ABSTRACT
ARID1a (BAF250), a component of human SWI/SNF chromatin remodeling complexes, is frequently mutated across numerous cancers, and its loss of function has been putatively linked to glucocorticoid resistance. Here, we interrogate the impact of siRNA knockdown of ARID1a compared to a functional interference approach in the HeLa human cervical cancer cell line. We report that ARID1a knockdown resulted in a significant global decrease in chromatin accessibility in ATAC-Seq analysis, as well as affecting a subset of genome-wide GR binding sites determined by analyzing GR ChIP-Seq data. Interestingly, the specific effects on gene expression were limited to a relatively small subset of glucocorticoid-regulated genes, notably those involved in cell cycle regulation and DNA repair. The vast majority of glucocorticoid-regulated genes were largely unaffected by ARID1a knockdown or functional interference, consistent with a more specific role for ARID1a in glucocorticoid function than previously speculated. Using liquid chromatography-mass spectrometry, we have identified a chromatin-associated protein complex comprising GR, ARID1a, and several DNA damage repair proteins including P53 binding protein 1 (P53BP1), Poly(ADP-Ribose) Polymerase 1 (PARP1), DNA damage-binding protein 1 (DDB1), DNA mismatch repair protein MSH6 and splicing factor proline and glutamine-rich protein (SFPQ), as well as the histone acetyltransferase KAT7, an epigenetic regulator of steroid-dependent transcription, DNA damage repair and cell cycle regulation. Not only was this protein complex ablated with both ARID1a knockdown and functional interference, but spontaneously arising DNA damage was also found to accumulate in a manner consistent with impaired DNA damage repair mechanisms. Recovery from dexamethasone-dependent cell cycle arrest was also significantly impaired. Taken together, our data demonstrate that although glucocorticoids can still promote cell cycle arrest in the absence of ARID1a, the purpose of this arrest to allow time for DNA damage repair is hindered.
Subject(s)
DNA Repair , Nuclear Proteins , Receptors, Glucocorticoid , Tumor Suppressor p53-Binding Protein 1 , Humans , Cell Cycle , Cell Cycle Checkpoints , Cell Line, Tumor , Chromatin/genetics , DNA Damage , DNA-Binding Proteins/genetics , DNA-Binding Proteins/metabolism , Histone Acetyltransferases/metabolism , Nuclear Proteins/metabolism , Transcription Factors/genetics , Receptors, Glucocorticoid/metabolism , Tumor Suppressor p53-Binding Protein 1/metabolismABSTRACT
Mineralocorticoid and glucocorticoid receptors (MRs and GRs) constitute a functionally important dual receptor system detecting and transmitting circulating corticosteroid signals. High expression of MRs and GRs occurs in the same cells in the limbic system, the primary site of glucocorticoid action on cognition, behavior, and mood; however, modes of interaction between the receptors are poorly characterized. We used chromatin immunoprecipitation with nucleotide resolution using exonuclease digestion, unique barcode, and single ligation (ChIP-nexus) for high-resolution genome-wide characterization of MR and GR DNA binding profiles in neuroblastoma cells and demonstrate recruitment to highly similar DNA binding sites. Expressed MR or GR showed differential regulation of endogenous gene targets, including Syt2 and Ddc, whereas coexpression produced augmented transcriptional responses even when MRs were unable to bind DNA (MR-XDBD). ChIP confirmed that MR-XDBD could be tethered to chromatin by GR. Our data demonstrate that MR can interact at individual genomic DNA sites in multiple modes and suggest a role for MR in increasing the transcriptional response to glucocorticoids.
Subject(s)
Glucocorticoids/pharmacology , Receptors, Glucocorticoid/metabolism , Receptors, Mineralocorticoid/metabolism , Transcription, Genetic/drug effects , Animals , Base Sequence , Binding Sites/genetics , Cell Line, Tumor , Chromatin Immunoprecipitation , DNA/genetics , DNA/metabolism , Mice , Protein Binding , RNA Interference , Rats , Receptors, Glucocorticoid/genetics , Receptors, Mineralocorticoid/genetics , Response Elements/geneticsABSTRACT
Since the 1950s, the systems level interactions between the hypothalamus, pituitary and end organs such as the adrenal, thyroid and gonads have been well known; however, it is only over the last three decades that advances in molecular biology and information technology have provided a tremendous expansion of knowledge at the molecular level. Neuroendocrinology has benefitted from developments in molecular genetics, epigenetics and epigenomics, and most recently optogenetics and pharmacogenetics. This has enabled a new understanding of gene regulation, transcription, translation and post-translational regulation, which should help direct the development of drugs to treat neuroendocrine-related diseases.
Subject(s)
Neuroendocrinology/instrumentation , Neuroendocrinology/methods , Neurosecretory Systems/physiology , Animals , Gene Editing , High-Throughput Nucleotide Sequencing , History, 20th Century , History, 21st Century , Humans , In Situ Hybridization, Fluorescence , Neuroendocrinology/history , Optogenetics , Receptors, SteroidABSTRACT
In a recent study, Jubb et al. used 3D DNA FISH to assess glucocorticoid-induced 'chromatin decompaction' at multiple loci. Determinants of the specificity, speed, and duration of this phenomenon further enhance our understanding of how the glucocorticoid receptor (GR) dynamically alters chromatin accessibility during acute-phase transcriptional regulation and beyond.
