Geneeskundige Stichting Koningin Elisabeth verslag - GSKE - FMRE

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To determine whether the XNAP gene is a direct target of Notch and to study the mechanisms
of its transcriptional activation, we isolated a mouse XNAP genomic clone and determined its
transcription start site. We created a luciferase reporter construct containing 4kb of the XNAP
gene upstream regions. This construct was transfected in 293T cells with or without an
expression vector for Notch ICD. As positive and negative controls, we used a wild-type and
Su(H) mutated HES1 promoter which is a well known effector of Notch. We could show that the
XNAP reporter construct is strongly activated by Notch ICD and are currently testing shorter
fragments to localize the sequences required for Notch ICD activation. The implication of the
Su(H) transcription factor in the induction of XNAP will be tested by analysing the effect of
overexpressing activated or dominant negative forms of Su(H) on the luciferase activity of the
XNAP reporter construct and by measuring its expression in Su(H) deficient cell lines.
3 DNA binding and transcriptional properties of XHRT1
The Hairy-related transcription factor (HRT) genes encode a subfamily of basic helix-loop-helix
(bHLH) transcription factors that show sequence similarity to the Hairy and Enhancer of split
(H/E(spl)) genes. Compared to the other H/E(spl) proteins, these HRT proteins are
characterized by an invariant glycine instead of a proline residue in the basic region and by the
substitution of the WRPW carboxy-terminal motif by a YxxW motif, followed by the TE(I/V)GAF
sequence (Davis and Turner, 2001). Together with the group of D. Christophe (ULB), we have
recently isolated one member of this HRT gene family, XHRT1. This gene marks in early embryos
floor plate and hypochord precursor cells and its expression in ectodermal cells is induced by
Notch signaling (Pichon et al., 2002).
In collaboration with the group of D. Christophe, we analysed its DNA-binding and
transcriptional activity. We found that, compared to HES1, XHRT1 binds in vitro to the
different E-box motifs but with distinct affinities and identified the sequence "ggCACGTGcc" as
the XHRT1 highest affinity binding site. In transiently transfected 3T3 cells, XHRT1 inhibits the
expression of a luciferase reporter gene under the control of a promoter containing
multimerized XHRT1 consensus binding sites and in embryos, inhibits the expression of chordin,
a notochord marker. The C-terminal YRPW motif is not involved in this repression and cannot
interact with the Groucho/TLE corepressor proteins. Deletion analysis of XHRT1 indicated that
the repression of transcriptional activity as well as the inhibition of axial mesoderm formation
require the Orange domain and the intermediate region between the Orange and YRPW Cterminal
motif. In a yeast two hybrid screen, we identified a novel protein, OIP (Orange
interacting protein), that associates with the orange domain of XHRT1. Overexpressed OIP
protein is found distributed uniformely within the cell and coexpression of XHRT1 caused
accumulation of OIP in the nucleus. The interaction with OIP appears functional as
cotransfection of OIP increases XHRT1 transcriptional repression activity in a dose dependent
manner. These results strongly suggest that OIP plays an important role in modulation of the
XHRT1 activity.