Fig. 7. Schematic diagram of the proposed mechanism that represses the HIF-1α transactivation by pVHL and the VHLaK protein. Based on the data presented in this study and previously published findings, we propose that pVHL not only mediates the ubiquitylation and degradation of HIF-1α in the cytoplasm, but also recruits the VHLaK protein, through interaction with the KRAB-A domain, to HIF-1α transcription factor complex. The VHLaK protein forms homo-oligomerization via its SCAN domain and can recruit KAP-1/KRIP-1/TIF-1β, a well- characterized transcriptional repressor, to repress the transcriptional activity of HIF-1α.

Fig. 4. VHLaK enhances pVHL-mediated repression of HIF-1α transcriptional activity. (A and B) GAL4-DBD-tHIF-1α expression plasmid (pM-tHIF-1α) together with various doses of pVHL expression plasmid (pcDNA4-VHL19) and/or VHLaK expression plasmids were transfected into HEK 293 cells (A) and A498 cells (B) along with 300 ng of pG5-LUC and 10 ng pRLSV40-LUC. Twenty-four hours after transfection, cells were harvested and measured for luciferase activity. The experiments were repeated three times to ensure consistency. (C) VHLaK can be recruited into pVHL–HIF-1α complex. VHLaK, pVHL19 and cMyc-tHIF-1α (amino acids 530–826) were expressed in the presence of [³⁵S]methionine. Co-immunoprecipitation was performed using an anti-c-Myc antibody. Note that the VHLaK protein can only be co-immunoprecipitated with cMyc- tHIF-1α in the presence of pVHL.

Fig. 1. Gene structure and alternative splicing of the ZnF197 locus, amino acid sequence alignment of VHLaK and ZnF197 proteins. (A) Exon–intron structure and alternative splicing of the ZnF197 gene. The ZnF197 gene consists of seven exons and generates VHLaK and ZnF197 transcripts by alternative splicing of exons 1–5 then exon 7, and exons 1–6, respectively. The corresponding coding regions for the SCAN domain, KRAB-A domain, and 22 C2H2 zinc finger motifs are indicated. (B) Amino acid sequence alignment of VHLaK and ZnF197 proteins. The arrowheads and numbers above and below the amino acids indicate the corresponding splicing junctions and exon numbers. The SCAN domain and KRAB-A domain are underlined using different lines. The 22 C2H2 zinc finger motifs of ZnF197 are shaded. Note that the VHLaK protein contains a short C-terminus and lacks all 22 C2H2 zinc finger motifs present in the ZnF197 protein.

Fig. 5. The VHLaK protein inhibits VEGF mRNA and protein expression in the presence of pVHL. (A and B) the VHLaK protein inhibits HIF-1α- induced VEGF mRNA expression in the presence of pVHL. HEK 293 cells (A) and A498 cells (B) were transfected with the plasmids as indicated. Thirty-six hours after transfection, total RNA was extracted and semi-quantitative RT–PCR was performed using primers specific for VEGF or GAPDH. Note that four bands were observed representing four VEGF isoforms (VEGF 121, 165, 189 and 206). The densities of VEGF bands were quantified and normalized with the density of GAPDH bands. Molecular size markers were loaded in lane M. (C and D) The VHLaK protein inhibits HIF-1α-induced VEGF protein expression in the presence of pVHL. HEK 293 cells (C) and A498 cells (D) were transfected with the plasmids as indicated. Thirty-six hours after transfection, aliquots of medium were collected for VEGF ELISA and results were normalized for cell numbers. Histograms represent the results of three independent experiments.

Fig. 6. The VHLaK protein can recruit KAP1/TIF-1β through its KRAB-A domain and both endogenous VHL and KAP1/TIF-1β proteins interact VHLaK simultaneously. (A) Analysis of the interaction between VHLaK and KAP1/TIF-1β by GST pull-down assay. Five microliters of in vitro translated [³⁵S]methionine-labeled KAP1/TIF-1β protein were incubated with GST–VHLaK, GST–KRAB-A or GST-coated beads. The eluted solutions and 10% of input protein were resolved on an 8% SDS–PAGE gel. (B) The interaction between VHLaK and KAP1/TIF-1β in mammalian two-hybrid assay. Lanes 4 and 5, GAL4-DBD-KAP1/TIF-1β and pVP16-VHLaK or pVP16-KRAB-A were co-transfected into COS-7 cells, respectively. Thirty-six hours after transfection, cells were harvested and lysed for luciferase assay. (C) In vivo co-immunoprecipitation of KAP1/TIF-1β with VHLaK. Lysates prepared from COS-7 cells transfected with GAL4-DBD-KAP1/TIF-1β and pEGFP-C3 or pEGFP-C3-VHLaK were subjected to immunoprecipitation (IP) with an anti-GAL4-DBD antibody followed by anti-GFP or anti-GAL4-DBD immunoblotting. (D) Both pVHL and KAP1 can interact with VHLaK simultaneously. VHLaK, pVHL19, and KAP1 were expressed in the presence of [³⁵S]methionine. Co-immunoprecipitation was performed using an anti-VHL antibody. Note that the KAP1 protein can only be co-immunoprecipitated with pVHL in the presence of VHLaK. (E) The endogenous VHLaK protein can recruit endogenous VHL and KAP1 proteins. The top panel shows a western blot analysis of crude extracts (lanes 1 and 2), control immunoprecipitates with the pre-immune serum (lane 3) or immunoprecipitates with anti-VHLaK antibody (lane 4) from HeLa cells immunoblotted with anti-KAP1 antibody; the bottom panel shows similar analysis of same crude extracts and immunoprecipitates immunoblotted with anti-VHL antibody.

Fig. 2. The VHLaK protein interacts with pVHL through its KRAB-A domain. (A) Analysis of the interaction between the VHLaK protein and pVHL by GST pull-down assay. Five microliters of in vitro translated [³⁵S]methionine-labeled VHLaK protein were incubated with GST–pVHL19 or GST-coated beads. The eluted solutions and 10% of input protein were resolved on a 10% SDS–PAGE gel. (B) In vitro co-immunoprecipitation of c-Myc-pVHL19 with the VHLaK protein. Five microliters of in vitro translated c-Myc-pVHL19 and VHLaK (lane 2) or 5 µl of in vitro translated VHLaK protein alone (lane 3) were incubated at 4°C. The proteins were immunoprecipitated with anti-c-Myc antibody and analyzed on a 10% SDS–PAGE gel. In lane 1, 0.5 µl of in vitro translated VHLaK protein were loaded into the gel as a marker. (C) Analysis of the interaction between the VHLaK protein and pVHL in a mammalian two-hybrid assay. In lane 5, pM-pVHL19 and pVP16-VHLaK were co-transfected into COS-7 cells together with reporter plasmid pG5CAT and cells were lysed for chloramphenicol acetyltransferase (CAT) assay. CAT activity was measured using a thin-layer chromatogram and ¹⁴C-labeled chloramphenicol as substrate. Other lanes served as negative controls. (D) In vivo co-immunoprecipitation of pVHL with GFP–VHLaK fusion protein. Lysates prepared from COS-7 cells transfected with pCMX-VHL19 and pEGFP-C3 or pEGFP-C3-VHLaK were subjected to immunoprecipitation (IP) with an anti-VHL antibody followed by anti-GFP or anti-pVHL immunoblotting. (E) Mapping pVHL-interacting region in the VHLaK protein by in vivo co-immunoprecipitation. Lysates prepared from COS-7 cells transfected with pCMX-VHL19 and pEGFP-C3 or pEGFP-C3-VHLaK (amino acids 1–189) were subjected to immunoprecipitation (IP) with an anti-VHL antibody followed by anti-GFP or anti-VHL immunoblotting. (F) Mapping pVHL-interacting region in VHLaK by GST pull-down assay. Five microliters of in vitro translated [³⁵S]methionine- labeled His₆-KRAB-A protein were incubated with GST–pVHL19 or GST-coated beads. The eluted solutions and 10% of input protein were resolved on a 15% SDS–PAGE gel.

Fig. 3. The KRAB-A domain of VHLaK exhibits transcriptional repression activity independent of HDAC activity and the homo-oligomerization mediated by the SCAN domain enhances this repressive activity. (A and B) Transcriptional repression activity of the VHLaK protein and its KRAB-A domain. Various doses of GAL4-DBD-VHLaK (pM-VHLaK) (A) and of GAL4-DBD-KRAB-A (pM-KRAB) (B), along with pVHL expression plasmid (pcDNA4-VHL19), 300 ng of pGAL4-tk-LUC and 10 ng pRLSV40-LUC, were transfected into COS-7 cells. Twenty-four hours after transfection, cells were harvested and measured for firefly and Renilla activity. The firefly luciferase activity was normalized against Renilla luciferase activity for each sample, and mean values of relative luciferase activity were calculated from triplicate wells. The experiments were repeated three times to ensure consistency. (C) Transcriptional repression activity of VHLaK protein is independent of HDAC activity. Lanes 3 and 4, 18 h after transfection, the cells were treated with 300 nM of trichostatin A (TSA) for another 18 h and then the cells were lysed for luciferase activity assay. (D) Analysis of self-interaction of the SCAN domain in mammalian two-hybrid assay. Lane 5, pM-SCAN and pVP16-SCAN were co-transfected into COS-7 cells together with reporter plasmid pG5-LUC and cells were lysed for luciferase activity assay. Lane 6, pM3-VP16 served as a positive control. (E) Analysis of the self-interaction of the SCAN domain by GST pull-down assay. Five microliters of in vitro translated [³⁵S]methionine-labeled His₆-SCAN protein were incubated with GST–SCAN or GST-coated beads. The eluted solutions and 10% of input protein were resolved on a 15% SDS–PAGE gel.