Abstract

The ETV6 gene is involved in many chromosomal translocations forming different fusion genes in both myeloid and lymphoid leukemias as well as in some solid tumors. ETV6 is a member of the ets family of transcription factors and has been shown to be a transcriptional repressor. There is also evidence that ETV6 is a tumor suppressor gene in certain leukemias. ETV6 interacts with TIP60β and PIN1. TIP60 (HIV-1 Tat interacting protein, 60 kDa), a member of the MYST family of histone acetyltransferases, is a transcriptional co-activator or co-repressor depending on the interacting transcription factor. PIN1, an important peptidyl-prolyl cis/trans isomerase, is the only known enzyme to specifically isomerize phosphorylated serine or threonine - proline bonds. This isomerization reaction changes the conformation of the protein substrate and is a central signaling mechanism controlling normal cell proliferation and malignant transformation. In this work the interactions between ETV6 and TIP60β and between ETV6 and PIN1 were analyzed in detail. Using the yeast two hybrid system the TIP60β interacting domain of ETV6 was mapped to the C terminal half of the central domain of ETV6. The interaction between full length ETV6 and TIP60β was shown to be dependent on an intact acetyltransferase activity (HAT) of TIP60β. Interestingly, the HAT deficient TIP60β mutant is still able to interact with the C terminal half of the ETV6 central domain. These results imply a complicated mechanism of ETV6-TIP60β interaction which might involve acetylation of ETV6 by TIP60β and a subsequent conformational change of ETV6. In transient co-transfection experiments using fluorescently tagged proteins ETV6 and TIP60β were co-localized in the nucleus. Reporter gene assays demonstrated that TIP60β is a corepressor of ETV6. This co-repressor activity of TIP60β was especially noticable when a reporter plasmid based on the ETV6-responsive stromelysin-1 promoter was used. TIP60β corepressor activity was dependent on an intact TIP60 acetyl- transferase domain. TIP60β did not exhibit corepessor activity for a serine to alanine mutant at position 257 of ETV6. This result could be caused by a lack of interaction between ETV6(S257A) and TIP60β which might be due to a requirement for a phosphorylation / isomerization dependent conformational change of ETV6 in this region for TIP60β interaction. The expression of GFP-ETV6 had no discernible effect on the proportion of cells in the different phases of the cell cycle as compared to the expression of GFP or YFP. The expression of a TIP60β-YFP construct led to a marked reduction of the proportion of cells in the S/G2/M phase of the cell cycle. Coexpression of both GFP-ETV6 and TIP60β-YFP restored the normal distribution of cycling cells. Similar studies were performed to analyze the interaction between ETV6 and PIN1. The PIN1 interaction domain of ETV6 was mapped to the pointed domain and to the C-terminal half of the central region of ETV6 in the yeast two hybrid system. In both regions, PIN1 consensus binding sites containing a serine/threoine – proline sequence are present. It could be shown that both the WW domain and the PPIase domain of PIN1 participate in the interaction. Co-localization of ETV6 and PIN1 was seen in the nucleus of transiently transfected cells. On a functional level, co-expression of PIN1 with ETV6 markedly reduced the repressor activity of ETV6 when ETV6 was fused to a heterologous DNA binding domain (GAL4DBD). When the stromelysin-1 promoter based reporter gene system was used, co-expression of PIN1 not only relieved ETV6 mediated repression but actually turned ETV6 from a transcriptional repressor to a transcriptional activator. A serine to alanine mutant at position 106 of ETV6 was completely unresponsive to PIN1 coexpression which indicates that S106 of ETV6 is the main site of PIN1 interaction. These findings show that ETV6 activity can be regulated in very decisive ways by interaction with PIN1. It could be that this regulation of ETV6 through PIN1 is a result of ETV6 peptidyl-prolyl cis-trans isomerization and hence a conformational change of ETV6. Expression of YFP-PIN1 led to a slight increase in the proportion of cells in the S/G2/M phase. Like for TIP60β, coexpression of GFP-ETV6 led to a restoration of the distribution of cycling cells.