Montantes

3.2.1 HIRA and Pax3 interact through the Pax3 homeodomain

At the start o f this project, the identification o f an interaction between Pax3 and HIRA in a yeast two-hybrid screen had been confirmed by GST in vitro interaction assays and co-immunoprecipitation (Magnaghi et a l, 1998). The next step was to map the precise region(s) required for the interaction.

The Pax3 protein contains two DNA-binding domains - a paired domain and a homeodomain, as well as an octapeptide motif. Functional dissection o f the protein (Chalepakis et al., 1994c) has revealed an N-terminal transcription inhibitory domain, and a C-terminal transcriptional activation domain (see figure 4).

219 278 479 III P A IR E D D O M A IN O C T H O M E O - D O M A IN R E P R E S S IO N A C T IV A T IO N D O M A IN D O M A IN

Figure 4. Schematic representation o f the Pax3 protein. The paired domain, homeodomain (with each o f the three a-helices I to III) and octapeptide (OCT) m otif are shown, as well as putative transcriptional regulatory domains. Numbers indicate amino acid positions.

Sequence analysis of the twelve Pax3 clones and one Pax7 clone isolated in the two-hybrid screen suggested that the homeodomain plays an important role in the interaction with HIRA, since this represents the only region common to all o f the clones. The three-dimensional crystal structure o f a prototypic paired-like homeodomain on DNA has been solved (Wilson et al., 1995), and consists o f three a - helices preceded by a short N-terminal arm. On binding DNA, a-helix III, the recognition helix, sits in and makes direct contacts with the major groove, while the N- terminal arm makes contacts with the minor groove, a-helices I and II lie outside the DNA binding site and are potentially accessible for binding by other nuclear factors.

The following experiments were carried out using a series o f constructs designed to map the region o f interaction between HIRA and the Pax3 homeodomain more precisely.

3.2.1.1 Production o f PaxS homeodomain constructs

In order to determine whether the Pax3 homeodomain was sufficient for HIRA interaction, and to further define the subdomain required, preliminary experiments were designed to assess the binding o f the isolated homeodomain and its constituent a - helices. A number o f Pax3 homeodomain constructs were produced by PCR. Primers were designed to amplify (i) the N-terminal arm and putative first a-helix, (ii) the third a-helix and C terminal o f the homeodomain, and (iii) the full length o f the homeodomain. Further constructs were also made that extended the region N-terminal to the homeodomain by -2 0 residues to include an additional region also common to the two-hybrid clones. The various a-helices were cloned into the pVP16 vector, suitable for testing in yeast two-hybrid assays and for in vitro translation as VP 16 fusion proteins to be tested in GST affinity capture assays. Pfu DNA polymerase was used in the PCR reactions for its proof-reading ability. Constructs were sequenced to verify that the DNA was in frame and contained no mutations.

Interaction between HIRA and the Pax3 homeodomain constructs was first tested with the yeast two-hybrid system, using the yeast strain L40 (Hollenberg et a l,

1995) in which the HIRA-Pax3 interaction had first been identified (Magnaghi et al.,

1998). Each o f the pVP16-Pax3 homeodomain constructs were co-expressed with pLexA-HIRA but activation o f the reporter genes, lacZ and HIS3, was not detected suggesting little or no interaction had occurred (data not shown). This may have been because the small homeodomain fragments could not fold correctly, and so were not adopting their native conformation required for an interaction, or because multiple points o f contact within Pax3 are required for stable interaction. However, expression o f the Pax3 fusion proteins was not verified due to the lack o f an appropriate antibody at that time, and lack o f expression or reduced stability could be an alternative

When assessed by GST affinity capture assay using GST HIRA FL as the bait protein (see section 3.1.1), the opposite result was observed. In this case all the in vitro

translated fusion proteins were found to weakly interact w ith HIRA (data not shown). It seems likely that this interaction may have been non-specific however, since the VP 16 control protein also bound GST HIRA with similar affinity. Altering the stringency o f the wash buffer did not improve the specificity o f binding. Non-specific binding to HIRA may reflect the problems encountered in producing the bait protein, GST HIRA FL as mentioned in section 3.1.1, and this is discussed further in section 3.2.1.3. Thus in this experiment it was not possible to form a conclusion regarding the interaction o f homeodomain fragments with HIRA, and due to the technical problems o f working with such small proteins, coupled to the contradictory results obtained, this approach was not pursued further.

S. 2.1.2 Production o f Pax deletion constructs

In order to overcome some o f the difficulties o f the previous approach an alternative strategy was devised, using Pax3 mutants containing internal deletions. Several Pax3 constructs were therefore made from which the corresponding regions had been deleted; a-helix I, a-helices II and III or the entire homeodomain. These constructs were made using the technique o f ‘splicing by overlap extension’ or ‘SOEing’ (Horton et al., 1989). This PGR approach involved the production o f two separate products that overlapped in the region to be joined, so that a second PGR reaction could be used to ‘splice’ the two products together, thus deleting the desired region. As detailed in figure 5, Pax3 constructs were made that lacked the entire homeodomain from amino acids 217 to 282 (Pax3AHD), just a-helix I contained within amino acids 217 to 247 (Pax3AHI) or a-helices II, III and the entire G-terminus

(Pax3AG), a truncation after amino acid 248.

In addition to Pax3, other Pax proteins were incorporated into the experimental series to identify regions necessary for HIRA interaction and to determine which Pax family members HIRA might interact with (see figure 5). Pax7 is placed in a paralogous group with Pax3 based on its similar structure (see section 1.5.1), and was the only other Pax protein identified as a HIRA-interactor in the initial yeast two-hybrid

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