Preanalytical impacts on protein stability by IHC evaluation
Ontology highlight
ABSTRACT:
PROVIDER: PRJNA701506 | ENA |
REPOSITORIES: ENA
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Project description:The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-M-NM-1 (ERM-NM-1)-positive breast tumors in which it participates with ERa and FOXA1 in a complex transcriptional regulatory program driving tumor growth. Paradoxically, GATA3 mutations are frequent in breast cancer and have been classified as drivers. To elucidate the contribution(s) of GATA3 alterations to oncogenesis, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERa and FOXA1 following estrogen stimulation. Thus, mutant GATA3 uncouples protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified stronger accumulation of GATA3 in cells bearing the mutation, albeit with a similar distribution across the genome. We propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of mammary epithelial cells to hormone signaling, thus conferring a selective growth advantage. Genome-wide mapping of GATA3 in two cell lines. There were two biological replicates and unchipped (input) DNA was used as reference.
2014-04-01 | E-GEOD-51274 | biostudies-arrayexpress
Project description:The genetic architecture of protein stability
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Project description:The transcription factor GATA3 is a favorable prognostic indicator in estrogen receptor-α (ERα)-positive breast tumors in which it participates with ERa and FOXA1 in a complex transcriptional regulatory program driving tumor growth. Paradoxically, GATA3 mutations are frequent in breast cancer and have been classified as drivers. To elucidate the contribution(s) of GATA3 alterations to oncogenesis, we studied two breast cancer cell lines, MCF7, which carries a heterozygous frameshift mutation in the second zinc finger of GATA3, and T47D, wild-type at this locus. Heterozygosity for the truncating mutation conferred protection from regulated turnover of GATA3, ERa and FOXA1 following estrogen stimulation. Thus, mutant GATA3 uncouples protein-level regulation of master regulatory transcription factors from hormone action. Consistent with increased protein stability, ChIP-seq profiling identified stronger accumulation of GATA3 in cells bearing the mutation, albeit with a similar distribution across the genome. We propose that this specific, cancer-derived mutation in GATA3 deregulates physiologic protein turnover, stabilizes GATA3 binding across the genome and modulates the response of mammary epithelial cells to hormone signaling, thus conferring a selective growth advantage.
2014-04-01 | GSE51274 | GEO
Project description:DZNep (3-deazaneplanocin A) is commonly used to reduce lysine methylation. DZNep inhibits S-adenosyl-L-homocysteine hydrolase (AHCY), preventing the conversion of S-adenosyl-L-homocysteine (SAH) into L-homocysteine and reducing the level of S-adenosylmethionine (SAM). As a result, the SAM to SAH ratio decreases, an indicator of the methylation potential within a cell. Many studies have characterized the impact of DZNep on histone lysine methylation or in specific cell or disease contexts. Recently, protein thermal stability has provided a new dimension for studying the mechanism of action of small molecule inhibitors. In addition to ligand binding, post-translational modifications and protein-protein interactions impact thermal stability. Here, we sought to characterize protein thermal stability changes induced by DZNep treatment in HEK293T cells using the Protein Integral Solubility Alteration (PISA) assay. DZNep treatment altered the thermal stability of 135 proteins, with over half previously reported to be methylated at lysine residues. In addition to thermal stability, we identify changes in transcript and protein abundance after DZNep treatment to distinguish between direct and indirect impacts on thermal stability. Nearly one-third of the proteins with altered thermal stability had no changes at the transcript or protein level. Of these thermally altered proteins, CDK6 had a stabilized methylated peptide, while its unmethylated counterpart was unaltered. Multiple methyltransferases were among the proteins with thermal stability alteration, including DNMT1, potentially due to changes in SAM/SAH levels. This study systematically evaluates DZNep’s impact on the transcriptome, the proteome, and the thermal stability of proteins.
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Project description:Prep1 is a tumor-suppressor, whereas Meis1 is an oncogene. We show that to perform these activities in MEFs both proteins competitively hetero-dimerize with Pbx1. Meis1 alone transforms Prep1-deficient fibroblasts while Prep1 overexpression inhibits Meis1 tumorigenicity. Pbx1 can therefore alternatively act as oncogene or tumor-suppressor. Prep1 post-translationally controls the level of Meis1 decreasing its stability by sequestering Pbx1. The different levels of Meis1 and the presence of Prep1 are followed at the transcriptional level by the induction of specific transcriptional signatures. The decrease of Meis1 prevents Meis1 interaction with Ddx3x and Ddx5, which are essential for Meis1 tumorigenesis, and modifies the growth promoting DNA binding landscape of Meis1 to the growth controlling landscape of Prep1. Hence the key feature of Prep1 tumor inhibiting activity is the control of Meis1 stability. Examination of Prep1 and Meis1 in three cell type
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