Analysis of the interaction of TIP60β and PIN1 with the ets family transcription factor ETV6

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.