Project description:A single nucleotide polymorphism (SNP) in the human glucocorticoid receptor (GR), N363S, has been the focus of several clinical studies, and some epidemiological data link this SNP to increased glucocorticoid sensitivity, coronary artery disease and increased body mass index (BMI). However, molecular studies in vitro using reporter gene expression systems have failed to define a link between this polymorphism and altered glucocorticoid receptor function. To address the biological relevancy of N363S in glucocorticoid receptor mechanisms and function, we established stable U-2 OS (human osteosarcoma) cell lines expressing wild type hGR or N363S using a tetracycline-regulated expression system. Functional assays with reporter gene systems revealed only minor differences between the wild type hGR and N363S receptors under a variety of conditions that probe for GR function. However, examination of this polymorphism by human gene microarray analysis showed, for the first time, that there are significant differences between wild type hGR and the N363S SNP in their ability to selectively regulate gene expression. Several of these genes may define the link between the N363S SNP and human disease. Experiment Overall Design: U-2 OS cells were transfected with the BD Clontech pTET-OFF regulatory plasmid to establish the U-OFF parental cell line. MluI and EcoRV ends were generated onto the coding region of hGRa using PCR amplification of the pCMVhGRa plasmid. The pTRE2hyg vector was digested with MluI and EcoRV and the two DNAs were ligated to form the pTRE2hGRa plasmid (Lu and Cidlowski). Site-directed mutagenesis was then performed to make pTRE2N363S. The wild type hGR and the N363S mutant were individually transfected into the U-OFF cells and clones were selected which stably expressed either hGRa or N363S using 200 mg/ml of geneticin and 500 mg/ml of hygromycin. Several clones were obtained for each receptor, and the receptor levels were compared using western blot analyses. In these cell lines, the expression of hGR can be repressed by the addition of tetracycline or the derivative doxycycline to the media. U-2 OS (human osteosarcoma) cells were maintained in DMEM/F-12 supplemented with 10% FCS:CS, 2 mM glutamine and pen-strep and selected clones were maintained in the same media with the addition of 200 mg/ml Geneticin and 200 mg/ml hygromycin. All cells were maintained in a humidified, 5% CO2 atmosphere. For the Microarray: U-2 OS cells stably expressing either wild type hGR or N363S were treated for six hours with 10 nM dexamethasone or vehicle, and total RNA was isolated using the Qiagen RNeasy midi kit (Qiagen, Valencia, CA). Experiment Overall Design: Gene expression analysis was conducted using Agilent Human1Av2 arrays (Agilent Technologies, Palo Alto, CA). Total RNA was amplified using the Agilent Low RNA Input Fluorescent Linear Amplification Kit protocol. Starting with 500ng of total RNA, Cy3 or Cy5 labeled cRNA was produced according to manufacturerâs protocol. For each two color comparison, 750ng of each Cy3 and Cy5 labeled cRNAs were mixed and fragmented using the Agilent In Situ Hybridization Kit protocol. Hybridizations were performed for 17 hours in a rotating hybridization oven using the Agilent 60-mer oligo microarray processing protocol. Slides were washed as indicated in this protocol and then scanned with an Agilent Scanner. Data was obtained using the Agilent Feature Extraction software (v7.5), using defaults for all parameters. For each comparison, two biological replicates were used, each with a dye reversal (totaling four arrays per comparison).

Project description:LC-MS data and DIA-NN search files relating to: PU.1 Eviction at Lymphocyte-Specific Chromatin Domains Mediates Glucocorticoid Response in Acute Lymphoblastic Leukemia. Abstract: The epigenetic landscape plays a critical role in the onset and evolution of various malignancies, but its therapeutic utility remains underutilized. Glucocorticoids are an essential part of many multi-agent treatment regimens for lymphoid malignancies. However, the emergence of glucocorticoid resistance is a significant barrier to cure, which is in part due to epigenetic alterations, including aberrant chromatin accessibility and hypermethylation at lymphocyte-specific glucocorticoid-response elements (GREs). To gain a deeper understanding of regulatory mechanisms leading to these epigenetic alterations, we conducted a multi-omics study, including chromosome conformation capture sequencing (HiC), to examine changes in the 3D genome structure following the in vivo treatment of acute lymphoblastic leukemia (ALL) patient-derived xenografts (PDXs) with glucocorticoid. We found that glucocorticoid treatment led to distinct patterns of topologically associated domains (TADs) in glucocorticoid sensitive compared to resistant PDXs. Furthermore, we show that these TADs were primed by the development-related pioneer transcription factor PU.1, which extensively interacts with the glucocorticoid receptor (GR) exclusively in glucocorticoid-sensitive ALL PDXs. An integrative analysis of rapid immunoprecipitation mass spectrometry of endogenous protein (RIME) and ChIP-seq revealed that PU.1 binding was associated with lymphocyte-specific activation of GREs and GRE-interacting super-enhancers. The PU.1-associated TADs modulated epigenetic marks, and particularly the eviction of PU.1 promoted GR binding and the expression of signature genes, including BIM, ZBTB16 and RASA1, mediating glucocorticoid-induced apoptosis in ALL. These findings were phenocopied using a PU.1 inhibitor DB2313 to restore glucocorticoid sensitivity in ALL. Taken together, this study identified a new epigenetic pathway integrating PU.1 priming and PU.1-GR interaction which ultimately leads to PU.1 eviction in ALL. This pathway provides the first link between the activity of a lineage-specific transcription factor and epigenetic modulators mediating the response to glucocorticoids and thus offers a new avenue to translate fundamental epigenetic research into the clinic.

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