The Pax3-FKHR oncoprotein is unresponsive to the Pax3-associated repressor hDaxx

Abstract

The Pax3-FKHR fusion protein is present in alveolar rhabdomyosarcoma and results from the t(2;13) (q35;q14) chromosomal translocation. Its oncogenic activity is dependent on a combination of protein-DNA and protein-protein interactions mediated by the Pax3 homeodomain recognition helix. In this report we demonstrate that human Daxx (hDaxx) interacts with Pax3 in vivo and with DNA-bound Pax3 in vitro. This interaction is mediated primarily through the homeodomain recognition helix with the additional involvement of the octapeptide domain and its N-terminal flanking amino acids. Through this interaction hDaxx represses the transcriptional activity of Pax3 by approximately 80%. The Pax3-FKHR fusion is unresponsive to this repressive effect despite an observed endogenous interaction with hDaxx in a rhabdomyosarcoma tumor cell line. Therefore, these data support the model that fusion of FKHR to Pax3 not only adds a strong transactivation domain, but also deregulates transcriptional control of Pax3 by overriding the natural repressive effect of hDaxx.

Regulation of c-met expression by transcription repressor Daxx

“…To date, endogenous targets of Daxx repression are not well characterized and the mechanism by which Daxx exerts its repression activity is poorly understood. The transiently expressed c-met promoter was identified recently as one of the targets of Daxx repression (Michaelson and Leder, 2003) Previously others and we have demonstrated association of Daxx with chromatin (Hollenbach et al, 1999(Hollenbach et al, , 2002Ishov et al, 2004); however, it was not known whether Daxx is associated with specific DNA sites, and whether this association results in transcription repression. To confirm the specificity of the mouse c-met gene as a direct target of Daxx, we have shown by ChIP assay that Daxx is associated with the c-met promoter; moreover, it preferentially binds to the proximal (activation-associated) part of the c-met promoter and to the beginning of the transcription region of the gene.…”

“…It has been shown by several groups that Daxx can act as a transcriptional repressor (Hollenbach et al, 1999;Li et al, 2000a, b;Michaelson and Leder, 2003); however, a major limitation of these studies is that they are mostly based on artificial transcription assays using overexpressed Daxx which can lead to nonspecific side effects. To date, endogenous targets of Daxx repression are not well characterized and the mechanism by which Daxx exerts its repression activity is poorly understood.…”

“…Daxx was purified as a component of a multiprotein repression complex that includes HDAC1 and HDAC2, Dek and the core histones (Hollenbach et al, 1999(Hollenbach et al, , 2002Li et al, 2000a); it also interacts with DNMT1 and DMAP1 (Michaelson et al, 1999;Muromoto et al, 2004). In addition, Daxx forms a complex with protein ATRX and this complex displays chromatin-remodeling activity (Xue et al, 2003;Ishov et al, 2004;Tang et al, 2004).…”

“…They have shown that Daxx associates with HDAC2 and the acetylated form of histone H4, but does not bind to DNA itself (Hollenbach et al, 1999(Hollenbach et al, , 2002. By modifying histone deacetylase activity at sites of active transcription, Daxx may facilitate the histone deacetylation, thus preventing the access of promoter elements by the transcription machinery.…”

“…First, Pax3 and Ets1 activate c-met transcription (Epstein et al, 1996;Gambarotta et al, 1996), while Daxx represses Pax3 and Ets1 transactivation (Hollenbach et al, 1999;Li et al, 2000b). Second, IFNa upregulates Daxx (Shimoda et al, 2002) that may lead to the observed downregulation of c-met expression upon IFNa treatment (Radaeva et al, 2002).…”