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Daxx-beta and Daxx-gamma, two novel splice variants of the transcriptional co-repressor Daxx.

ABSTRACT: Daxx is involved in transcriptional control and apoptosis. It comprises several domains, including a regulatory C terminus that is responsible for the interaction with numerous proteins such as p53, promyelocytic leukemia protein (PML), and Hsp27. Here, we describe the identification and characterization of two novel variants of Daxx termed Daxx-? and Daxx-?, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a consequence, Daxx-? and Daxx-? show a markedly decreased affinity to PML, which in turn is associated with a different subnuclear localization of these proteins compared with Daxx. Although Daxx is localized mainly in PML-oncogenic domains (PODs) Daxx-? and Daxx-? display a distinct distribution pattern. Furthermore, Daxx-? and Daxx-? show a decreased affinity to p53 also due to the truncated C terminus. We provide evidence that the p53 recruitment into PODs is Daxx isoform-dependent. The decreased affinity of Daxx-?/-? to p53 and PML results in a diffuse localization of p53 throughout the nucleus. In contrast to Daxx, Daxx-? and Daxx-? are unable to repress p53-mediated transcription. Therefore, alternative splicing of Daxx might indicate an additional level in the cellular apoptosis network.

SUBMITTER: Wethkamp N

PROVIDER: S-EPMC3103337 | biostudies-literature | 2011 Jun

REPOSITORIES: biostudies-literature

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Daxx-beta and Daxx-gamma, two novel splice variants of the transcriptional co-repressor Daxx.

Wethkamp Nils N Hanenberg Helmut H Funke Sarah S Suschek Christoph V CV Wetzel Wiebke W Heikaus Sebastian S Grinstein Edgar E Ramp Uwe U Engers Rainer R Gabbert Helmut E HE Mahotka Csaba C

The Journal of biological chemistry 20110410 22

Daxx is involved in transcriptional control and apoptosis. It comprises several domains, including a regulatory C terminus that is responsible for the interaction with numerous proteins such as p53, promyelocytic leukemia protein (PML), and Hsp27. Here, we describe the identification and characterization of two novel variants of Daxx termed Daxx-β and Daxx-γ, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a cons ...[more]

PMID: 21482821

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Project description:Peroxisome proliferator activated receptor-gamma (PPAR-gamma) is abundantly expressed in atherosclerotic lesions and is implicated in atherogenesis. The existence of three splice variants, PPAR-gamma 1, PPAR-gamma 2, and PPAR-gamma 3 has been established. Using monocyte-derived macrophages from cynomolgus monkeys, we demonstrate here the identification of two new PPAR-gamma exons, exon C and exon D, which splice together with already established exons A1, A2, and B in the 5(') terminal region to generate four novel PPAR-gamma subtypes, PPAR-gamma 4, -gamma 5, -gamma 6, and -gamma 7. PPAR-gamma 4 and gamma 5 were detected only in macrophages whereas gamma 6 and gamma 7 were expressed both in macrophages and adipose tissues. None of these novel isoforms were detected in muscle, kidney, and spleen from monkeys. We found sequences identical to exons C and D in the human genome database. These and all PPAR-gamma exons known to date are encoded by a single gene, located from region 10498 K to 10384 K on human chromosome 3. We cloned and expressed PPAR-gamma 1, PPAR-gamma 4, and PPAR-gamma 5 proteins in yeast using the expression vector pPICZB. As expected, all recombinant proteins showed a molecular weight of approximately 50 kDa. We also investigated the effect of a high-fat diet on the level of macrophage PPAR-gamma expression in monkeys. RT-PCR showed a significant increase in total PPAR-gamma and ABCA1 mRNA levels in macrophages of fat-fed monkeys (n=7) compared to those maintained on a normal diet (n=2). However, none of the novel isoforms seemed to be induced by fat-feeding. We used tetracycline-responsive expression vectors to obtain moderate expression of PPAR-gamma 4 and -gamma 5 in CHO cells. In these cells, expression of PPAR-gamma 5 but not -gamma 4 repressed the expression of ABCA1. Neither isoform modulated the expression of lipoprotein lipase. Our results suggest that individual PPAR-gamma isoforms may be responsible for unique tissue-specific biological effects and that PPAR-gamma 4 and -gamma 5 may modulate macrophage function and atherogenesis.

Project description:BACKGROUND:Orphan nuclear receptors ERR?, ERR? and ERR? that belong to NR3B or type IV nuclear receptor family are well studied for their role in breast cancer pathophysiology. Their homology with the canonical estrogen receptor dictates their possible contributing role in mammary gland development and disease. Although function and regulation of ERR?, ERR? and less about ERR? is reported, role of histone methylation in their altered expression in cancer cells is not studied. Transcriptional activity of nuclear receptors depends on co-regulatory proteins. The present study for the first time gives an insight into regulation of estrogen-related receptors by histone methylation specifically through methyltransferase EZH2 in breast cancer. METHODS:Expression of ERR?, ERR?, ERR? and EZH2 was assessed by immunohistochemistry in four identical tissue array slides that were prepared as per the protocol. The array slides were stained with ERR?, ERR?, ERR? and EZH2 simultaneously. Array data was correlated with expression in MERAV expression dataset. Pearson correlation coeficient r was calculated from the partial matrix expression values available at MERAV database to study the strength of association between EZH2 and three orphan nuclear receptors under study. By western blot and real time PCR, their correlated expression was studied in breast cancer cell lines MCF-7, MDA-MB-231, T47D and MDA-MB-453 including normal breast epithelial MCF-10A cells at both protein and RNA level. Regulation of ERR?, ERR?, ERR? by EZH2 was further investigated upon overexpression and silencing of EZH2. The interaction between ERRs and EZH2 was validated in vivo by CHIP-qPCR. RESULTS:We found a negative correlation between estrogen-related receptors and Enhancer of Zeste Homolog 2, a global repressor gene. Immunohistochemistry in primary breast tumors of different grades showed a correlated expression of estrogen-related receptors and EZH2. Their correlated expression was further validated using online MERAV expression dataset where a negative correlation of variable strengths was observed in breast cancer. Ectopic expression of EZH2 in low EZH2-expressing normal breast epithelial cells abrogated their expression and at the same time, its silencing enhanced the expression of estrogen-related receptors in cancerous cells. Global occupancy of EZH2 on ERR? and ERR? was observed in-vivo. CONCLUSION:Our findings identify EZH2 as a relevant coregulator for estrogen-related receptors in breast carcinoma.

Dataset Information

Daxx-beta and Daxx-gamma, two novel splice variants of the transcriptional co-repressor Daxx.

Publications

Daxx-beta and Daxx-gamma, two novel splice variants of the transcriptional co-repressor Daxx.

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