Daxx-β and Daxx-γ, Two Novel Splice Variants of the Transcriptional Co-repressor Daxx

Nils Wethkamp,Helmut Hanenberg,Sarah Funke,Christoph V Suschek,Wiebke Wetzel,Sebastian Heikaus,Edgar Grinstein,Uwe Ramp,Rainer Engers,Helmut E Gabbert,Csaba Mahotka

doi:10.1074/jbc.m110.196311

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-β and Daxx-γ, which are generated by alternative splicing. Alternative splicing results in a truncated regulatory C terminus in both proteins. As a consequence, Daxx-β and Daxx-γ 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-β and Daxx-γ display a distinct distribution pattern. Furthermore, Daxx-β and Daxx-γ 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-β/-γ to p53 and PML results in a diffuse localization of p53 throughout the nucleus. In contrast to Daxx, Daxx-β and Daxx-γ are unable to repress p53-mediated transcription. Therefore, alternative splicing of Daxx might indicate an additional level in the cellular apoptosis network.

Highlights

Stiftung fur Krebsforschung. 1 The results of this work are in partial fulfillment of the Ph.D. thesis at the Heinrich Heine University. 2 To whom correspondence should be addressed: Heinrich Heine University (PML)3 in speckled subnuclear structures called PML oncogenic domains (PODs) or PML nuclear bodies [7,8,9,10]
Identification of Two Novel Daxx Splice Variants Daxx-␤ and Daxx-␥ in Various Cell Lines—We investigated the role of Daxx in human carcinomas (Fig. 1A) and found that Daxx mRNA expression could be detected in all tested renal cell carcinoma (RCC) cell lines
The smaller fragment was designated as Daxx-␥ whereas usage of the regular splice donor (SD) site at the exon 5/intron boundary with the alternative splice acceptor (SA) in exon 6 leads to the lack of the entire first 170 nucleotides of exon 6

Summary

EXPERIMENTAL PROCEDURES

Cell Culture and Transient Transfection—All renal cell carcinoma (RCC) cell lines were derived from typical representatives of the clear cell and chromophilic/papillary types of RCC, as established in our laboratory. To create HA-tagged Daxx splice variants a HA tag encoding doublestranded DNA cassette was made by annealing the two complementary oligonucleotides 5Ј-GAT CTA CCG GTC GCC ACC ATG GCT TAC CCA TAC GAT GTT CCA GAT TAC GCG G-3Ј and 5Ј-TCG ACC GCG TAA TCT GGA ACA TCG TAT GGG TAA GCC ATG GTG GCG ACC GGT A-3Ј which contain an internal AgeI site (underlined) and flanking XhoI and SalI “sticky ends,” respectively. The latter were used to subclone the HA tag encoding the DNA cassette into the pLEGFP-Daxx vector. After 48 h, cells were lysed and Renilla and firefly luciferase activity was monitored using the Dual Luciferase kit (Promega) according to the manufacturer’s instructions

RESULTS

DISCUSSION

Gabbert and Csaba Mahotka