The Neuregulin 1-HER axis as a key mediator of hyperglycemic memory effects in breast cancer
ABSTRACT: Poor outcomes in diabetic patients are observed across a range of human tumors, suggesting that cancer cells develop unique characteristics under diabetic conditions. Cancer cells exposed to hyperglycemic insults acquire permanent aggressive traits of tumor growth, even after a return to euglycemic conditions. Comparative genome-wide mapping of hyperglycemia-specific open chromatin regions and concomitant mRNA expression profiling revealed that neuregulin-1 gene, an established endogenous ligand for the HER3 receptor, is activated through a putative distal enhancer. Our findings highlight the targeted inhibition of NRG1-HER3 pathways as a potential target for the treatment breast cancer patients with associated diabetes Chromatin was extracted from hyperglycemic (HyG) and euglycemic (Control) cancer cells; FAIRE DNA and input DNA from each sample were used to generate libraries for single end sequencing on the the SOLiD 4 HQ system
Project description:Hyperglycemic memory is part of the pathogenesis of diabetic retinopathy. We established a novel mouse model of intermediate-term hyperglycemic memory and demonstrated that changes in gene expression and microvascular damage in the neurovascular unit of the diabetic retina persist after euglycemic reentry, indicating memory. Using microarrays and functional annotation clustering of full genome expression data, genes meeting the criteria for hyperglycemic memory were attributed to the cytoskeletal and nuclear compartments of cells of the neurovascular unit. Overall design: 12-weeks-old STZ-diabetic animals (DC12W), 6-weeks-old STZ-diabetic animals receiving isogenic pancreatic islet transplantation for further 6 weeks (DC+Tx@12W) and age-matched controls (NC) were analyzed after 6 (@6W) and 12 weeks (@12W).
Project description:The goal of this study was to identify chromatin regulatory sites by FAIRE-seq under conditions of basal and increased dosage of transcription factor SF-1 in the H295R human adrenocortical tumor cell line. 4 samples: input DNA in basal SF-1 expression conditions - FAIRE-seq in basal SF-1 expression conditions - input DNA in SF-1 overexpression conditions - FAIRE-seq in SF-1 overexpression conditions
Project description:Chromatin accessibility is a hallmark of active regulatory function in the genome and variation of chromatin accessibility across individuals has been shown to contribute to complex traits and disease susceptibility. However, the mechanisms responsible for chromatin variation among different individuals and how this variation contributes to phenotypic diversity remain poorly understood. We examined chromatin accessibility variation in liver tissue from seven strains of adult mice that have phenotypic diversity in response to a high-fat/high-sucrose diet. Remarkably, nearly 40% of the loci with the greatest degree of chromatin variability across the strains are associated with transposable elements (TEs), with evolutionarily younger TEs being particularly enriched for regions of chromatin variation. We found that evolutionary younger and older TEs have differential chromatin accessibility profiles and are enriched for binding sites of different transcription factors, indicating the role of TEs in the evolution of regulatory networks in the liver. We also demonstrate that TE polymorphisms and epigenetic regulation of TEs contribute to regulatory variation across different strains through providing binding sites for liver transcription factors. Intriguingly, variable chromatin loci that are associated with liver metabolism are primarily TE-associated. We demonstrate that TEs contribute to regulatory variation in liver and have downstream effects on metabolism. Our data reveal TEs as a novel and important contributor to regulatory and phenotypic variation in the liver and suggest that regulatory variation at TEs is a major contributor to phenotypic variation in populations. Examination of chromatin accessibility with FAIRE-seq in livers of male mice (A/J, AKR/J, BALB/cJ, C57BL/6J, C3H/HeJ, CBA/J, DBA/2J, BXH2/TyJ, and BXH19/TyJ) fed a high-fat, high-sucrose diet.
Project description:We report the open chromatin landscape in primary human macrophages and foam cells using FAIRE-seq CD14+ monocytes were isolated from the blood of 3 healthy volunteers. Monocytes were differentiated into macrophages by culture for 7 days with 50ng/ml macrophage colony stimulating factor and then treated for 48 hours with either oxidized low density lipoprotein (oxLDL) to induce foam cell formation or with a control buffer that lacked oxLDL. The resulting six samples were then subjected to FAIRE-seq using an established protocol (Simon JM, Giresi PG, Davis IJ, Lieb JD. Using formaldehyde-assisted isolation of regulatory elements (FAIRE) to isolate active regulatory DNA. Nature protocols 2012;7:256-67).
Project description:These experiments were performed to identify differentially expressed genes in the pancreatic lymph nodes of hyperglycemic NOD mice with high levels of destructive insulitis compared to euglycemic mice with lower levels of insulitis. The pancreatic lymph nodes of 16-week-old hyperglycemic and euglycemic NOD mice were isolated and homogenized in Trizol reagent (Invitrogen). Total RNA was extracted from the aqueous phase using the RNeasy mini kit (Qiagen). RNA quality was assessed using the Agilent 2100 Bioanalyzer and the RNA 6000 Nano Reagent Kit (Agilent). Total RNA from the PLN of hyperglycemic mice (n=2) were labelled with Cy5 and run individually against a pool of Cy3-labeled total RNA isolated from euglycemic mice (n=2). Microarrays were performed using the Whole Mouse Genome Microarray Kit, 4×44K 2-color arrays (Agilent Technologies).
Project description:Using the estrogen receptor alpha (ERalpha) as a model ligand inducible transcription factor, we sought to explicitly define parameters that determine transcription factor binding site selection on a genomic scale in an inducible system that minimizes confounding chromatin effects by the transcription factor itself. By examining several genetic and epigenetic parameters, we find that an energetically favorable estrogen response element (ERE) motif sequence, evidence of occupancy of a "pioneering" transcription factor FOXA1, the presence of the enhancer mark, H3K4me1, and an open chromatin configuration (FAIRE) at the pre-ligand state provide specificity for ER binding. Genome-wide ChIP-sequencing was done in MCF-7 cancer cell line for the following histone H3 modifications: monomethylation H3K4me1, trimethylation H3K4me3, H3K9me3, H3K27me3, acetylation H3K9ac, H3K14ac. In addition sequencing of RNA Pol II was done at same treatment conditions (E2 and DMSO). In addition, we assessed the chromatin configuration of ERα binding sites by deeply sequencing fragments isolated by Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) (Giresi et al, 2007) which enriches for nucleosome free genomic DNA in the aqueous phase of a phenol extraction. The analysis histone modifications in MCF-7 cancer cells was done by ChIP-seq data obtained either with E2 stimulation or without stimulation using vehicle as a control. Using the ERα binding sites defined by ChIP-seq (separate submission), we analyzed the population characteristics of the chromatin configuration of the ERα binding sites. To this end, we performed ChIP-seq analysis for the occupancy configuration of each of the following marks before and after E2 exposure: RNA Pol II, the activation marks H3K4me1, H3K4me3, H3K9ac and H3K14ac, and the repression marks H3K9me3 and H3K27me3. We assessed the chromatin configuration of ERα binding sites by deeply sequencing fragments isolated by Formaldehyde-Assisted Isolation of Regulatory Elements (FAIRE) (Giresi et al, 2007) which enriches for nucleosome free genomic DNA in the aqueous phase of a phenol extraction. The tag count of FAIRE fragments reflects the nucleosome depletion at any given site. RNA Pol II - Cat# ab5408, Abcam; H3K9me3 - Cat# ab8898, Abcam; H3K27me3 - Cat# 07-449, Upstate Biotechnology Inc.; H3K4me1 - Cat# ab8895, Abcam; H3K4me3 - Cat# ab8580, Abcam; H3K9ac - Cat# 07-352, Upstate Biotechnology Inc.; H3K14ac - Cat# 07-353, Upstate Biotechnology Inc.
Project description:Identifying cis-regulatory elements is important to understand how human pancreatic islets modulate gene expression in physiologic or pathophysiologic (e.g., diabetic) conditions. We conducted genome-wide analysis of DNase I hypersensitive sites, histone H3 lysine methylation marks (K4me1, K4me3, K79me2), and CCCTC factor (CTCF) binding in human islets. This identified ~18,000 putative promoters (several hundred novel and islet-active). Surprisingly, active promoter marks were absent at genes encoding islet-specific hormones, suggesting a distinct regulatory mechanism. Of 34,039 distal (non-promoter) regulatory elements, 47% are islet-unique and 22% are CTCF-bound. These findings present a global snapshot of the human islet epigenome and should provide functional context for non-coding variants emerging from genetic studies of T2D and other pancreatic islet disorders. Three different islet samples were tested for DNase I hypersensitivity by DNase-Seq. Five different primary pancreatic islet samples were evaluated for several chromatin modifications (H3K4me3, H3K4me1, H3K79me2) by ChIP-seq. One islet sample was evaluated for CTCF binding via ChIP-seq, All ChIP-seq samples have both non-specific IP (GFP) and input DNA controls.
Project description:The goal of this study was to analyse the effect of a 12 weeks treatment with rosiglitazone on insulin sensitivity in the adipose tissue of type 2 diabetic patients. Diabetic patients were submitted to a 3 hours hyperinsulinemic- euglycemic clamp. Adipose tissue biopsies were taken before and after the clamp, labelled A & B respectively. Then, the patients were treated with rosiglitazone, agonist of PPAR gamma, during 12 weeks. After the treatment, all the patients were submitted to a second 3 hours hyperinsulinemic-euglycemic clamp. Adipose tissue biopsies were taken before and after the clamp, labelled C & D respectively. Overall design: Adipose tissues Biopsies from 7 diabetic patients were used in this study. The samples from the same patients (obtained before and after the hyperinsulinemic-euglycemic clamp) were compared on the same microarray. Biopsie taken before the clamp was considered as the control. A dye switch protocol was applied.
Project description:The tyrosine kinase receptors HER2 and HER3 play an important role in breast cancer. The HER2/HER3 heterodimer is a critical oncogenic unit associated with reduced relapse-free and decreased overall survival. We provide gene expression profile of the mammary epithelial cells MCF10A expressing HER2, HER3 or HER2/HER3 and grown in three-dimensional cultures for 15 days in the presence of heregulin, a known HER3-ligand that stabilizes and activates the HER2/HER3 heterodimer. The mammary epithelial cells MCF10A were transduced with retroviral vectors expressing HER2, HER3 or both. Cells from each group were grown for 15 days in three-dimensional cultures. At the end of the experiment, RNA was extracted for gene expression analysis.