The Drosophila Huntington's disease gene ortholog dhtt influences chromatin regulation during development.

Huntington's disease is an autosomal dominant neurodegenerative disorder caused by a CAG expansion mutation in HTT, the gene encoding huntingtin. Evidence from both human genotype-phenotype relationships and mouse model systems suggests that the mutation acts by dysregulating some normal activity of huntingtin. Recent work in the mouse has revealed a role for huntingtin in epigenetic regulation during development. Here, we examine the role of the Drosophila huntingtin ortholog (dhtt) in chromatin regulation in the development of the fly. Although null dhtt mutants display no overt phenotype, we found that dhtt acts as a suppressor of position-effect variegation (PEV), suggesting that it influences chromatin organization. We demonstrate that dhtt affects heterochromatin spreading in a PEV model by modulating histone H3K9 methylation levels at the heterochromatin-euchromatin boundary. To gain mechanistic insights into how dhtt influences chromatin function, we conducted a candidate genetic screen using RNAi lines targeting known PEV modifier genes. We found that dhtt modifies phenotypes caused by knockdown of a number of key epigenetic regulators, including chromatin-associated proteins, histone demethylases (HDMs) and methyltransferases. Notably, dhtt strongly modifies phenotypes resulting from loss of the HDM dLsd1, in both the ovary and wing, and we demonstrate that dhtt appears to act as a facilitator of dLsd1 function in regulating global histone H3K4 methylation levels. These findings suggest that a fundamental aspect of huntingtin function in heterochromatin/euchromatin organization is evolutionarily conserved across phyla.

Phosphorylation of serine 205 by the protein kinase CK2 persists on Pax3-FOXO1, but not Pax3, throughout early myogenic differentiation.

The myogenic transcription factor Pax3 plays an essential role in early skeletal muscle development and is a key component in alveolar rhabdomyosarcoma (ARMS), a childhood solid muscle tumor. ARMS is characterized by a t(2;13) chromosomal translocation resulting in the fusion of the 5' Pax3 sequences to the 3' FOXO1 sequences to encode the oncogenic fusion protein, Pax3-FOXO1. Posttranslational modifications such as phosphorylation are common mechanisms by which transcription factors are regulated. Consistent with this fact, we demonstrated in a previous report that Pax3 is phosphorylated on Ser205 in proliferating, but not differentiated, primary myoblasts. However, the kinase that mediates this phosphorylation event has yet to be identified. In addition, it is not known whether Pax3-FOXO1 is phosphorylated at this site or how the phosphorylation of the fusion protein changes during early myogenic differentiation. In this report we identify CK2 (formerly termed "casein kinase II") as the kinase responsible for phosphorylating Pax3 and Pax3-FOXO1 at Ser205 in proliferating mouse primary myoblasts. Furthermore, we demonstrate that, in contrast to wild-type Pax3, phosphorylation at Ser205 persists on Pax3-FOXO1 throughout early myogenic differentiation. Finally, we show that Pax3-FOXO1 is phosphorylated at Ser205 in a variety of translocation-containing ARMS cell lines. The results presented in this report not only suggest a possible mechanism by which the disregulation of Pax3-FOXO1 may contribute to tumorigenesis but also identify a novel target for the development of therapies for the treatment of ARMS.

Identification of serines 201 and 209 as sites of Pax3 phosphorylation and the altered phosphorylation status of Pax3-FOXO1 during early myogenic differentiation.

Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development and is key in the development of the childhood solid muscle tumor alveolar rhabdomyosarcoma (ARMS). ARMS is primarily characterized by a t(2;13)(q35;q14) chromosomal translocation, which fuses the 5'-coding sequences of Pax3 with the 3'-coding sequence of the forkhead transcription factor FOXO1 generating the oncogenic fusion protein Pax3-FOXO1. We previously demonstrated that Pax3 and Pax3-FOXO1 are phosphorylated by the protein kinase CK2 at serine 205 in proliferating primary myoblasts and that this phosphorylation event is rapidly lost from Pax3, but not Pax3-FOXO1 upon the induction of differentiation. However, reports suggested that additional sites of phosphorylation might be present on Pax3. In this report we use in vitro and in vivo analyses to identify serines 201 and 209 as additional sites of phosphorylation and along with serine 205 are the only sites of phosphorylation on Pax3. We provide solid evidence supporting the role of the protein kinase GSK3ß as phosphorylating Pax3 at serine 201. Using phospho-specific antibodies we demonstrate a changing pattern of phosphorylation at serines 201, 205, and 209 throughout early myogenic differentiation and that this pattern of phosphorylation is different for Pax3-FOXO1 in primary myoblasts and in several ARMS cell lines. Taken together, our results allow us to propose a molecular model to describe the changing pattern of phosphorylation for Pax3 and the altered phosphorylation for Pax3-FOXO1 during early myogenic differentiation.

Identification of serine 205 as a site of phosphorylation on Pax3 in proliferating but not differentiating primary myoblasts.

Pax3, a member of the paired class homeodomain family of transcription factors, is essential for early skeletal muscle development. Previously, others and we have shown that the stability of Pax3 is regulated on a post-translational level. Evidence in the literature and from our laboratory suggests that phosphorylation, a common form of regulation, may play a role. However, at present, the sites of Pax3 phosphorylation are not known. We demonstrate here the first evidence that Pax3 exists as a phosphoprotein in proliferating mouse primary myoblasts. Using an in vitro kinase assay, deletion, and point mutant analysis, we conclusively identify Ser205 as a site of phosphorylation. The phosphorylation of Ser205 on endogenously expressed Pax3 was confirmed in vivo using antibodies specific for phosphorylation at Ser205. Finally, we demonstrate for the first time that the phosphorylation status of endogenous Pax3 changes rapidly upon the induction of myogenic differentiation. The presence of phosphorylation in a region of Pax3 important for mediating protein-protein interactions, and the fact that phosphorylation is lost upon induction of differentiation, allow for speculation on the biological relevance of phosphorylation.