Mapping Transcription Factor Nucleosome Dynamics in Plasma
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE171434 | GEO | 2021/05/24
REPOSITORIES: GEO
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Project description:Current methods for mapping the tissue-of-origin of circulating cell-free DNA (cfDNA) are still insufficient. Here, we have extended a previously developed methylated CpG tandems amplification and sequencing (MCTA-Seq) method for quantitative analysis of tissue-of-origin of plasma cfDNA. By comparing paired plasma cfDNA and white blood cell genomic DNA, we have demonstrated that the liver is the major non-hematopoietic tissue contributing to plasma cfDNA in healthy adults, accounting for approximately 2%. Furthermore, we have detected changes in liver-derived DNA in patients with benign liver diseases and increases in pancreas-derived DNA in acute pancreatitis patients. Interestingly, our results suggest that DNA derived from pathological tissues makes a minor contribution to the increased cfDNA in many clinical cases. Finally, we have identified a tissue-specific hypermethylated cfDNA marker located in the intragenic regions of tissue-specifichighlyexpressed genes. This study represents valuable progress in the field of cfDNA and offers promise for clinical research and medical diagnostics using the described method.
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Project description:The early detection of tissue and organ damage associated with autoimmune diseases (AID) has been identified as key to improve long-term survival, but non-invasive biomarkers are lacking. Elevated cell-free DNA (cfDNA) levels have been observed in AID and inflammatory bowel disease (IBD), prompting interest to use cfDNA as a potential non-invasive diagnostic and prognostic biomarker. Despite these known disease-related changes in concentration, it remains impossible to identify AID and IBD patients through cfDNA analysis alone. By using unsupervised clustering on large sets of shallow whole-genome sequencing (sWGS) cfDNA data, we uncover AID- and IBD-specific genome-wide patterns in plasma cfDNA in both the obstetric and general AID and IBD populations. Supervised learning of the genome-wide patterns allows AID prediction with 50% sensitivity at 95% specificity. Importantly, the method can identify pregnant women with AID during routine non-invasive prenatal screening. Since AID pregnancies have an increased risk of severe complications, early recognition or detection of new onset AID can redirect pregnancy management and limit potential adverse events. This method opens up new avenues for screening, diagnosis and monitoring of AID and IBD.
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Project description:Methods for identifying protein-protein interactions have mostly been limited to tagged exogenous expression approaches. We now establish a rapid, robust and comprehensive method for finding interacting proteins using endogenous proteins from limited cell numbers. We apply this approach called ‘Rapid IP-Mass Spectrometry of Endogenous proteins (RIME)’ to identify ER, FoxA1 and E2F4 interacting proteins in breast cancer cells. From small numbers of starting cells, we find a comprehensive collection of known ER, FoxA1 and E2F4 targets, plus a number of novel unexpected interactors. One of the most ER (and FoxA1) associated interactors is GREB1, an estrogen induced gene with almost no known function. We apply RIME, in parallel with ER ChIP-seq, to identify ER protein interactors and ER binding events from solid tumor xenografts, resulting in the validation of the ER-GREB1 interactions. Furthermore, we establish a method for identifying endogenous interacting proteins from solid primary breast cancer samples, whih we apply to validate ER interactions with GREB1 and additional co-factors. Mechanistically, we show that GREB1 is recruited with ER to the chromatin where it functions as an essential estrogen-mediated regulatory factor required for effective ER transcriptional activity. Our novel approach enables, for the first time, the ability for discovery and validation of protein-protein interactions in whole tissue and solid tumors, revealing significant insight into ER regulatory factors. Cells were transfected with siControl or siGREB1. The cells were treated with 10nM ICI 182780 for 24 hr to further deplete ER. 48 hr after transfection, the cells were treated with 100nM of Estrogen or vehicle for 6 hr and total RNA was collected from four biological replicates
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Project description:Aberrant activation of the forkhead protein FOXA1 is observed in advanced hormone-related cancers. However, to date, key mediators of high FOXA1 signaling remain elusive. We demonstrate that ectopic high FOXA1 (H-FOXA1) expression promotes estrogen receptor-positive (ER+) breast cancer (BC) metastasis in a xenograft mouse model. Mechanistically, H-FOXA1 reprograms ER-chromatin binding to elicit a core gene signature (CGS) and an ER-dependent secretome highly enriched in ER+ endocrine-resistant (EndoR) cells. ER+ BC MCF7-parental (P) cells treated with estrogen deprivation or estrogen, and with doxycycline (Dox)-inducible FOXA1 overexpression were used in this study. We found that a majority of the lost or retained ER binding induced by H-FOXA1 overlapped with the ER binding sites in P cells induced by estrogen vs. tamoxifen treatment. The lost ER binding sites were also significantly enriched for the ER binding under E2 vs. ED, which corresponds to 11% of the total E2-stimulated ER binding, suggesting that H-FOXA1 induces a loss of selective E2-stimulated ER binding sites. In addition, we found that the proportion of H-FOXA1-induced ER-loss regions were significantly enriched for the unique ER binding sites in P vs. tamoxifen-resistant (TamR) cells, yet no enrichment of the ER-gain regions was observed in the unique ER binding in TamR vs. P cells. Our findings uncover H-FOXA1-induced ER reprogramming driving EndoR and metastasis, possibly via a H-FOXA1/ER-dependent secretome that warrants further studies to clarify its involvement in disease progression of ER+ metastatic breast cancer.
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Project description:Methods for identifying protein-protein interactions have mostly been limited to tagged exogenous expression approaches. We now establish a rapid, robust and comprehensive method for finding interacting proteins using endogenous proteins from limited cell numbers. We apply this approach called ‘Rapid IP-Mass Spectrometry of Endogenous proteins (RIME)’ to identify ER, FoxA1 and E2F4 interacting proteins in breast cancer cells. From small numbers of starting cells, we find a comprehensive collection of known ER, FoxA1 and E2F4 targets, plus a number of novel unexpected interactors. One of the most ER (and FoxA1) associated interactors is GREB1, an estrogen induced gene with almost no known function. We apply RIME, in parallel with ER ChIP-seq, to identify ER protein interactors and ER binding events from solid tumor xenografts, resulting in the validation of the ER-GREB1 interactions. Furthermore, we establish a method for identifying endogenous interacting proteins from solid primary breast cancer samples, whih we apply to validate ER interactions with GREB1 and additional co-factors. Mechanistically, we show that GREB1 is recruited with ER to the chromatin where it functions as an essential estrogen-mediated regulatory factor required for effective ER transcriptional activity. Our novel approach enables, for the first time, the ability for discovery and validation of protein-protein interactions in whole tissue and solid tumors, revealing significant insight into ER regulatory factors.
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Project description:Methods for identifying protein-protein interactions have mostly been limited to tagged exogenous expression approaches. We now establish a rapid, robust and comprehensive method for finding interacting proteins using endogenous proteins from limited cell numbers. We apply this approach called ‘Rapid IP-Mass Spectrometry of Endogenous proteins (RIME)’ to identify ER, FoxA1 and E2F4 interacting proteins in breast cancer cells. From small numbers of starting cells, we find a comprehensive collection of known ER, FoxA1 and E2F4 targets, plus a number of novel unexpected interactors. One of the most ER (and FoxA1) associated interactors is GREB1, an estrogen induced gene with almost no known function. We apply RIME, in parallel with ER ChIP-seq, to identify ER protein interactors and ER binding events from solid tumor xenografts, resulting in the validation of the ER-GREB1 interactions. Furthermore, we establish a method for identifying endogenous interacting proteins from solid primary breast cancer samples, whih we apply to validate ER interactions with GREB1 and additional co-factors. Mechanistically, we show that GREB1 is recruited with ER to the chromatin where it functions as an essential estrogen-mediated regulatory factor required for effective ER transcriptional activity. Our novel approach enables, for the first time, the ability for discovery and validation of protein-protein interactions in whole tissue and solid tumors, revealing significant insight into ER regulatory factors.
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2010-11-17 | E-GEOD-25314 | biostudies-arrayexpress
Project description:Estrogen Receptor-a (ER) is the key feature in the majority of breast cancers and ER binding to the genome correlates with the Forkhead protein FOXA1 (HNF3a), but mechanistic insight is lacking. We now show that FOXA1 is the defining factor that governs differential ER-chromatin interactions. We show that almost all ER-chromatin interactions and gene expression changes are dependent on the presence of FOXA1 and that FOXA1 dictates genome-wide chromatin accessibility. Furthermore, we show that CTCF is an upstream negative regulator of FOXA1-chromatin interactions. In ER responsive breast cancer cells, the dependency on FOXA1 for tamoxifen-ER activity is absolute and in tamoxifen resistant cells, ER binding occurs independently of ligand, but in a FOXA1 dependent manner. Importantly, expression of FOXA1 in non-breast cancer cells is sufficient to alter ER binding and response to endocrine treatment. As such, FOXA1 is the primary determinant that regulates estrogen-ER activity and endocrine response in breast cancer cells and is sufficient to program ER functionality in non-breast cancer contexts.
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