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. 2003 Dec 19;278(51):51116-24.
doi: 10.1074/jbc.M303470200. Epub 2003 Oct 6.

HCF-1 functions as a coactivator for the zinc finger protein Krox20

Randy L Luciano  1 , Angus C Wilson
Affiliations

HCF-1 functions as a coactivator for the zinc finger protein Krox20

Randy L Luciano et al. J Biol Chem. .

Abstract

HCF-1 is a transcriptional cofactor required for activation of herpes simplex virus immediate-early genes by VP16 as well as less clearly defined roles in cell proliferation, cytokinesis, and spliceosome formation. It is expressed as a large precursor that undergoes proteolysis to yield two subunits that remain stably associated. VP16 uses a degenerate 4-amino acid sequence, known as the HCF-binding motif, to bind to a six-bladed beta-propeller domain at the N terminus of HCF-1. Functional HCF-binding motifs are also found in LZIP and Zhangfei, two cellular bZIP transcription factors of unknown function. Here we show that the HCF-binding motif occurs in a wide spectrum of DNA-binding proteins and transcriptional cofactors. Three well characterized examples were further analyzed for their ability to use HCF-1 as a coactivator. Krox20, a zinc finger transcription factor required for Schwann cell differentiation, and E2F4, a cell cycle regulator, showed a strong requirement for functional HCF-1 to activate transcription. In contrast, activation by estrogen receptor-alpha did not display HCF dependence. In Krox20, the HCF-binding motif lies within the N-terminal activation domain and mutation of this sequence diminishes both transactivation and association with the HCF-1 beta-propeller. The activation domain in the C-terminal subunit of HCF-1 contributes to activation by Krox20, possibly through recruitment of p300. These results suggest that HCF-1 is recruited by many different classes of cellular transcription factors and is therefore likely to be required for a variety of cellular processes including cell cycle progression and development.

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Figures

Fig. 1
Fig. 1. Candidate HCF-binding motifs are found in many human transcription factors
Comparison of known or candidate HBM sequences. For each, the core motif ((E/D)HXY, where X can be any residue), is highlighted. The first 8 sequences correspond to experimentally verified HBM proteins (see text for details). SWISS-PROT or EMBL accession numbers and amino acid coordinates are given.
Fig. 2
Fig. 2. Temperature-dependent transactivation by Krox20 and E2F4 in tsBN67 cells
A, schematic showing structure of murine Krox20 with sequences of the HBM region from the following orthologs: Zebrafish (SWISS-PROT accession Q05159), Xenopus (Q08427), chicken (Q98T82), rat (P51774), mouse (P08152), and human (P11161). Three functional domains have been defined: an N-terminal activation domain (residues 1–184), a repression domain (193–229), and a DNA-binding domain composed of three zinc fingers (331–418). Residues mutated to alanine in Krox20 HBMKO are indicated by arrowheads. B, hamster tsBN67 cells were cotransfected with an expression plasmid (500 ng) encoding full-length mouse Krox20 together with a Krox20 responsive reporter (GC-luc, 5 μg) and incubated at 33.5 or 39.5 °C for 40 h. Each assay was performed in triplicate and the mean ± S.D. are shown. C, as in panel B, except that tsBN67 cells were cotransfected with 500 ng of expression plasmid encoding Gal4DBD or Gal4-Krox20N170 together with a Gal4 reporter (5xGal4-E1B-luc, 500 ng). D, domain structure of human E2F4. The HBM (filled box) lies within the transactivation domain (33). For the reporter assay tsBN67 cells were cotransfected with 250 ng of Gal4DBD or Gal4-E2F4-(240–412) expression plasmid and 500 ng of Gal4-luciferase reporter. E, in the estrogen receptor (ERα) the putative HBM (filled box) lies in the ligand-binding domain. 500 ng of ERα expression plasmid were cotransfected into tsBN67 cells together with an ER-responsive reporter plasmid (ERE-luc, 500 ng). Note that the calf serum used to culture the cells contains sufficient levels of natural ligands to activate the transfected receptors.
Fig. 3
Fig. 3. Mutation of the HBM prevents transactivation by Krox-20
A, GC-luc reporter; B, HCF-dependent activation of the bFGF-2 promoter. C, schematic showing the human bFGF-2 promoter that lacks a TATA box but contains multiple GC-rich boxes (i–v) that serve as binding sites for Sp1 and Krox24/Egr-1. Egr-1 was shown to bind to human bFGF-2 promoter at two sites (sites i and iii) (36). D, 293T cells were cotransfected with increasing amounts of wild type (filled circles) or HBMKO (open squares) Krox20 expression plasmids (0.1, 0.5, and 1 μg) and a bFGF-2 promoter reporter plasmid (bFGF-2-luc, 100 ng). Luciferase activity was measured after 40 h. E, in a separate experiment, protein extracts were prepared from cells transfected with 0.5 μg of expression plasmid, resolved by 10% SDS-PAGE, and immunoblotted with αT7 antibody.
Fig. 4
Fig. 4. The Krox-20 activation domain interacts with the β-propeller of HCF-1
A, 293T cells were cotransfected with 5xGal4- E1B-luc reporter (100 ng) together with expression plasmids (500 ng) encoding Gal4-HCF-1N380 WT or P134S and full-length Krox20 (Krox20FL). Luciferase activity was measured after 40 h. B, as in the previous panel except that only the N terminus of Krox20 (Krox20N170) was used.
Fig. 5
Fig. 5. HCF-1AD is required for transactivation by Krox-20
A, structure of wild type (WT) and activation domain deleted (ΔAD) versions of HCF-1 used to complement tsBN67 cells (13). Major structural features of HCF-1 are indicated. Note the central HCF-1PRO repeats were removed (HCF-1Δrep) to prevent processing and subunit exchange. B, equivalent cultures of tsBN67 cells that have been complemented with a derivative of human HCF-1 (HCF-1Δrep) with (R-tsBN67-HCF-1ΔrepWT) or without (R-tsBN67-HCF-1ΔrepΔAD) the C-terminal activation domain (HCF-1AD) were transiently transfected with 5xGal4-E1B-luc (500 ng) together with expression plasmids (500 ng) encoding Gal4DBD or Gal4-Krox20N170 and assayed for luciferase activity after incubation at 39.5 °C for 40 h. C, equal numbers of WT and ΔAD cells were transfected with the GC-luc reporter (500 ng) together with expression plasmids (500 ng) encoding WT or HBMKO full-length Krox20 and assayed after incubation for 40 h at 39.5 °C. D, R-tsBN67-HCF-1ΔrepWT and R-tsBN67-HCF-1ΔrepΔAD cells were transfected with bFGF-2-luc reporter (500 ng) with or without cotransfected wild type Krox20 expression plasmid (100 ng and 1 μg).
Fig. 6
Fig. 6. Expression of p300 stimulates activation by Krox-20
A, 293T cells were cotransfected with the GC-luc reporter (5 μg) together with expression plasmids encoding full-length Krox20 (500 ng) and p300 (6 μg). Luciferase activity was assayed 40 h post-transfection. B, model showing recruitment of HCF-1 and p300 to the activation domain of Krox20.
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