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In this study a specific biochemical interaction between HIRA and the DNA- binding transcription factor Pax3 has been demonstrated. This interaction is mediated through the C-terminal region o f HIRA excluding the WD40 repeats, and the Pax3 homeodomain. Domain mapping using GST affinity capture assays has failed to narrow down the interacting region o f the homeodomain to a particular a-helix (section 3.2.1). This suggests that HIRA may make contacts with residues throughout the homeodomain, and/or may require the homeodomain to be in a specific structural conformation, requiring the presence o f all three a-helices, for stable interaction with Pax3. This result contrasts with that o f other Pax3-interactors for which specific regions o f the Pax3 homeodomain have been implicated in the interaction. For example, Pax3 interacts with the product o f the retinoblastoma gene pRB, and the related protein p i 07, through a-helices I and II o f the homeodomain, an interaction unaffected by deletion o f a-helix III (Wiggan et a l, 1998). Similarly, Pax3 has been demonstrated to bind the co­ repressor hDaxx, an interaction mediated primarily through a-helix III (Hollenbach et al., 1999). This requirement of a complete homeodomain structure for HIRA interaction is supported by the fact that only those Pax proteins that contain such a homeodomain appear to bind HIRA.

In addition to Pax proteins, and consistent with the evidence that HIRA can bind homeodomain-containing proteins, Otx2 was also isolated in a yeast two-hybrid screen. Although this interaction has not been studied in great detail, in vitro analysis indicates that, as for Pax3, the interaction is mediated through the C-terminal region o f HIRA, and that it is likely that the Otx2 homeodomain is critical (section 3.3.1). The homeodomain is the only region common to both Pax3 and Otx2 and shows significant similarity between the two proteins along its length.

Two independent yeast two-hybrid screens identified core histones H2B, H3 and H4 as HIRA interactors (Magnaghi et al., 1998; Lorain et al., 1998). This result has been confirmed by both GST affinity capture assay and Far W estern analysis (R. Buckle, unpublished data). Interaction o f HIRA with H4 is mediated through the second a-helix (Lorain et al., 1998) which lies in the central globular domain o f the histone, a region not exposed when histones are assembled into a nucleosomal structure. This suggests HIRA binds either to free histone H4, or to H4 within an abnormal nucleosomal structure, for example unfolded by the SWI/SNF remodelling complex. Consistent with this is the observation that GST HIRA is unable to bind to a reconstituted nucleosome or core particle (G. Almouzni, personal communication). Histone H2B, however, interacts with HIRA through its N terminus (Lorain et al.,

1998), a region that extends from the histone octamer core o f an assembled nucleosome. HIRA could therefore bind to H2B whether free or within a nucleosome. H2B and H4 have been shown to interact with HIRA through overlapping but distinguishable domains in the C-terminal region o f HIRA (Lorain et al., 1998). The H3 interaction with HIRA has also been mapped to the C-terminus o f HIRA (R. Buckle, unpublished data), but as yet the H3 domain required is unknown.

The identification o f proteins with which HIRA interacts, as well as its similarity to other proteins, provide clues as to the possible function o f HIRA. HIRA may act as a core histone chaperone, suggested by its similarity over the WD40 repeat region to the p60 subunit o f human Chromatin assembly factor 1 (CAF-1), and the interaction o f HIRA with core histones. CAF-1 binds newly synthesised, acetylated histones H3 and H4 in the cytoplasm, then deposits them at sites o f DNA replication (Smith and Stillman, 1991; Kaufrnan et al., 1995; Verreault et a l, 1996). HIRA may play a similar role in the assembly o f histones onto DNA. Alternatively HIRA may function in the disassembly o f nucleosomes. HIRA is capable o f binding histones, and thus could facilitate the destabilisation or removal o f nucleosomes from DNA. This may be as part o f its function as a transcriptional regulator for example, working in conjunction with chromatin remodelling factors to allow increased transcription factor binding to DNA or to enhance or maintain open chromatin by removal o f histones from active regions.

HIRA is presumed to be the homologue o f the yeast HIR proteins, which act as transcriptional co-regulators o f histone gene transcription during the cell cycle (Sherwood et al., 1993; Spector et a l, 1997). It is proposed that they function by remodelling chromatin structure, since H IR l and HIR2 interact with components o f the SWI/SNF complex, and HIR2 with core histones (Dimova et a l, 1999). Like HIRA, the HIR proteins have no obvious RNA- or DNA-binding motifs and are proposed to be targeted to promoters via as yet unidentified DNA-binding factors. In many respects HIRA also resembles the yeast TUPl transcriptional co-repressor, which is recruited as a complex (TUP1/SSN6) to target promoters via interaction with transcription factors, including homeodomain-containing proteins (for example a 2 , Komachi et a l, 1994). Like HIRA and the HIR proteins, TUPl contains WD40 repeats. It interacts with core histones (H3 and H4) through a domain outside o f its WD40 repeats, and is thought to repress transcription through modulation o f chromatin structure and/or interactions with the general transcriptional machinery (reviewed by Smith and Johnson, 2000). Whilst the homology o f HIRA with the yeast HIR proteins suggests a role for HIRA in the regulation o f histone synthesis, the dynamic expression pattern o f HIRA and similarity to other proteins such as TUPl suggest it may act as a more global regulator o f transcription via transcription factor interactions. These observations suggest a model for HIRA function whereby HIRA-containing complexes could be recruited to target gene promoters through interactions with homeodomain transcription factors such as Pax3 and Otx2. Once there, HIRA could modulate transcriptional activity either through modification o f chromatin surrounding the target gene, as suggested by its interactions with histones, or by direct action on RNA polymerase II.