INTRODUCTION

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Lytic infection of human cells by herpes simplex virus is characterized by a cascade of viral gene expression initiated by the virion protein VP16 (reviewed in reference 22). Following infection of the host cell, the viral transactivator VP16 (also known as Vmw65 and TIF) is released from the virion, whereupon it associates with two preexisting nuclear factors, Oct-1 and HCF-1, to form a multiprotein-DNA complex on VP16-responsive cis-regulatory elements in the viral immediate-early promoters (8).

Oct-1 is a broadly expressed POU homeodomain-containing transcription factor (24). The function of HCF-1 (also known as HCF, C1, VCAF, and CFF) in the uninfected cell is less well understood. The principal HCF-1 translation product is a large protein of 2,035 amino acids (12, 27), but only an amino-terminal region of about 380 amino acids is essential for HCF-1 interaction with VP16 and stabilization of the VP16-induced complex with Oct-1 (10, 15, 26). Characterization of a temperature-sensitive hamster cell line called tsBN67 has indicated that HCF-1 is involved in cell proliferation (7): at nonpermissive temperature, tsBN67 cells stop proliferating owing to a missense mutation within the HCF-1 VP16 interaction region, which also inhibits HCF-1 association with VP16 (7, 26). The molecular mechanisms, however, by which HCF-1 regulates cell proliferation are not known.

HCF-1 is a member of a protein family that includes the recently described human protein HCF-2 (11), and both HCF-1 and HCF-2 are related to a protein in the worm Caenorhabditis elegans called CeHCF (16). These three proteins have conserved the VP16 interaction region and associate with VP16 to differing extents (11, 16).

In mammalian cells, HCF-1 undergoes an unusual maturation process. Its primary translation product is cleaved at a series of six conserved 26-amino-acid repeats called HCF_PRO repeats located near the center of the primary translation product (12, 27, 29). The resulting fragments, called HCF-1_N and HCF-1_C, are stable, and they remain noncovalently associated, creating a mature HCF-1 complex (29). Although the majority of HCF-1_N and HCF-1_C subunits remain noncovalently associated after proteolysis, there is a variant HCF-1 translation product called HCF-1_382-450, which results from removal of an exon encoding amino-terminal HCF-1 residues 382 to 450 through alternative pre-mRNA processing; this smaller HCF-1 protein undergoes proteolytic processing, but the resulting HCF-1_N and HCF-1_C subunits do not remain associated (29). Unlike the HCF-1 protein, HCF-2 and CeHCF do not possess HCF_PRO repeats, and HCF-2 is not proteolytically cleaved like HCF-1 (11).

HCF-1 processing and the subsequent association of the resulting HCF-1_N and HCF-1_C subunits are unusual, and little is known concerning the mechanisms by which they occur. Here, we describe aspects of human HCF-1_N and HCF-1_C subunit association. Our results show that the HCF-1_N and HCF-1_C subunits contain two matched pairs of subunit association elements and that subunit association can permit recruitment of HCF-1_N subunits to the nucleus via a nuclear localization signal (NLS) located at the carboxyl terminus of the HCF-1_C subunit. Unexpectedly, HCF-2, which is not proteolyzed, shares one of the HCF-1 subunit association elements, suggesting that the role of such an element is not solely to maintain HCF-1_N and HCF-1_C subunit association.

	MATERIALS AND METHODS

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Mammalian expression plasmids. The hemagglutinin (HA)-epitope-tagged human HCF expression constructs pCGNHCF_N1011382-450, pCGNHCF_N450-1011, pCGNHCF_N450, and pCGNHCF_N380 have been described previously (26). pCGTHCF_C (C-terminal residues 1436 to 2035) is identical to pCGNHCF_C (26) except that the influenza virus HA epitope is replaced by a bacteriophage T7 gene 10 (T7) epitope. To ensure detection and protein stability, HCF-1 residues 348 to 450 or smaller derivatives were expressed as fusions to the GAL4 DNA-binding domain (residues 1 to 94) by subcloning into pCGNGAL4(1-94). pCGTHCF_SAS2C encodes HCF-1 residues 1436 to 1756, and pCGTHCF_SAS1C encodes residues 1758 to 2035. Truncations were generated either by using suitable restriction sites or by PCR or oligonucleotide-mediated mutagenesis. A fragment spanning residues 341 to 394 of human HCF-2 (11) was amplified using PCR and subcloned into pCGNGAL4(1-94). The nucleotide sequences of PCR-generated fragments were verified by DNA sequencing.

Sequence analysis. Searches of the protein databases were performed using BLASTP (1) and SMART (Simple Modular Architecture Research Tool) (23). Sequence alignments were compiled using ZEGA and CLUSTALW (provided on-line by the Molecular Modeling and Bioinformatics Group, Skirball Institute, New York University School of Medicine).

Transfections, immunoprecipitations, and immunoblotting. Human 293 and 293T cells were transfected by electroporation or with Lipofectamine (Gibco BRL, Inc.), respectively (11, 26). Preparation of whole-cell nuclear extracts, immunoprecipitation, and immunoblotting were performed as described previously (26).

Immunofluorescence. 293 cells were transfected by electroporation and seeded onto sterile coverslips. After 36 h, the coverslips were washed in phosphate-buffered saline (PBS); the cells were fixed in 4% paraformaldehyde for 20 min and permeabilized for 5 min with PBS containing 0.1% Triton X-100. The samples were then washed three times with PBS and blocked for 30 min in PBS containing 2% dry milk. After washing in PBS, coverslips were incubated for 45 min with the primary antibody, washed, and incubated with the secondary antibody for an additional 45 min. Fluorescence was observed with a Nikon fluorescence microscope or a Zeiss confocal microscope.

	RESULTS

Top Abstract Introduction Materials and Methods Results Discussion References

Figure 1A shows a schematic of the structure of the primary human HCF-1 translation product called HCF-1₃₀₀. HCF-1₃₀₀ contains two previously described sets of repeat elements: centrally located HCF-1_PRO repeats and six amino-terminal kelch-like repeats called HCF-1_KEL, which likely fold into a six-bladed -propeller structure (residues 17 to 360 [15, 26]). Here, we analyzed the sequence of the carboxy-terminal region of HCF-1, which was originally described as rich in charged residues and the large hydrophobic residues tryptophan, tyrosine, and phenylalanine (27). This region has been conserved in evolution: both HCF-2 (11) and CeHCF (16) share extensive sequence similarity to residues 1812 to 2001 of HCF-1 (Fig. 1B). This region also exhibits internal sequence similarity, which indicates a repeat of HCF-1 residues 1812 to 1875 and 1910 to 1992. These two sequences share a conserved tryptophan (W) residue (positions 1812 and 1910) and the interdigitated sequence FRXXXXNXXGXG, where "X" indicates any amino acid (residues 1864 to 1875 and 1981 to 1992 [Fig. 1B]).

View larger version (37K): [in this window] [in a new window]   FIG. 1.   HCF-1 contains two Fn3-like repeats. (A) Structural features of human HCF-1 (hHCF-1). The six near-perfect HCF-1PRO repeats (filled arrowheads) and two degenerate nonfunctional repeats (open arrowheads) are indicated. The amino-terminal -propeller domain involved in association with VP16 is represented as a shaded box. A carboxy-terminal domain rich in charged or bulky hydrophobic residues (tryptophan, tyrosine, and phenylalanine) is shown in greater detail. This region contains two putative Fn3 repeats (HCF-1Fn31 and HCF-1Fn32), indicated by filled arrows. A bipartite NLS (filled box) lies at the very carboxyl terminus (13). Regions with overall basic or acidic charge are also indicated. The region 382-450 is deleted in a natural form of HCF-1 that results from alternative splicing of the HCF-1 transcript. (B) Alignment of the tandem HCF-1Fn31 and HCF-1Fn32 repeats from the carboxy termini of HCF-1, HCF-2, and CeHCF. The six perfectly conserved positions (invariant residues) are highlighted in black, while residues that are identical in each of the three HCF proteins are shaded. Small arrows denote the limits of the HCF-1SAS1C element; dots over the HCF-1 sequence indicate 10-amino-acid segments. (C) Alignment of the four Fn3 repeats from the intracellular domain of integrin 4. The positions of  strands A through G2 (open arrows) are derived from the crystal structure of the tandem Fn3-1 and Fn3-2 modules from integrin 64 (3). In close agreement with other Fn3 structures,  strands A, B, and E and  strands C, C', F, G1, and G2 form two  sheets that pack as a  sandwich, enclosing a hydrophobic core (2). The invariant residues identified in HCFFn31 and HCFFn32 (shown below the sequences) are to a large extent conserved in all four modules of integrin 4.

A comparison of each carboxy-terminal repeat with other protein sequences using the BLASTP program (1) and also the SMART database (23) revealed significant similarity to fibronectin type 3 (Fn3) repeats. Fn3 repeats are typically 90 to 100 residues long and, although exhibiting relatively low primary amino acid sequence similarity among different proteins, display considerable structural similarity (reviewed in reference 2). Figure 1C shows a sequence alignment of four Fn3 repeats from the ₄ subunit of ₆₄ integrin. The first two (Fn3-1 and Fn3-2) and second two (Fn3-3 and Fn3-4) represent tandem repeats as with the two repeats in HCF-1. The structure of the first set of tandem repeats has been determined (3), and the positions of  strands in the structure are shown in Fig. 1C. Together, Fig. 1B and C show the similarity of the two carboxy-terminal HCF-1 repeats to Fn3 repeats. The predicted Fn3 repeats in HCF-1 have also been identified independently by the SMART database (http://smart.embl-heidelberg.de). These sequence analyses identify for the first time a predicted structural element within the carboxy-terminal region of HCF-1. We therefore refer to these carboxy-terminal HCF-1 repeats as HCF-1_Fn31 and HCF-1_Fn32.

The HCF-1_N and HCF-1_C subunits readily associate when synthesized separately. In the mature endogenous HCF-1 complex, the noncovalently associated HCF-1_N and HCF-1_C subunits result from proteolytic processing of the HCF-1₃₀₀ precursor (29); thus, the HCF-1_N and HCF-1_C subunits are in close proximity for coassociation prior to proteolytic cleavage. HCF-1_N and HCF-1_C subunit association might, however, be regulated through sequential dissociation and reassociation. To test the feasibility of such a hypothesis, we asked whether dissociated HCF-1_N and HCF-1_C subunits can associate without prior tethering as a single translation product.

View larger version (47K): [in this window] [in a new window]   FIG. 2.   HCF-1 contains two N-terminal domains that can mediate association with the C terminus. Extracts were prepared from 293 cells transfected with 5 µg of expression plasmids encoding HA-tagged derivatives of the HCF-1N subunit and with 2 µg of expression plasmids encoding a T7 epitope-tagged version of either VP16C (VP16) or the HCF-1C subunit. HA-tagged polypeptides were recovered by immunoprecipitation with an HA monoclonal antibody, resolved on an SDS-8% polyacrylamide gel, and immunoblotted with an T7 tag antibody (Novagen) to detect coimmunoprecipitated VP16 or HCF-1C protein. The HA-tagged polypeptides were as follows: mock (lanes 1 to 3), HCF-1N1011 (lanes 4 and 5), HCF-1N1011382-450 (lanes 6 and 7), HCF-1N450 (lanes 8 and 9), HCF-1N450-1011 (lanes 10 and 11), and HCF-1N380 (lanes 12 and 13). The structures of HCF-1N and its derivatives used for coimmunoprecipitation are shown schematically below. The ability (+) or inability () of each amino-terminal fragment to interact with VP16 or HCF-1C is indicated.

The HCF-1_N subunit contains two HCF-1_C subunit association regions. We have previously shown that the natural HCF-1 variant HCF-1_382-450 (Fig. 1A) is processed normally, but the resulting HCF_N382-450 and HCF_C subunits do not remain associated (29). Consistent with this observation, when synthesized separately as shown in Fig. 2, an HCF-1_N subunit lacking residues 382 to 450 but retaining the VP16 interaction region (HCF-1_N1011382-450) bound VP16 (lane 6) but not the engineered HCF-1_C subunit (lane 7), suggesting that loss of residues 382 to 450 disrupts sequences involved in HCF-1_N association with the HCF-1_C subunit.

N382-450

N382-450

The HCF-1_C subunit contains two independent HCF-1_N association elements. Given the identification of two regions within the HCF-1_N subunit that can associate independently with the HCF-1_C subunit, HCF-1_SAS1N and HCF-1_SAS2N, we asked whether these regions might associate with the same region or different regions of the HCF-1_C subunit. In addition to the Fn3 repeats, the HCF-1_C subunit contains a region with more acidic than basic residues (reference 37 and Fig. 1A). To determine whether either the acidic or Fn3 repeat regions are involved in HCF-1_N subunit association, we divided the HCF-1_C subunit in tworesidues 1436 to 1756 covering the acidic region (HCF-1_C1436-1756) and residues 1758 to 2035 covering the HCF-1_Fn3 repeats (HCF-1_C1758-2035)and tested each individually for association with the HCF-1_N subunit as shown in Fig. 3. In addition to associating with the full-length HCF-1_C subunit (lane 1) as expected, the full-length HCF-1_N subunit associated with both the acidic HCF-1_C1436-1756 (lane 2) and Fn3 repeat-containing HCF-1_C1758-2035 (lane 3) fragments, although recovery of the HCF-1_C1436-1756 fragment was considerably less efficient, which may reflect inhibition by the amino-terminal region of HCF-1 (see Discussion). In contrast, the alternative-splice variant HCF-1_N382-450 subunit associates neither with the full-length HCF-1_C subunit (lane 4), as expected, nor with either HCF-1_C subfragment (lanes 5 and 6) effectively. Thus, the full-length but not the splice-variant HCF-1 can associate with two independent regions within the carboxy-terminal HCF-1_C subunit. We refer to the most carboxy-terminal HCF-1_C self-association sequence in the HCF-1_C1758-2035 segment as HCF-1_SAS1C and the more internal HCF-1_C self-association sequence in the HCF-1_C1436-1756 segment as HCF-1_SAS2C.

View larger version (34K): [in this window] [in a new window]   FIG. 3.   HCF-1SAS1N and HCF-1SAS2N interact with different regions of the HCFC subunit. HA-tagged HCF-1N fragments and T7-tagged HCFC fragments were coexpressed in transfected 293 cells, and association was assayed by immunoprecipitation with the HCF-1 antibody followed by immunoblotting with the T7 antibody. The HA-tagged polypeptides were as follows: HCF-1N1011 (lanes 1 to 3), HCF-1N1011382-450 (lanes 4 to 6), HCF-1N450 (lanes 7 to 9), and HCF-1N450-1011 (lanes 10 to 12). The T7-tagged polypeptides were as follows: HCF-1C (lanes 1, 4, 7, and 10), HCF-1C1436-1756 (lanes 2, 5, 8, and 11), and HCF-1C1758-2035 (lanes 3, 6, 9, and 12).

Mapping of the amino-terminal HCF-1_SAS1N and HCF-1_SAS2N elements. Using the strategy shown in Fig. 3, in experiments not shown here we refined the location of the HCF-1_SAS1N and HCF-1_SAS2N elements. The results of these experiments are summarized in Fig. 4. As shown in Fig. 3, HCF-1_N1011 associated with both HCF-1_SAS1C and to a lesser extent with HCF-1_SAS2C, whereas HCF-1_N450 associated effectively only with the HCF-1_SAS1C element. Consistent with the inability of the minimal VP16 interaction region in HCF-1_N380 to associate with the entire HCF-1_C subunit (Fig. 2), HCF-1_N380 did not exhibit association with either the HCF-1_SAS1C or HCF-1_SAS2C element. In these experiments, we mapped the HCF-1_SAS1N element to within HCF residues 348 to 450 (which we fused to the yeast GAL4 DNA-binding domain owing to our inability to detect this and smaller HCF fragments of this region on their own in our assay). Residues 382 to 450 alone, however, which represent the region removed in the alternative-splice variant of HCF, failed to associate with any of the HCF-1_C fragments (Fig. 4). Consistent with this mapping of the HCF-1_SAS1N element, a fragment covering residues 348 to 1011 (HCF-1_N348-1011) associated with all three of the HCF-1_C fragments used in the coimmunoprecipitation assay.

View larger version (22K): [in this window] [in a new window]   FIG. 4.   Summary of mapping of the HCF-1N SAS elements. Each HCF-1N derivative was assayed for association with HCF-1C (residues 1436 to 2035), HCF-1SAS1C (residues 1758 to 2035), and HCF-1SAS2C (residues 1436 to 1756) by coimmunoprecipitation from transfected 293 cell extracts. The interactions were scored as strong (+), weak (+/), or not detected (). HCF-1N348-450 and HCF-1N382-450 were unstable as isolated polypeptides and were assayed as fusions to the GAL4 DNA-binding domain (residues 1 to 94).

Fine mapping of the paired HCF-1_SAS1N and HCF-1_SAS1C elements. To map one pair of SAS elements in detail, we performed a deletion analysis of the HCF-1_SAS1N and HCF-1_SAS1C elements as shown in Fig. 5A. The results of the analysis shown in Fig. 4 located the HCF-1_SAS1N element to within the 103-amino-acid HCF-1_N348-450 fragment. This region contains the carboxy-terminal residues of the HCF-1_KEL6 repeat (residues 348 to 360), which are conserved among the HCF-1, HCF-2, and CeHCF proteins (11, 16), and an additional region conserved among these three proteins (residues 361 to 396) as shown in Fig. 5B. To locate the HCF-1_SAS1N element within residues 348 to 450, we performed a more extensive deletion analysis of this region fused to the GAL4 DNA-binding domain. As shown in Fig. 5A (lanes 1 to 3), deletion of residues 348 to 360, but not to 382, had no apparent effect on association with the HCF-1_SAS1C-containing HCF-1_C1758-2035 fragment. Also, deletion of residues 450 to 402, but not to 392, had no apparent effect on association with the HCF-1_C1758-2035 fragment (lanes 4 to 6), suggesting that the 43-amino-acid segment from 360 to 402 is sufficient to associate with the HCF-1_SAS1C element. Indeed, the 360-402 sequence fused to the GAL4 DNA-binding domain is sufficient to associate with the HCF-1_C1758-2035 fragment (lane 7). Thus, the SAS1N element resides within residues 360 to 402. Interestingly, this region, which lies immediately adjacent to the HCF-1_KEL6 region, coincides with the 361-401 segment that is conserved among HCF-1, HCF-2, and CeHCF (Fig. 5B).

View larger version (48K): [in this window] [in a new window]   FIG. 5.   Fine mapping of the HCF-1SAS1N and HCF-1SAS1C regions. (A) HA-tagged HCF-1N fragments (fused to GAL4 residues 1 to 94) and T7-tagged HCF-1C fragments were coexpressed in transfected 293T cells, and noncovalent association was assayed by immunoprecipitation (IP) with the HA antibody followed by immunoblotting with the T7 antibody. Truncations of the HA-tagged HCF-1SAS1N domain were assayed by cotransfection with pCGTHCF-1SAS1C (residues 1758 to 2035) and were as follows: HCF-1N348-450 (lane 1), HCF-1N360-450 (lane 2), HCF-1N382-450 (lane 3), HCF-1N348-420 (lane 4), HCF-1N348-402 (lane 5), HCF-1N348-392 (lane 6), and HCF-1N360-402 (lane 7). Truncations of T7-tagged HCF-1SAS1C domain were assayed by cotransfection with the HA-tagged HCF-1SAS1N fragment (residues 348 to 450, pCGNGalHCF-1N348-450) and were as follows: HCF-1C1758-2014 (lane 8), HCF-1C1758-2002 (lane 9), HCF-1C1758-1990 (lane 10), HCF-1C1812-2035 (lane 11), HCF-1C1819-2035 (lane 12), and HCF-1C1812-2002 (lane 8). The two smallest functional fragments HCF-1N360-402 and HCF-1C1812-2002 were combined in lane 13. (B) Alignment of the carboxy-terminal end of the HCF-1KEL6 and HCFSAS1N sequences with the corresponding regions of HCF-2 (11) and CeHCF (16). Identical residues in all three sequences are highlighted in black. The conservation extends beyond the last  strand (3) of HCF-1KEL6, across the length of the HCF-1SAS1N domain. Amino acid numbers below the sequence refer to HCF-1.

HCF-2 contains a conserved SAS1N element. Unlike the HCF-1 protein, HCF-2 and CeHCF do not contain any HCF-1_PRO processing repeats and there is no evidence that HCF-2 and CeHCF are proteolytically processed (11) (Y. Liu and W. Herr, unpublished results). Thus, there is no a priori reason to expect the SAS elements of HCF-1 to be conserved in HCF-2 and CeHCF. We were surprised, therefore, to find that the sequences of both the small SAS1N (residues 360 to 402) and larger SAS1C (residues 1812 to 2002) elements are conserved in the HCF-2 and CeHCF proteins (Fig. 1B and 5B). The sequence conservation led us to ask whether HCF-2 contains a functional SAS1 element. Unfortunately, the carboxy-terminal HCF-2 segment corresponding to HCF-1_SAS1C could not be synthesized effectively in transfected cells. We therefore tested the ability of the putative amino-terminal SAS1 element (HCF-2 residues 341 to 394) to associate with the HCF-1_SAS1C region as shown in Fig. 6. Consistent with an HCF-2_SAS1N function, this HCF-2_341-394 segment could associate effectively with HCF-1_SAS1C (compare lanes 1 to 3). Thus, HCF-2 and perhaps CeHCF are likely to possess HCF self-association activity.

View larger version (25K): [in this window] [in a new window]   FIG. 6.   HCFSAS1 function is conserved between HCF-1 and HCF-2. T7-tagged HCF-1C1758-2035 was coexpressed in transfected 293T cells with HA-tagged fragments (fused to GAL4 residues 1 to 94) corresponding to residues 341 to 394 of HCF-2 (lane 1), 348 to 450 of HCF-1 (lane 2), and 382 to 450 of HCF-1 (lane 3). HCF-1SAS1N and HCF-1SAS1C association was assayed by coimmunoprecipitation (IP) with the HA antibody followed by immunoblotting with the T7 antibody (upper panel). Protein expression was confirmed by immunoblotting extracts directly with the T7 (center panel; HCF-1C polypeptides) or HA (lower panel; HCF-1C polypeptides) antibody.

Association with the carboxy-terminal HCF-1_C subunit results in nuclear localization of the HCF-1_N subunit. Immunofluorescence and subcellular fractionation studies have shown that HCF-1 is predominantly a nuclear protein (12-14, 29) and that nuclear localization is determined by a consensus bipartite NLS (5) at its carboxyl terminus (KRPMSSPEMKSAPKKSK; residues 2015 to 2031) (14). Thus, when expressed separately, the HCF-1_C600 fragment accumulates in the nucleus (Fig. 7B) like the endogenous protein (Fig. 7F), and the HCF-1_N1011 fragment accumulates largely in the cytoplasm (Fig. 7A). Deletion of the NLS sequence results in HCF-1_C remaining largely in the cytoplasm (Fig. 7B and C).

View larger version (50K): [in this window] [in a new window]   FIG. 7.   Nuclear localization of the HCF-1N subunit through association with the HCF-1C subunit, visualized by Immunofluorescence of transfected 293 cells using the HA (A, D, and E) or T7 (B and C) primary antibody. Cells were transfected with plasmids pCGNHCF-1N (A), pCGTHCF-1C (B), pCGTHCF-1CNLS (C), pCGNHCF-1N1011 and pCGTHCF-1C (D), and pCGNHCF-1N1011 and pCGTHCF-1CNLS (E). For comparison, mock-transfected cells were probed with HCF-M2 (endog. HCFC [F]), a monoclonal antibody against HCF-1 (28).

C600NLS

	DISCUSSION

Top Abstract Introduction Materials and Methods Results Discussion References

We have characterized the sequences required for HCF-1_N and HCF-1_C subunit association. To our surprise, this characterization revealed two matched pairs of HCF-1_N and HCF-1_C SAS elements: HCF-1_SAS1, consisting of HCF-1_SAS1N and HCF-1_SAS1C partners, and HCF-1_SAS2, consisting of HCF-1_SAS2N and HCF-1_SAS2C partners. The positions of the two sets of HCF-1 SAS elements are shown in Fig. 8A. The HCF-1_SAS1 pair of elements has been mapped with considerable precision and represents a small 43-amino-acid amino-terminal HCF-1_SAS1N sequence (residues 360 to 402) immediately adjacent to the -propeller-containing VP16 interaction domain and a larger 191-amino-acid carboxy-terminal HCF-1_SAS1C sequence (residues 1812 to 2002), which contains a pair of Fn3-type repeats. The HCF-1_SAS2 element has been less rigorously mapped: the HCF-1_SAS2N sequence lies within residues 491 to 755 in the so-called, basic region and the HCF-1_SAS2C sequence lies within residues 1436 to 1756, which contains the so-called acidic region.

View larger version (25K): [in this window] [in a new window]   FIG. 8.   Summary of HCF-1 functional elements and model for HCF-1 and HCF-2 self-association and association with putative effector proteins. (A) Schematic of human HCF-1 (hHCF-1) showing the locations of the two matched pairs of SAS elements (SAS1 and SAS2). The HCF-1SAS1 pair have been mapped to residues 360 to 402 (SAS1N) and 1812 to 2002 (SAS1C). The HCF-1SAS2 pair are less well defined and lie between residues 491 and 755 (SAS2N) and residues 1436 and 1756 (SAS2C). Features of the HCF-1 illustration are as described in the legend to Fig. 1A. (B) Self-association is likely to be regulated in vivo. The proposed general structure of HCF-1 is shown in cartoon form. Following proteolytic cleavage at the central HCFPRO repeats, the HCF-1N and HCF-1C subunits remain associated through noncovalent interactions mediated by the two self-association domains present in each subunit. Subunit interaction is required for the nuclear localization of HCF-1N, which does not contain its own NLS. The presence of two intersubunit contacts also allows for limited dissociation. For example, following dissociation of the SAS1 pairing, contacts between the two HCF-1 subunits may be maintained by the SAS2 interaction. In this scenario, the SAS1 sequences are available for interaction with other cellular proteins (X and Y), a dual function that might explain the strong sequence conservation of the SAS1 sequences in HCF-2 and CeHCF. Because HCF-2 is not proteolytically cleaved, there is no requirement for a second noncovalent SAS2-like association.

Regulation of HCF-1_N and HCF-1_C association. The identification of two separate SAS elements raises a paradox because deletion of just one half of one of these sets, HCF-1_SAS1N, inactivates HCF-1 subunit association. These results suggest that amino-terminal HCF-1 sequences can inhibit the activity of the HCF-1_SAS2N element. First, the entire amino-terminal region of HCF-1 (residues 1 to 1011) associates less well with the HCF-1_SAS2C region than residues 450 to 1011 alone (Fig. 3); second, deletion of residues 380 to 452 in the alternative-splice HCF-1 variant (Fig. 1), which impinges on the HCF-1_SAS1N but not the HCF-1_SAS2N sequence, prevents detectable HCF-1_C association. Thus, inactivation of HCF-1_SAS1N in this natural deletion variant also functionally inactivates HCF-1_SAS2N. Perhaps, the region encompassing the HCF-1_SAS1N sequence inhibits the activity of the HCF-1_SAS2N element, particularly when residues 382 to 450 are removed, providing an efficient mechanism for regulation of HCF subunit association. Whatever the case, this result shows that HCF-1_SAS2 element is not simply due to nonspecific association of the basic and acidic regions of HCF.

The HCF-1_SAS1 association element is conserved in HCF family members. The SAS elements in HCF-1 were identified because HCF-1 is processed to produce amino-terminal and carboxy-terminal HCF subunits that remain associated (27). We were very much surprised, therefore, to find that the sequence of one of the paired HCF-1 SAS elements, HCF-1_SAS1, is conserved in human HCF-2 and C. elegans HCF (Fig. 1B and 5B), proteins that do not display any evidence of proteolytic cleavage. Furthermore, the sequence in HCF-2 corresponding to the HCF-1_SAS1N sequence can associate with the HCF-1_SAS1C sequence (Fig. 6). Thus, even though there is no evidence that HCF-1 and HCF-2 associate with one another in vivo (K. M. Johnson and A. C. Wilson, unpublished results), both proteins have conserved HCF_SAS1N activity. As illustrated in Fig. 8B, we hypothesize that the HCF_SAS1 element has been conserved in both proteins and retains self-association activity in HCF-2. Given the sequence conservation in C. elegans HCF, we also suggest that the C. elegans HCF protein possesses an active HCF-1_SAS1 element, although this possibility has not been tested directly.

Fn3 repeats form a protein-protein interaction module. The sequence of the HCF-1_SAS1C element exhibits similarity to a tandem pair of Fn3 repeats. Fn3 repeats from different proteins generally exhibit limited sequence similarity (2). Structurally, however, all Fn3 repeats fold into two  sheets packed against each other like a sandwich (3, 4, 9, 18). Thus, Fn3 repeats represent a conserved structure that can vary considerably in sequence to create many different types of functional surfaces. This flexibility may account for their widespread use (2).

Implications of the two-subunit composition of HCF-1 for its function. Consistent with earlier results (14), we have identified a classical bipartite NLS at the extreme carboxyl terminus of HCF-1. We have also shown that the HCF-1_N subunit does not contain an NLS and that HCF-1_SAS1N subunits can migrate to the nucleus through association with the HCF-1_SAS1C subunit. This arrangement resembles polymerase  primase, which is also composed of two stably associated subunits. In this case, a single NLS in the p54 subunit targets the smaller p46 subunit to the nucleus (19). Pulse-chase analysis of the HCF-1₃₀₀ precursor has shown that it migrates to the nucleus prior to its initial proteolytic cleavage (29). Thus, HCF-1 subunit association is more likely to be required for the nuclear retention of the HCF-1_N subunit rather than its initial nuclear import. Because HCF-1 is stable (29), it may persist through multiple rounds of cell division. Perhaps HCF-1 subunit association is required to maintain the nuclear location of the HCF-1_N subunit following nuclear breakdown and resynthesis as a result of mitosis.