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Expression data from MDA-MB-231 breast cancer cell lines with DAXX overexpression (OE) and DAXX SUMO interacting motif mutant (DSM) overexpression (DSM OE)

ABSTRACT: DAXX was originally discovered as a context-dependent regulator of cell death or survival. Emerging evidence suggests that DAXX has an oncogenic role in diverse cancer types. However, the molecular mechanisms underlying DAXX's oncogenic function remain to be defined. We used Affymetrix Human Transcriptome Array 2.0 (HTA-2_0) to analyze genes that were up or downregulated upon DAXX OE and DSM OE.

ORGANISM(S): Homo sapiens

PROVIDER: GSE223583 | GEO | 2023/01/28

REPOSITORIES: GEO

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Project description: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).

Project description:Objective: Daxx is a protein with multiple functions and is essential for embryonic development. Daxx knockout embryos fail to develop properly and exhibit lethal phenotype around E6.5. One of the important functions is as a histone chaperone for the histone H3 variant, H3.3. Daxx interacts with Atrx to form a protein complex that deposits H3.3 into heterochromatic regions of the genome, including centromeres, telomeres and repeat loci. Here, we investigated how histone chaperone function of Daxx contributes to the embryonic development. Methods: We developed two Daxx mutant alleles in the mouse germline which abolish the interactions between Daxx and Atrx (DaxxY130A), Daxx and H3.3 (DaxxS226A). We set up mating between either heterozygous DaxxY130A or heterozygous DaxxS226A individually and looked for the viability of homozygous mutants at different development stages. We also performed bulk RNA-seq on tissues from the two mutant embryos and analyzed the changes in gene expression and transposable elements (TE). Results: We found that the interaction between Daxx and Atrx is dispensable for viability in both the pre- and post-natal setting as homozygous Daxx-Y130A mutants are both viable and fertile. The loss of the Atrx interaction, however, does cause dysregulated expression of both endogenous retroviruses and nearby protein coding genes. On the contrary, the interaction between Daxx and H3.3 is not required for embryonic development but is essential for postnatal viability. Transcriptome analysis of embryonic tissues demonstrates that this interaction is important for silencing endogenous retroviruses and for maintaining proper hematopoiesis. Conclusions: The histone chaperone function of Daxx is dispensable for embryonic development but important for hematopoiesis, which is independent of the interaction with Atrx. Moreover, both the interactions with Atrx and with H3.3 is important for regulation of ERV expression. Overall, these results clearly demonstrate that Daxx and H3.3 have both Atrx-dependent and independent functions, advancing our understanding of this epigenetic regulatory complex.

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Expression data from MDA-MB-231 breast cancer cell lines with DAXX overexpression (OE) and DAXX SUMO interacting motif mutant (DSM) overexpression (DSM OE)

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