Unknown,Transcriptomics,Genomics,Proteomics

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Stress-dependent CHIP/Daxx interaction suppresses the p53 apoptotic program

ABSTRACT: Our previous studies have implicated CHIP as a co-chaperone/ubiquitin ligase, whose activities yield protection against stress-induced apoptotic events. In this report, we demonstrate a stress-dependent interaction between CHIP (carboxyl terminus of Hsp70-interacting protein) and Daxx, death domain-associated protein. This interaction interferes with the stress-dependent association of HIPK2 with Daxx, blocking phosphorylation of serine 46 in p53 and inhibiting the p53-dependent apoptotic program. Microarray analysis confirmed suppression of the p53-dependent transcriptional portrait in CHIP (+/+) but not in CHIP (-/-) heat shocked MEFs. The interaction between CHIP and Daxx results in ubiquitination of Daxx which is then partitioned to an insoluble compartment of the cell. In vitro ubiquitination of Daxx by CHIP revealed that Ub chain formation utilizes non canonical lysine linkages associated with resistance to proteasomal degradation. CHIP's ubiquitination of Daxx utilizes lysines 630 and 631 and competes with the cell's sumoylation machinery at these residues. These studies implicate CHIP as a stress-dependent regulator of Daxx that counters Daxx's pro-apoptotic influence in the cell. By abrogating p53-dependent apoptotic pathways and by ubiquitination competitive with Daxx sumoylation, CHIP integrates the cell's proteotoxic stress response with cell cycle pathways that influence cell survival. Keywords: p53, apoptosis, cell stress, ubiquitination We utilized a âsample x referenceâ experimental design strategy in which RNA extracted from mouse embryonic fibroblasts was hybridized to the microarray slide in the presence of labeled Universal Mouse Reference RNA (UMRR, Stratagene, LaJolla, CA). A total of 24 RNA samples were used in this analysis. Briefly, five hundred nanograms of total RNA were used for gene expression profiling following reverse transcription and T-7 polymerase-mediated amplification/labeling with Cyanine-5 CTP. Labeled subject cRNA was co-hybridized to Agilent G4112F Whole Mouse Genome 4x44K oligonucleotide arrays with equimolar amounts of Cyanine-3 labeled UHRR. Slides were hybridized, washed, and scanned on an Axon 4000b microarray scanner. The data were processed using Feature Extaction software (Agilent, Santa Clara, CA).

ORGANISM(S): Mus musculus

SUBMITTER: Andrea Portbury

PROVIDER: E-GEOD-14339 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Stress-dependent Daxx-CHIP interaction suppresses the p53 apoptotic program.

McDonough Holly H Charles Peter C PC Hilliard Eleanor G EG Qian Shu-Bing SB Min Jin-Na JN Portbury Andrea A Cyr Douglas M DM Patterson Cam C

The Journal of biological chemistry 20090522 31

Our previous studies have implicated CHIP (carboxyl terminus of Hsp70-interacting protein) as a co-chaperone/ubiquitin ligase whose activities yield protection against stress-induced apoptotic events. In this report, we demonstrate a stress-dependent interaction between CHIP and Daxx (death domain-associated protein). This interaction interferes with the stress-dependent association of HIPK2 with Daxx, blocking phosphorylation of serine 46 in p53 and inhibiting the p53-dependent apoptotic progra ...[more]

PMID: 19465479

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Project description:Endogenous retroviruses (ERVs) comprise a significant portion of mammalian genomes. Although specific ERV loci feature regulatory roles for host gene expression, most ERV integrations are transcriptionally repressed by Setdb1 mediated H3K9me3 and DNA methylation. However, the protein network which regulates the deposition of these chromatin modifications is still incompletely understood. Here, we performed a genome-wide sgRNA screen for genes involved in ERV silencing and identified the GHKL ATPase protein Morc3 as a top-scoring hit. Morc3 knock-out cells display de-repression, reduced H3K9me3, and increased chromatin accessibility of distinct ERV families. We found that the Morc3 ATPase cycle and Morc3 SUMOylation are important for ERV chromatin regulation. Proteomic analysis revealed that Morc3 mutant proteins fail to interact with the histone H3.3 chaperone Daxx. This interaction depends on Morc3 SUMOylation and Daxx SUMO binding. Notably, in Morc3 ko cells, we observed strongly reduced histone H3.3 on Morc3 binding sites. Thus, our data demonstrate Morc3 as a critical regulator of Daxx-mediated histone H3.3 incorporation to ERV regions. This dataset comprises several experiments addressing different questions: 1. ChIP-MS experiment to determine the protein interaction context of Morc3 using a Morc3-3xFLAG knock-in ES cell line compared to wild type ES cells (Experiment 20200408). 2. ChIP-MS experiments to investigate changes in the protein interaction context of the Morc3 mutant rescue cell lines. Comparison of Morc3 knock-out cell lines with re-expression of Morc3-CW-3xFLAG mutant (Ref. #3111), Morc3-ATP-binding-3xFLAG and Morc3-SUMOylation-3xFLAG mutants (Ref. #3635), and Morc3-deltaN-3xFLAG mutant (Ref. #5174) compared to wt Morc3-3XFLAG rescue. 3. ChIP-MS experiment to determine if the interaction between Morc3 and Daxx is mediated through this C-terminal SIM, comparing Daxx knock-out cell lines with re-expression of wild type 3xFLAG-Daxx protein or 3xFLAG-Daxx ∆SIM, which lacks the C-terminal SIM domain. (Ref. #3301)

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Stress-dependent CHIP/Daxx interaction suppresses the p53 apoptotic program

Publications

Stress-dependent Daxx-CHIP interaction suppresses the p53 apoptotic program.

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