopsis thaliana Col-0 and 35S::FLAG-GR-KAN1 plants grown in ambient light or shade. 8 samples (Col-0 wildtype, 35S::FLAG-GR-KAN1 each in shade or ambient light, each with mock or Dexamethasone treatment) with two technical replicates were sequenced.
Project description:In eukaryotes, inositol polyphosphates perform essential metabolic and signaling functions. Using human fungal pathogen Cryptococcus neoformans as a model, we created mutants in three inositol polyphosphates kinases: Arg1, Ipk1 and Kcs1. Each of the mutants produces a unique repertoire of inositol polyphosphates, different from the wild type strain. Comparative phenotypic and transcriptome analyses of wild type and mutant strains indicates that inositol polyphosphate PP-IP5 (IP7) is the key regulator of gene expression, fitness and virulence in C. neoformans. Comparison of WT and mutants (Darg1, Dkcs1 and Dipk1) grown in broth culture in the absence of stress.
Project description:Characterization of gene expression changes 72 hours after withdrawal of tamoxifen in murine hematopoietic progenitors transformed by Hoxa9-ER/Meis1 using RNAseq. In the presence of tamoxifen (4OHT), Hoxa9-ER localizes to the nucleus of cells allowing for transformation, while withdrawal of 4OHT (culture in EtOH) leads to loss of nuclear Hoxa9-ER. Loss of Hoxa9-ER leads to a decrease in cellular proliferation and differentiation along the myeloid lineage. Examination of gene expression by RNAseq in two conditions in biological replicates.
Project description:Mitochondrial dysfunction is implicated in aging and aging-related disorders, such as neurodegenerative diseases and stroke. To study the effects of progressive mitochondrial dysfunction, a homozygous knock-in mouse expressing a proof-reading deficient version of the nucleus-encoded catalytic subunit of mitochondrial DNA (mtDNA) polymerase (PolgA) has been developed. In the mtDNA mutator mouse the proofreading activity of PolgA has been abolished by a single amino acid change. PolgA is the catalytic subunit of the polymerase gamma, which is involved in replicating and proofreading the mitochondrial DNA. As a result, mtDNA mutator mice develop high levels of point mutations and linear deletions, which lead to several human-like phenotypes associated with aging, including reduced lifespan (42-44 weeks), weight loss, alopecia, anemia, kyphosis, osteoporosis, sarcopenia, loss of subcutaneous fat, and reduced fertility. We investigate the molecular mechanism through which exercise may improve the phenotype of the mtDNA mutator mouse, which is a model of premature aging induced by mitochondrial dysfunction. Remarkably, forced endurance exercise has been shown to rescue the progeroid aging phenotypes of the mtDNA mutator mice, and to induce systemic mitochondrial rejuvenation. Here, using voluntary, rather than forced exercise, we investigate the molecular mechanisms underlying such a dramatic improvement, and also assess the effect of exercise on brain tissues, such as cortex and striatum in our model. The complete proteome of key tissues (muscle, brain cortex, brain striatum) from exercising and sedentary mtDNA mutator mice as well as exercising and sedentary wild type mice is quantified using peptide high-resolution isoelectric focusing (HiRIEF) coupled with liquid chromatography tandem mass spectrometry (LC-MS/MS) with an isobaric tag (TMT10plex) strategy.
Project description:Tamoxifen, an antagonist to estrogen receptor (ER), is a first line drug used in breast cancer treatment. However, this therapy is complicated by the fact that a substantial number of patients exhibit either de novo or acquired resistance. To characterize the signaling mechanisms underlying the resistance to tamoxifen, we established a tamoxifen-resistant cell line by treating the MCF7 breast cancer cell line with tamoxifen for over 6 months. We showed that this cell line exhibited resistance to tamoxifen both in vitro and in vivo. In order to quantify the phosphorylation alterations associated with tamoxifen resistance, we performed SILAC-based quantitative phosphoproteomic profiling on the resistant and vehicle-treated sensitive cell lines where we identified >5,600 unique phosphopeptides. We found phosphorylation levels of 1,529 peptides were increased (>2 fold) and 409 peptides were decreased (<0.5-fold) in tamoxifen resistant cells compared to tamoxifen sensitive cells. Gene set enrichment analysis revealed that focal adhesion pathway was the top enriched signaling pathway activated in tamoxifen resistant cells. We observed hyperphosphorylation of the focal adhesion kinases FAK1 and FAK2 in the tamoxifen resistant cells. Of note, FAK2 was not only hyperphosphorylated but also transcriptionally upregulated in tamoxifen resistant cells. Suppression of FAK2 by specific siRNA knockdown could sensitize the resistant cells to the treatment of tamoxifen. We further showed that inhibiting FAK activity using the small molecule inhibitor PF562271 repressed cellular proliferation in vitro and tumor formation in vivo. More importantly, our survival analysis revealed that high expression of FAK2 significantly associated with short metastasis-free survival of ER-positive breast cancer patients treated with tamoxifen-based hormone therapy. Our studies suggest that FAK2 is a great potential target for the development of therapy for the treatment of hormone refractory breast cancers.
Project description:Damage to the mitochondrial genome (mtDNA) severely affects the cell and causes disease. Mutations to mtDNA also accumulate throughout the lifespan of many organisms and may be a proximal cause of aging. There is no effective treatment for ailments caused by mtDNA mutation. Since mitochondrial function and biogenesis are controlled by the nutrient environment of the cell, it is possible that perturbation of conserved, nutrient-sensing pathways may successfully treat mitochondrial disease. Experiments using the tractable eukaryote Saccharomyces cerevisiae allow us to investigate the connection between nutrient-sensing and mitochondrial function. We have focused our current studies on the protein kinase A (PKA) pathway, which controls S. cerevisiae behavior according to glucose availability. We found that reduced PKA signaling can lead, in a background-dependent manner, to improved fitness after mtDNA loss. Specifically, over-expression of the cyclic AMP phosphodiesterase Pde2p, removal of PKA isoform Tpk3p, or ablation of other proteins promoting PKA activity leads to improved proliferation of cells deleted of the mitochondrial genome. Over-expression of Pde2p also suppresses the inviability of several mutants that normally cannot survive mtDNA loss. Moreover, Pde2p over-expression diminishes the nuclear transcriptional response to mtDNA damage, further supporting the idea that glucose sensation is harmful for cells lacking the mitochondrial genome. These findings are heavily dependent upon yeast genetic background. Interestingly, robust import of mitochondrial polytopic membrane proteins may be required in order for cells with no mtDNA to receive the full benefits of PKA reduction. Our findings support the idea that perturbation of nutrient-sensing pathways, and specifically the sensation of glucose, may benefit cells with dysfunctional mitochondria. Four experimental conditions were used: BY4743 (WT) cells containing empty pRS426 vector and containing mtDNA because EtBr was not used, BY4743 (WT) cells containing empty pRS426 vector and lacking mtDNA after 24 hours of treatment with 25 µg/ml ethidium bromide, BY4743 (WT) cells overexpressing TIP41 from plasmid pRS426 and lacking mtDNA after 24 hours of treatment with 25 µg/ml ethidium bromide, and BY4743 (WT) cells overexpressing PDE2 from plasmid pRS426 and lacking mtDNA after 24 hours of treatment with 25 µg/ml ethidium bromide. Two replicates were performed for each sample type.
Project description:Candida albicans BWP17 Wild-type strain was grown at 39oC to perform RNA deep sequencing analysis at the study: The chromatin state of Candida albicans pericentromeric repeats bears features of both euchromatin and heterochromatin. The aim of the study is to analyse differential gene expression at 30 oC and 39 oC of centromere proximal genes.