INTRODUCTION

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The transcription of eukaryotic genes is predominantly controlled through the assembly of transcription factors into macromolecular complexes on cis-acting DNA target sequences. The frequency with which multicomponent regulatory complexes (sometimes referred to as enhancesomes) are used predicts the existence of specialized proteins that regulate gene expression by coordinating the recruitment and ordered assembly of the various components into an active complex. One example of this new class of regulatory protein is host cell factor 1 (HCF-1; also known as C1 factor). First identified through its role in assembly of the VP16-induced complex (VIC), a multiprotein-DNA complex that coordinates the activation of the herpes simplex virus immediate-early genes (31, 41), HCF-1 is likely to perform a similar function in the assembly of cellular transcription complexes.

VP16 (also known as Vmw65 or TIF) is a potent transcriptional activator encoded by herpes simplex virus and packaged into the infective virion particle. Once released into the newly infected cell, VP16 binds directly to HCF-1, allowing translocation to the nucleus and interaction with the POU domain transcription factor Oct-1 and a DNA sequence element known as the TAATGARAT motif that is found upstream of each viral immediate-early gene. VIC assembly is highly selective and is achieved through a combination of specific protein-protein and protein-DNA contacts (12, 14, 23, 32, 39, 44). Both HCF-1 and Oct-1 belong to multiprotein families, and VP16 has evolved mechanisms to recruit single members of each family. VP16 can distinguish Oct-1 from other POU proteins, including the very similar Oct-2, through recognition of differences on the solvent-exposed surface of the POU homeodomain (23, 32). Similarly, VP16 preferentially targets HCF-1 rather the closely related protein HCF-2 through recognition of a limited number of amino acid differences in blades 5 and 6 of the -propeller domain (14). Once the VIC is formed, the carboxy-terminal activation domain of VP16 activates transcription by recruiting coactivators (42) and by making direct contacts with components of the general transcription machinery (16, 43).

The function of HCF-1 is poorly understood but is likely to yield information of general significance. HCF-1 comprises a series of polypeptides derived from a >2,000-amino-acid precursor through proteolytic processing (19, 48). Cleavage occurs at a series of six centrally located 26-amino-acid repeats (called HCF_PRO repeats) producing multiple amino- and carboxy-terminal fragments that remain tightly, but noncovalently, associated following cleavage (49). VP16 interacts with a discrete amino-terminal domain (HCF_VIC) composed of six kelch-like repeats that fold into a six-bladed -propeller. The HCF-1 -propeller is in itself sufficient for VIC assembly in vitro and in vivo (13, 34, 47), although the carboxy terminus of HCF-1 contributes to the efficiency of complex formation and to translocation of VP16 to the nucleus (21, 22).

HCF-1 is expressed in all mammalian cell types and is essential for cell proliferation (reviewed in reference 14). The role in cell cycle progression was demonstrated through studies of tsBN67 cells, a temperature-sensitive hamster cell line that undergoes a G₀/G₁ arrest at the nonpermissive temperature (39.5°C) (8). The cell cycle arrest is reversible, and cells will reenter the proliferative cycle if returned to the permissive temperature (33.5°C). The tsBN67 phenotype is due to a single proline-to-serine change in the -propeller domain of HCF-1 (8, 47). The mutation apparently does not alter the stability or processing of HCF-1 but prevents association with VP16. The fact that a single mutation in HCF-1 could disrupt both known functions (transactivation by VP16 and cell proliferation) led to the idea that VP16 may have copied a preexisting interaction between HCF-1 and an unknown cellular protein involved in G₁ progression (5, 8, 47). This hypothesis is supported by the strong conservation of the amino acid sequence of the -propeller during evolution (14, 27) and the fact that HCF from invertebrates such as insects and nematodes can readily support VIC formation (18, 27, 46).

To date, the only known cellular target of the HCF-1 -propeller is a ubiquitous basic leucine zipper transcription factor known as LZIP or Luman (5, 28). Mammalian LZIP and a related protein from Drosophila, called dCREB-A or BBF-2, interacts with HCF-1 through a short tetrapeptide motif known as the HCF-binding motif (HBM) that is also found in VP16 (5, 29). The interaction can be disrupted by point mutations in the HBM (5, 9, 29) or by competition with short peptides derived from VP16 that span the motif (10, 34, 50). Both LZIP and dCREB-A function as potent transcriptional activators (1, 29, 35), indicating that HCF-1 is likely to be involved in the regulation of cellular as well as viral transcription.

In this study, we used mutagenesis to define the surfaces on the HCF-1 -propeller involved in the association with VP16 and LZIP. Our results show that all six blades of the -propeller contribute to recognition of the relatively simple HBM. Surprisingly, we find major differences between LZIP and VP16 in terms of their sensitivities to individual HCF-1 mutants, implying a significant contribution by the nonconserved sequences flanking the HBM. In general, mutations that disrupt VIC assembly affect the HCF-1-VP16 interaction; however, we identify a mutation in the sixth blade of the -propeller that prevents complex formation without disrupting association with VP16. Last, our mutational analysis reveals a poor correlation between association with LZIP and ability to complement the tsBN67 proliferation defect, arguing that the HCF-1-LZIP interaction may not be required for G₁ progression.

	MATERIALS AND METHODS

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Plasmid construction and site-directed mutagenesis. Mammalian expression vectors encoding the wild-type and P134S mutant -propeller domains of HCF-1 (residues 1 to 380; pCGNHCF-1_N380 and pCGNHCF-1_N380P134S, respectively), the amino terminus of HCF-1 (residues 1 to 902; pCGNHCF-1_N902), VP16 (residues 5 to 411; pCGTVP16C), and (residues 1 to 154; pCGTLZIP) have been described previously (14, 47).

Transfections, coimmunoprecipitations, immunoblotting, and electrophoretic mobility shift assays. Human 293T cells were transfected with Lipofectamine (Life Technologies), using 20 µl lipid reagent per 6-cm-diameter dish. Whole cell and nuclear extracts were prepared after 24 h by lysing cells in high-salt lysis buffer (420 mM KCl, 10 mM Tris-HCl [pH 7.9], 5% glycerol, 0.25% NP-40, 0.2 mM EDTA, 0.5 mM phenylmethylsulfonyl fluoride, 0.2 mM sodium vanadate, 50 µM sodium fluoride, 1 mM dithiothreitol). Nuclei were extracted at 4°C for 30 min and removed by centrifugation. For immunoprecipitations, 100 µl of extract was incubated with 2.5 µl of antihemagglutinin (HA) antibody (12CA5)-coupled protein G-agarose beads at 4°C for 1 h. The beads were washed four times in 1 ml of wash buffer (200 mM KCl, 10 mM Tris-HCl [pH 7.9], 5% glycerol, 0.5 mM EDTA) before separation by sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE). Immunoblotting was performed by semidry transfer and detected by enhanced chemiluminescence (SuperSignal; Pierce). The HA antibody and T7 antibody (Novagen) were diluted 1:5,000 and 1:10,000, respectively.

Complementation of tsBN67 cells. Subconfluent tsBN67 cells were incubated at 33.5°C for 20 h and transfected with 1 µg of each HCF-1 expression vector together with 0.5 µg of pSV2neo, using Lipofectamine (Life Technologies). The DNA mixes were sterilized by ethanol precipitation prior to transfection. After 2 days at 33.5°C, transfected cells were split into two 15-cm-diameter dishes, and Geneticin (0.8 mg/ml) was included in the medium to select for stable transfectants. Following 1.5 to 2 weeks of incubation at 39.5°C, the plates were stained with crystal violet and colonies of proliferating cells were counted. In some cases, complementation was confirmed by subcloning individual colonies.

	RESULTS

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VP16 associates with an amino-terminal domain of HCF-1, called the HCF_VIC domain (13, 22, 34, 47) (Fig. 1A). Spanning approximately 380 residues, the HCF_VIC domain is comprised of six degenerate sequence repeats that were first identified in the Drosophila actin-associated protein Kelch and are referred to as HCF_KEL1 to HCF_KEL6 (47). A sequence alignment of the HCF_VIC domains from HCF-1, HCF-2, and Caenorhabditis elegans HCF is shown in Fig. 1B, illustrating the extensive sequence conservation across the domain (14, 27). By analogy to kelch repeat proteins of known structure, the HCF_KEL repeats are predicted to fold into four-stranded -sheets that stack pseudosymmetrically around a central axis, forming a structure known as a -propeller (2). This compact arrangement is decorated by loops of variable length clustered on one surface (36, 37, 45). We follow a standard nomenclature for mutants; for example, P134S indicates proline 134 changed to serine.

View larger version (60K): [in this window] [in a new window]   FIG. 1.   (A) Primary structure of HCF-1. The amino-terminal -propeller (HCFVIC) domain is shown as a shaded box. The eight HCFPRO repeats are located near the center of the polypeptide and represented by filled (functional) and open (nonfunctional) arrowheads. The first 902 residues of HCF-1 comprising the -propeller, amino-terminal self-association domain, and poorly defined basic region are required for complementation of the tsBN67 cell proliferation defect. (B) Alignment of the six kelch repeats that make up the -propeller domain of HCF-1 (h1), HCF-2 (h2) and C. elegans HCF (ce) (14, 27). The predicted -strands in each repeat unit are boxed. Residues that are conserved in two or more HCF proteins are shown in bold. The residues that have been mutated in this study are numbered and highlighted. , amino acids.

All six kelch repeats contribute to VIC formation. The tsBN67 cell cycle arrest phenotype results from a single proline-to-serine substitution at position 134 in the fourth residue in HCF_KEL3 (Fig. 1B). In addition to blocking G₁ progression, this single mutation abolishes the interaction of HCF-1 with VP16 and LZIP but does not alter the overall stability or folding of the -propeller domain itself (5, 8, 47). Remarkably, there is a proline residue at the equivalent position in each of the six kelch repeats of HCF-1, HCF-2 and C. elegans HCF (14, 27, 47), suggesting a critical role in domain function. By analogy to other -propeller domains, the universally conserved proline lies within a loop connecting the fourth strand (4) of one -sheet (or kelch repeat unit) to the first strand (1) on the next sheet. These 4-1 loops are positioned on the "top" surface of the structure, lining the central cavity, and are thus ideally positioned to contribute to protein-protein interactions.

View larger version (38K): [in this window] [in a new window]   FIG. 2.   Proline-to-serine substitutions at position 4 in each HCFKEL repeat. (A) HCF-1 polypeptides were coexpressed with VP16C by transfection of 293T cells. Extracts were prepared after 40 h and assayed for VIC formation in an electrophoretic mobility shift assay. The first three lanes are controls showing probe alone (lane 1), Oct-1 POU domain protein alone (lane 2), and cell extract transfected with 1 µg of VP16C (lane 3). Each HCF-1N380 expression plasmid (1 µg) was cotransfected with 0.1 and 1.0 µg of VP16C expression plasmid. The HCF-1N380 proteins used are indicated above the lanes. Positions of the free probe, Oct-1 POU domain complex, and VIC containing native (endogenous) human HCF-1 (endog.VIC) or truncated HCF-1 (mini-VIC) are indicated. (B) Coimmunoprecipitation of VP16. To measure the direct association between HCF-1N380 and VP16C, the extracts shown in panel A were subject to coimmunoprecipitation assay. Extracts were immunoprecipitated (IP) with an HA antibody (12CA5) coupled to agarose beads and resolved on an SDS-12% polyacrylamide gel, and coimmunoprecipitated VP16 was detected by immunoblotting with an T7 epitope tag antibody. Direct immunoblotting of the extracts (lower two panels) showed that each HA-tagged HCF-1 polypeptide was expressed at equivalent levels and that the expression of T7-tagged VP16 was proportional to amount of input plasmid. Irrelevant cross-reacting polypeptides are indicated with an asterisk.

The conserved arginine at position 7 in each HCF_KEL repeat is critical for activity. With the exception of HCF_KEL2, the amino acid residue at position 7 of each HCF_KEL repeat corresponds to an arginine residue, again implying a critical role in domain function. In HCF_KEL2 (or, interestingly, in the analogous position in HCF-2), this position is occupied by a cysteine residue. To address the importance of this semiconserved position with respect to association with VP16, we made a set of radical substitution mutants, replacing the residue at position 7 with an aspartic acid. We chose this substitution because C. elegans HCF uses an alanine at position 7 in HCF_KEL2 (27), suggesting that a noncharged residue may be more readily tolerated. Unfortunately, we were unable to generate a mutation at R33 in HCF_KEL1. Our analysis of these point mutants is shown in Fig. 3A. Exchanging the cysteine at position 82 for aspartic acid led to only a moderate reduction in complex formation (Fig. 3A, compare lane 6 with lane 8). In contrast, R137D (lane 9), R200D (lane 10), R255D (lane 11), and R322D (lane 12) disrupted VIC formation. The experiment shown in Fig. 3A and B used HCF-1_N380 polypeptides expressed by in vitro translation. Equivalent results were obtained for HCF-1 expressed by transient transfection, and we have chosen to show the in vitro translation experiment simply because P134S (lane 7) retains some activity when expressed in a cell-free system and is actually more active than R137D, R200D, R255D, and R322D. This implies that the arginine-to-aspartic acid substitution is extremely severe, perhaps resulting in a gross disruption of the -propeller structure.

View larger version (69K): [in this window] [in a new window]   FIG. 3.   Radical substitution of the conserved arginine residue at position 7 of each HCFKEL repeat has a severe effect on VIC formation. (A) Electrophoretic mobility shift assay of HCF-1 proteins expressed by in vitro translation. The first three lanes are controls showing probe alone (lane 1), Oct-1 POU domain protein alone (lane 2), bacterially produced glutathione S-transferase-VP16C (lane 3), or unprogrammed reticulocyte lysate (lane 4). In the remaining lanes, unprogrammed lysate (lane 5) or lysates expressing recombinant HCF-1N380 polypeptides were mixed with glutathione S-transferase-VP16C (lanes 6 to 12). The HCF-1N380 proteins used are indicated above the lanes. Positions of the free probe, Oct-1 POU domain complex, and VIC containing rabbit HCF-1 from the lysate (endog.VIC) or truncated HCF-1N380 (mini-VIC) are indicated. A nonspecific complex is indicated with an asterisk. (B) In vitro translation products were resolved on an SDS-12% polyacrylamide gel and detected by fluorography. (C) Electrophoretic mobility shift assay. Extracts were prepared from transfected 293T cells expressing wild-type or mutant versions of HCF-1N380 together with 0.01 µg (lanes 4, 7, and 10), 0.1 µg (lanes 5, 8, and 11), and 1.0 µg (lanes 6, 9, and 12) of VP16C expression plasmid. The HCF-1N380 proteins used are indicated above the lanes. (D) The extracts shown in panel C were used in a coimmunoprecipitation assay as described for Fig. 2B. The upper panel shows the HA-immunoprecipitated (IP) proteins probed with T7 antibody; the lower two panels show direct immunoblots of the extracts. Each HCF-1N380 expression plasmid (1 µg) was cotransfected with 0.01 µg (lanes 2, 5, and 8), 0.1 µg (lanes 3, 6, and 9), and 1.0 µg (lanes 4, 7, and 10) of VP16C expression plasmid. The transfections were as follows: VP16 alone (1.0 µg, lane 1), wild-type HCF-1N380 (lanes 2 to 4), P134S (lanes 5 to 7), and C82D (lanes 8 to 10).

Residues within the 2-3 loops also contribute to VIC assembly. Next we examined the role of the 2-3 loops (connecting 2 to 3), which also contribute to the predicted top surface of the -propeller. In HCF-1, the 2-3 loops show relatively little sequence homology to each other and also differ significantly in length (Fig. 1B). To determine whether the 2-3 loops contribute to interaction with VP16, we generated substitutions in the 2-3 loops of HCF_KEL2 (K105D), HCF_KEL4 (R228D), HCF_KEL5 (EWK289A3), and HCF_KEL6 (RK344A2). Each mutant was expressed in transfected 293T cells and assayed for association with VP16 by the electrophoretic mobility shift and coimmunoprecipitation assays (Fig. 4). Substitutions in HCF_KEL2 (K105D) and HCF_KEL5 (EWK289A3) had a minor affect on both VIC assembly (Fig. 4A, lanes 8, 9, 12, and 13) and association with VP16 (Fig. 4B, lanes 6, 7, 10, and 11), suggesting that these residues do not make a significant contribution to complex assembly. In contrast, mutation in HCF_KEL4 (R228D) and HCF_KEL6 (RK344A2) disrupted VIC assembly (Fig. 4A, lanes 10, 11, 14, and 15). This result indicates that residues in the 2-3 loops of HCF_KEL4 and HCF_KEL6 are critical for VIC assembly. In contrast to a previous report (34), the K105D mutation had only a minor effect on VIC assembly.

View larger version (36K): [in this window] [in a new window]   FIG. 4.   Residues within the 2-3 loops also participate in association with VP16. (A) HCF-1 polypeptides were expressed with VP16C by cotransfection of 293T cells and assayed for VIC formation. The first three lanes are controls showing probe alone (lane 1), Oct-1 POU domain protein alone (lane 2), and cell extract transfected with 1.0 µg of VP16C (lane 3). Each HCF-1N380 expression plasmid (1.0 µg) was cotransfected with 0.1 and 1.0 µg of VP16C expression plasmid. The HCF-1N380 proteins used are indicated above the lanes. (B) Coimmunoprecipitation assay using extracts from panel A. The upper panel shows the HA-immunoprecipitated proteins (IP) probed with T7 antibody; the lower two panels show direct immunoblots of the extracts. The samples were as indicated above the lanes.

View larger version (42K): [in this window] [in a new window]   FIG. 5.   RK344A2 associates with VP16 and yet fails to support VIC formation. (A) Transfected 293T cells extracts were prepared and assayed by electrophoretic mobility shift assay as described for Fig. 2. The first three lanes are controls showing probe alone (lane 1), Oct-1 POU domain protein alone (lane 2), and cell extract transfected with 1.35 µg of VP16C (lane 3). Each HCF-1N380 expression plasmid (1.0 µg) was cotransfected with 0.05, 0.15, 0.45, or 1.35 µg of VP16C expression plasmid. The HCF-1N380 proteins used are indicated above the lanes. (B) Coimmunoprecipitation assay using extracts from panel A. The upper panel shows the HA-immunoprecipitated (IP) proteins probed with T7 antibody; the lower two panels show direct immunoblots of the extracts. The samples are as follows: VP16 alone (lane 1) or increasing amounts of VP16 expression plasmid cotransfected with wild-type HCF-1N380 (lanes 2 to 5), K105D (lanes 6 to 9), and RK344A2 (lanes 10 to 13).

View larger version (41K): [in this window] [in a new window]   FIG. 6.   The RK344A2 mutation interferes with coimmunoprecipitation of Oct-1. Full-length Oct-1 and HA-tagged HCF-1N380 were expressed in vitro and mixed, and coassociation was assayed by coimmunoprecipitation using HA antibody beads. Immunoprecipitates (IP) were fractionated by SDS-PAGE (10% gel), and radiolabeled proteins detected by fluorography (upper panel). The input translations (prior to mixing) are shown in the lower panel.

VP16 and LZIP show different sensitivities to individual point mutants in the HCF-1 -propeller. VP16 and LZIP contain a tetrapeptide sequence (EHAY in VP16 [residues 361 to 364] and DHTY in LZIP [residues 78 to 81]), HBM, that is essential for association with HCF-1 (5, 29). In both VP16 and LZIP, individual mutation of the acidic, histidine, or tyrosine residue was sufficient to prevent association with HCF-1 (5, 29). This similarity suggested that the two proteins interact with HCF-1 equivalently. To examine this further, we used a cell-based recruitment assay to compare the sensitivities of VP16 and LZIP binding to each of the HCF_VIC domain mutants (Fig. 7). We used this recruitment assay because LZIP is expressed at very low levels in transfected cells, making it difficult to quantitate association with cotransfected HCF-1 mutants by coimmunoprecipitation. The HCF-1 -propeller domain was expressed in 293T cells as a Gal4 fusion together with either VP16 (residues 5 to 490) or the amino terminus of LZIP (residues 1 to 154). Recruitment was measured in terms of activation of a Gal4-responsive luciferase reporter gene and the amounts of each expression plasmid chosen to give a linear response (data not shown). The recruitment of VP16 was generally consistent with the results of the immunoprecipitation assay described above. The one exception was P30S, which immunoprecipitated VP16 relatively efficiently (Fig. 2B, lanes 3 and 4) but failed to interact in the recruitment assay. This result was highly reproducible, and the reason for this single inconsistency is unclear.

View larger version (23K): [in this window] [in a new window]   FIG. 7.   Individual mutations in the HCFVIC domain have different effects on association with VP16 and LZIP. A cell-based recruitment assay was performed with transiently transfected 293T cells. A Gal4-responsive luciferase reporter gene (p5xGal-E1B-luc) was transfected into 293T cells by electroporation together with 1 µg of wild-type or mutant version of pCGNGal(1-94)HCF-1N380 and 500 ng of pCGTVP16C or pCGTLZIPN154, as indicated. The values are the average of three independent transfections, and the standard deviation from the mean is indicated by error bars. Fold activation was calculated relative to the activity of wild-type pCGNGal(1-94)HCF-1N380 cotransfected with pUC119.

Limited correlation between complementation of the tsBN67 proliferation defect and association with VP16 or LZIP. Inactivation of the HCF_VIC domain in tsBN67 cells leads to a G₁/G₀ cell cycle arrest. This defect can be complemented by stable expression of an amino-terminal fragment (residues 1 to 902) comprising the HCF_VIC domain, amino-terminal self-association domain (HCF_SASN), and basic region (Fig. 1A) (8, 14, 47). The association of LZIP with HCF-1 is prevented by the tsBN67 mutation, suggesting that LZIP is a candidate target of HCF-1 in controlling cell proliferation (5). To address this, we asked whether there is a close correlation between the ability of HCF_VIC to interact with LZIP and to support the growth of tsBN67 cells at the nonpermissive temperature. Each mutant was constructed in an expression plasmid encoding the amino-terminal 902 residues of HCF-1 (pCGNHCF-1_N902) and transfected into tsBN67 cells together with a selectable marker. After nearly 2 weeks at the nonpermissive temperature, the number of proliferating (or complemented) colonies was determined. Under the conditions used, wild-type HCF-1_N902 gave a large number of colonies (~120 to 200/dish), whereas an empty vector produced no more than one or two revertant colonies. Representative plates are shown in Fig. 8A, and complementation by each of the mutants is summarized in Fig. 8B. These fell into two categories: those capable of supporting cell proliferation and those that were incapable. Mutations at positions 4 (P30S, P79S, P134S, P197S, P252S, and P319S) and 7 (C82D, R137D, R200D, R255D, and R322D) of each HCF_KEL repeat abolished complementation. Although complementation appears to be an all-or-none event, subcloning reveals small differences in the relative growth rates of rescued colonies, presumably reflecting differences in the capability of individual mutations to promote G₁ progression (data not shown).

View larger version (34K): [in this window] [in a new window]   FIG. 8.   K105D, EWK289A3, and RK344A2 complement the tsBN67 cell proliferation defect. (A) Hamster tsBN67 cells were stably transfected with 1 µg of pCGNHCF-1N902 wild type, P134S, K105D, EWK298A2, and RK344A2. Following transfection, cells were incubated at 39.5°C with G418 for 2 weeks, and proliferating (rescued) colonies were stained with 0.5% crystal violet. (B) Quantitation of the number of rescued colonies after 2 weeks of selection at the nonpermissive temperature. Numbers are expressed relative to the value for wild-type HCF-1 (pCGNHCF-1N902).

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

In this study, we have used mutagenesis to probe the structure of the HCF-1 -propeller domain and have identified a number of mutants that disrupt the three known functions of the domain: VIC assembly, recruitment of HCF-1 to the cellular transcription factor LZIP, and complementation of the tsBN67 proliferation defect. The properties of each mutant (summarized in Table 1) can be grouped into three functional categories: inactive, partially active, and fully active. Eight of the mutants (P134S, P197S, P319S, R137D, R200D, R255D, R228D, and R322D), including the previously characterized tsBN67 mutant (P134S), were inactive for all three functions. This may indicate a shared role for each of these evolutionarily conserved residues or that these substitutions simply bring about a global change in domain structure. Separating these two options will require further analysis; however, the fact that all of these mutants were expressed at near-wild-type levels and were equally soluble argues against a global defect in folding of the -propeller.

View this table: [in this window] [in a new window]   TABLE 1.   Phenotypes associated with amino acid substitutions in the -propeller domain of HCF-1

Of the remaining eight mutants, two (K105D and S338A) showed little or no phenotype, while six (P30S, P79S, C82D, P252S, EWK289A2, and RK344A2) showed differential effects on each of the functions tested. This latter class of mutants is likely to be the most informative. Four mutants (P79S, C82D, EWK289A3, and RK344A2) showed significant differences in the ability to interact with LZIP compared to VP16. This was most striking in EWK289A3, which was similar to wild type for association with VP16 but inactive for association with LZIP. These results point to an important role for residues flanking the core HBM tetrapeptide. It should be noted that while mutagenesis of VP16 implicates three aspartic acids (residues 385 to 387) on the carboxy-terminal side of the HBM (361 to 364) in HCF-1 binding (24), both LZIP and its Drosophila homologue dCREB-A/BBF-2 lack an equivalent acidic stretch.

Association of HCF-1 with VP16 is not sufficient for VIC formation. HCF-1's role in VIC formation is poorly understood. HCF-1 is not essential for specific recognition of Oct-1 or the TAATGARAT element by high levels of VP16 in vitro (38, 44) but is required for VIC formation at lower protein concentrations and in vivo (8, 24). Deletion and mutagenesis studies of VP16 have mapped the primary determinants for interaction with Oct-1, HCF-1, and the TAATGARAT element to a small region between residues 331 and 391 of VP16 (9, 11, 24, 38). Although this region is unstructured in the VP16 crystal structure, it is thought that part of this loop may fold as an amphipathic -helix, capable of packing against helices 1 and 2 on the exposed surface of the Oct-1 homeodomain (25, 26). HCF-1 may facilitate the Oct-1-VP16 interaction by stabilizing this -helix (24). The predicted -helix (VP16 residues 376 to 387) lies between the HBM and three aspartic acid residues implicated in HCF-1 association (24). By clasping each end of the recognition -helix or by constraining the loop within a narrow groove, it seems reasonable to imagine that HCF-1 stabilizes the more ordered conformation and thus facilitates VIC formation.

The -propeller as a molecular work surface. -Propeller proteins perform a remarkable variety of functions (reviewed in reference 37). Some have enzymatic activity, while others serve as scaffolds upon which protein-protein interactions are built. Perhaps as a result of this inherent flexibility, -propeller-containing proteins are implicated in such diverse functions as protein trafficking (40), signal transduction (6), regulation of chromatin structure (30), transcriptional activation, and transcriptional repression (4, 15, 17). One emerging theme that unites these different examples is the ability of a single -propeller domain to associate with a variety of target molecules (17, 33, 40). Because the -propeller fold does not require a strict sequence pattern, it may be particularly amenable to the evolution of shallow grooves capable of trapping solvent-exposed flexible arms that are presented by partner proteins. At the same time, the compact nature of the domain provides an opportunity for regulation, either by steric hindrance or through the ability to bring prospective partners into close proximity to each other. For instance, a number of different regulatory cofactors interact with the -propeller subunit of heterotrimeric G proteins and use steric hindrance to prevent the GDP-binding G subunit from accessing its binding site on the -propeller (3, 6). The HCF_VIC domain provides a good example of how the compact architecture of the -propeller fold can be used to promote protein-protein interactions. Thus, HCF-1 is able to regulate viral immediate-early gene transcription, and ultimately the viral life cycle, by simply enabling VP16 and Oct-1 to come together on the TAATGARAT element.

The HCF-1 -propeller serves as an interaction site for multiple cellular components. The results presented in this study argue that HCF-1 does not need to recruit LZIP in order to promote G₁ progression. This is best illustrated by the behavior of mutants P252S and EWK289A3. Although the P252S mutation can still interact with LZIP (~80% of the wild-type level [Fig. 7]), it is unable to overcome the tsBN67 block to cell proliferation. One interpretation of this result is that the P252S mutation effects an undescribed interaction with another cellular component necessary for cell cycle progression. The behavior of EWK289A3 also brings into question the relevance of LZIP to understanding HCF-dependent proliferation: this mutant is able to complement the tsBN67 proliferation defect and yet appears to be severely impaired for association with LZIP. This observation argues that the HCF-1-LZIP interaction must not be essential for tsBN67 cell growth. While it is conceivable that on natural target promoters the weakened association between EWK289A3 and LZIP is stabilized through additional protein-protein or protein-DNA interactions, we favor the hypothesis that the cell cycle arrest reflects the disruption of an interaction between the HCF-1 -propeller and an additional cellular protein. To this end, we have recently identified an unrelated cellular polypeptide that interacts with the HCF-1 -propeller mutants in a manner more closely paralleling the results of the tsBN67 complementation assay. Specifically, the new candidate interacts in a yeast-based assay with EWK289A3 and RK344A2 but not with P252S or P134S (S. S. Mahajan, M. D. Little, and A. C. Wilson, unpublished data). Its role in promoting cell proliferation is currently under investigation.