TET2 is a component of the ER complex and controls 5mC to 5hmC conversion at ER cis-regulatory regions [RNA-Seq]
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE153250 | GEO | 2021/02/10
REPOSITORIES: GEO
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Project description:Estrogen receptor-alpha (ER) drives tumour development and metastasis in ER positive (ER+) breast cancer. GATA3 is a transcription factor that has been closely linked to ER function, but the role of GATA3 in ER-transcriptional activity is not clear. We sought to identify the contribution of GATA3 to the ER complex, by conducting quantitative multiplexed rapid immunoprecipitation mass spectrometry of endogenous proteins (qPLEX-RIME), to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, very few proteins were dissociated from the ER complex in the absence of GATA3, with the only major change being loss of TET2 in the ER complex. In breast cancer cells and Patient-Derived Xenograft (PDX) tissue, TET2 binding events were shown to constitute a near-total subset of ER binding events, and loss of TET2 was functionally associated with reduced activation of proliferative pathways. To investigate the TET2-ER relationship, the role of TET2 in regulating DNA modifications in ER+ breast cancer cells was examined. TET2 knockdown did not appear to result in changes to global DNA methylation, however, oxidation of methylated DNA to 5-hydroxymethylcytosine (5hmC) was significantly reduced after TET2 depletion and these events occurred at ER enhancers. These findings implicate TET2 in the production and maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.
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Project description:Estrogen receptor-a (ER) drives tumour development and metastasis in ER positive (ER+) breast cancer. GATA3 is a transcription factor that has been closely linked to ER function, but the role of GATA3 in ER transcriptional activity is not clear. We sought to identify the contribution of GATA3 to the ER complex by conducting quantitative multiplexed rapid immunoprecipitation mass spectrometry of endogenous proteins (qPLEX-RIME) to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, very few proteins were dissociated from the ER complex in the absence of GATA3, with the only major change being depletion of TET2 from the ER complex. In breast cancer cells and Patient-Derived Xenograft (PDX) tissue, TET2 binding events were shown to constitute a near-total subset of ER binding events, and loss of TET2 was functionally associated with reduced activation of proliferative pathways. To investigate the TET2-ER relationship, the role of TET2 in regulating DNA modifications in ER+ breast cancer cells was examined. TET2 knockdown did not appear to result in changes to global DNA methylation, however, oxidation of methylated DNA to 5-hydroxymethylcytosine (5hmC) was significantly reduced after TET2 depletion and these events occurred at ER enhancers. These findings implicate TET2 in the production and maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.
2021-02-10 | GSE153252 | GEO
Project description:Estrogen receptor-a (ER) drives tumour development and metastasis in ER positive (ER+) breast cancer. GATA3 is a transcription factor that has been closely linked to ER function, but the role of GATA3 in ER transcriptional activity is not clear. We sought to identify the contribution of GATA3 to the ER complex by conducting quantitative multiplexed rapid immunoprecipitation mass spectrometry of endogenous proteins (qPLEX-RIME) to assess changes to the ER complex in response to GATA3 depletion. Unexpectedly, very few proteins were dissociated from the ER complex in the absence of GATA3, with the only major change being depletion of TET2 from the ER complex. In breast cancer cells and Patient-Derived Xenograft (PDX) tissue, TET2 binding events were shown to constitute a near-total subset of ER binding events, and loss of TET2 was functionally associated with reduced activation of proliferative pathways. To investigate the TET2-ER relationship, the role of TET2 in regulating DNA modifications in ER+ breast cancer cells was examined. TET2 knockdown did not appear to result in changes to global DNA methylation, however, oxidation of methylated DNA to 5-hydroxymethylcytosine (5hmC) was significantly reduced after TET2 depletion and these events occurred at ER enhancers. These findings implicate TET2 in the production and maintenance of 5hmC at ER sites, providing a potential mechanism for TET2-mediated regulation of ER target genes.
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Project description:GATA3 transcription factor is considered critical for luminal development and differentiation in normal and malignant mammary epithelial cells (MECs). The androgen receptor (AR) has also been associated with luminal gene expression profiles in breast cancer, independent of the estrogen receptor (ER), and has been shown to promote a basal to luminal phenotype transition in mouse MECs. To date, the potential interaction of GATA3 and AR in transcriptional regulation of lineage driver genes in breast cancer has never been investigated. Our unbiased proteomic analysis identified GATA3 as a novel AR interacting protein in a variety of breast cancer cell types regardless of ER expression. We showed that AR and GATA3 interact in the cytoplasm and nucleus of normal and malignant breast epithelia, an interaction increased by androgen treatment. Androgen stimulation of breast cancer cells also induced nuclear translocation of AR and GATA3 and resulted in enrichment of co-localized AR and GATA3 chromatin binding events. Using ER+ and/or ER- breast cancer cell lines and ER+ patient-derived xenograft (PDX) models of breast cancer, we identified a conserved subset of AR agonist-induced AR and GATA3 co-occupied cis-regulatory elements across all models. Knockdown experiments indicated that GATA3 acts as an AR co-regulator to upregulate transcription of known luminal-lineage genes (e.g. EHF, AQP3, and KDM4B) and downregulate basal-lineage genes (e.g. SMAD3 and ELK3). Also, we showed an induction in the chromatin accessibility at those subsets of common AR-GATA3 cis-regulatory elements, which drive the luminal lineage identity in breast cancer upon androgen stimulation. Collectively, AR-GATA3 interaction and function provide a mechanism underpinning epithelial breast cancer cells that are classified as having a luminal mature transcriptome independent of ER status, through regulating the expression of lineage-restricted markers.
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Project description:GATA3 transcription factor is considered critical for luminal development and differentiation in normal and malignant mammary epithelial cells (MECs). The androgen receptor (AR) has also been associated with luminal gene expression profiles in breast cancer, independent of the estrogen receptor (ER), and has been shown to promote a basal to luminal phenotype transition in mouse MECs. To date, the potential interaction of GATA3 and AR in transcriptional regulation of lineage driver genes in breast cancer has never been investigated. Our unbiased proteomic analysis identified GATA3 as a novel AR interacting protein in a variety of breast cancer cell types regardless of ER expression. We showed that AR and GATA3 interact in the cytoplasm and nucleus of normal and malignant breast epithelia, an interaction increased by androgen treatment. Androgen stimulation of breast cancer cells also induced nuclear translocation of AR and GATA3 and resulted in enrichment of co-localized AR and GATA3 chromatin binding events. Using ER+ and/or ER- breast cancer cell lines and ER+ patient-derived xenograft (PDX) models of breast cancer, we identified a conserved subset of AR agonist-induced AR and GATA3 co-occupied cis-regulatory elements across all models. Knockdown experiments indicated that GATA3 acts as an AR co-regulator to upregulate transcription of known luminal-lineage genes (e.g. EHF, AQP3, and KDM4B) and downregulate basal-lineage genes (e.g. SMAD3 and ELK3). Also, we showed an induction in the chromatin accessibility at those subsets of common AR-GATA3 cis-regulatory elements, which drive the luminal lineage identity in breast cancer upon androgen stimulation. Collectively, AR-GATA3 interaction and function provide a mechanism underpinning epithelial breast cancer cells that are classified as having a luminal mature transcriptome independent of ER status, through regulating the expression of lineage-restricted markers.
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Project description:GATA3 transcription factor is considered critical for luminal development and differentiation in normal and malignant mammary epithelial cells (MECs). The androgen receptor (AR) has also been associated with luminal gene expression profiles in breast cancer, independent of the estrogen receptor (ER), and has been shown to promote a basal to luminal phenotype transition in mouse MECs. To date, the potential interaction of GATA3 and AR in transcriptional regulation of lineage driver genes in breast cancer has never been investigated. Our unbiased proteomic analysis identified GATA3 as a novel AR interacting protein in a variety of breast cancer cell types regardless of ER expression. We showed that AR and GATA3 interact in the cytoplasm and nucleus of normal and malignant breast epithelia, an interaction increased by androgen treatment. Androgen stimulation of breast cancer cells also induced nuclear translocation of AR and GATA3 and resulted in enrichment of co-localized AR and GATA3 chromatin binding events. Using ER+ and/or ER- breast cancer cell lines and ER+ patient-derived xenograft (PDX) models of breast cancer, we identified a conserved subset of AR agonist-induced AR and GATA3 co-occupied cis-regulatory elements across all models. Knockdown experiments indicated that GATA3 acts as an AR co-regulator to upregulate transcription of known luminal-lineage genes (e.g. EHF, AQP3, and KDM4B) and downregulate basal-lineage genes (e.g. SMAD3 and ELK3). Also, we showed an induction in the chromatin accessibility at those subsets of common AR-GATA3 cis-regulatory elements, which drive the luminal lineage identity in breast cancer upon androgen stimulation. Collectively, AR-GATA3 interaction and function provide a mechanism underpinning epithelial breast cancer cells that are classified as having a luminal mature transcriptome independent of ER status, through regulating the expression of lineage-restricted markers.
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Project description:GATA3 transcription factor is considered critical for luminal development and differentiation in normal and malignant mammary epithelial cells (MECs). The androgen receptor (AR) has also been associated with luminal gene expression profiles in breast cancer, independent of the estrogen receptor (ER), and has been shown to promote a basal to luminal phenotype transition in mouse MECs. To date, the potential interaction of GATA3 and AR in transcriptional regulation of lineage driver genes in breast cancer has never been investigated. Our unbiased proteomic analysis identified GATA3 as a novel AR interacting protein in a variety of breast cancer cell types regardless of ER expression. We showed that AR and GATA3 interact in the cytoplasm and nucleus of normal and malignant breast epithelia, an interaction increased by androgen treatment. Androgen stimulation of breast cancer cells also induced nuclear translocation of AR and GATA3 and resulted in enrichment of co-localized AR and GATA3 chromatin binding events. Using ER+ and/or ER- breast cancer cell lines and ER+ patient-derived xenograft (PDX) models of breast cancer, we identified a conserved subset of AR agonist-induced AR and GATA3 co-occupied cis-regulatory elements across all models. Knockdown experiments indicated that GATA3 acts as an AR co-regulator to upregulate transcription of known luminal-lineage genes (e.g. EHF, AQP3, and KDM4B) and downregulate basal-lineage genes (e.g. SMAD3 and ELK3). Also, we showed an induction in the chromatin accessibility at those subsets of common AR-GATA3 cis-regulatory elements, which drive the luminal lineage identity in breast cancer upon androgen stimulation. Collectively, AR-GATA3 interaction and function provide a mechanism underpinning epithelial breast cancer cells that are classified as having a luminal mature transcriptome independent of ER status, through regulating the expression of lineage-restricted markers.
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Project description:The TET proteins TET1, TET2 and TET3 constitute a new family of dioxygenases that utilize molecular oxygen and the cofactors Fe(II) and 2-oxoglutarate to convert 5-methylcytosine (5mC) to 5-hydroxy-methylcytosine (5hmC) and further oxidation products in DNA1-5. Here we show that Tet1 and Tet2 have distinct roles in regulating 5hmC deposition and gene expression in mouse embryonic stem cells (mESC). Tet1 depletion in mESC primarily diminishes 5hmC levels at transcription start sites (TSS), whereas Tet2 depletion is mostly associated with decreased 5hmC in gene bodies relative to TSS. 5hmC is enriched at exon start and end sites, especially in exons that are highly expressed, and is significantly decreased upon Tet2 knockdown at the boundaries of high-expressed exons that are selectively regulated by Tet2. In differentiating murine B cells, Tet2 deficiency is associated with selective exon exclusion in the gene encoding the transmembrane phosphatase CD45. Tet2 depletion is associated with increased 5hmC and decreased 5mC at promoters/ TSS regions, possibly because of the redundant activity of Tet1. Together, these data indicate a complex interplay between Tet1 and Tet2 in mESC, and show that loss-of-function of a single TET protein does not necessarily lead to loss of 5hmC and a corresponding gain of 5mC, as generally assumed. The relation between Tet2 loss-of-function and selective changes in exon expression could potentially explain the frequent occurrence of both TET2 loss-of-function mutations and mutations in proteins involved in pre-mRNA splicing in myeloid malignancies in humans. Gene and exon expression analysis in mESC, Tet1 knockdown mESC, and Tet2 knockdown mESC by RNA-sequencing. Mapping of 5-hydroxymethylcytosine in mESC, Tet1 knockdown mESC, and Tet2 knockdown mESC by anti-CMS-seq. Mapping of methylcytosine in mESC, and Tet2 kd mESC by MeDIP-seq.
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