The C-terminal region of cellular Qin oligomerizes: correlation with oncogenic transformation and transcriptional repression

Abstract

Expression of the oncoprotein Qin induces tumors in chickens and oncogenic transformation of chicken embryo fibroblasts in culture. We performed a detailed deletion analysis of the C-terminal region of Qin (amino acids 246-451, extending from the winged helix domain to the C-terminus) and identified amino acids 246-379 as important for transformation. The same region mediates homo-oligomerization of Qin as documented in vitro by GST pulldowns and in vivo by coimmunoprecipitation. A 60 amino-acid region within the oligomerization domain is necessary and sufficient for transcriptional repression induced by Qin.

Proteasomal degradation of the FoxO1 transcriptional regulator in cells transformed by the P3k and Akt oncoproteins

“…For reporter assays, the SfiI fragments were subcloned into pcDNA3.Sfi. The pGL3-cytomegalovirus (CMV)-3xIRS firefly luciferase reporter construct was built by subcloning annealed oligonucleotides 3xIRS-1 (5Ј-TCGATTCAAAATAAGTTTG-TTTTGCTTCAAAATAAGTTTGTTTTGCTTCAAAAT-AAGTTTGTTTTGCGTAC-3Ј) and 3xIRS-2 (5Ј-GCAAA-ACAAACTTATTTTGAAGCAAAACAAACTTATTTTG-AAGCAAAACAAACTTATTTTGAAT-3Ј) into XhoI͞KpnI-digested pGL3-CMV with a silent mutation in the luciferase gene to remove a putative Fox-binding site (32). Akt and FoxG1 plasmids have been described in refs.…”

Binding of the corepressor TLE1 to Qin enhances Qin-mediated transformation of chicken embryo fibroblasts

“…We used a 137 aa region of cQin including the part of the C-terminal region required for transformation (Sonderegger et al, 2003), fused to the Ga14 DBD, as a bait to screen 1.5 Â 10 5 clones of a cDNA library, expressing fusions to the Ga14 TAD. AH109 yeast cells (Clontech) were sequentially transfected with pGBKT7-cQin243-379 and an oligo(dT)-primed directional human fetal brain cDNA library in pACT2 (Clontech) using a lithium acetate transfection protocol (Gietz et al, 1997) and plated on minimal medium omitting leucine, tryptophan (selection for plasmids), and histidine (selection for interacting proteins), in the presence of 2.5 mm 3-aminotriazole.…”

FoxG1, a member of the forkhead family, is a corepressor of the androgen receptor

“…FoxG1 full-length strongly decreases AR signaling. At the highest concentration of FoxG1 expression plasmid used (0.1 g/well in a 6-well plate), a slight reduction of the reporter signal is measurable with control reporter gene vector, containing no consensus sequence site for FoxG1 binding, as reported elsewhere [45,46]. However, the repression of genes which are androgen receptor regulated or whose promoter contains FoxG1 binding sites are considerably stronger in the presence of FoxG1.…”