Sequence-specific transcriptional repression by KS1, a multiple-zinc-finger-Krüppel-associated box protein

Abstract

The vertebrate genome contains a large number of Krüppel-associated box-zinc finger genes that encode 10 or more C(2)-H(2) zinc finger motifs. Members of this gene family have been proposed to function as transcription factors by binding DNA through their zinc finger region and repressing gene expression via the KRAB domain. To date, however, no Krüppel-associated box-zinc finger protein (KRAB-ZFP) and few proteins with 10 or more zinc finger motifs have been shown to bind DNA in a sequence-specific manner. Our laboratory has recently identified KS1, a member of the KRAB-ZFP family that contains 10 different C(2)-H(2) zinc finger motifs, 9 clustered at the C terminus with an additional zinc finger separated by a short linker region. In this study, we used a random oligonucleotide binding assay to identify a 27-bp KS1 binding element (KBE). Reporter assays demonstrate that KS1 represses the expression of promoters containing this DNA sequence. Deletion and site-directed mutagenesis reveal that KS1 requires nine C-terminal zinc fingers and the KRAB domain for transcriptional repression through the KBE site, whereas the isolated zinc finger and linker region are dispensable for this function. Additional biochemical assays demonstrate that the KS1 KRAB domain interacts with the KAP-1 corepressor, and mutations that abolish this interaction alleviate KS1-mediated transcriptional repression. Thus, this study provides the first direct evidence that a KRAB-ZFP binds DNA to regulate gene expression and provides insight into the mechanisms used by multiple-zinc-finger proteins to recognize DNA sequences.

FIG. 1

ROB assay for KS1. ROB oligonucleotides with a 35-bp degenerate core were end labeled with ³²P, incubated with GST alone or with GST-KS1, and separated by nondenaturing polyacrylamide gel electrophoresis. After seven rounds of ROB purification, 31 oligonucleotides were cloned, sequenced, and aligned. Uppercase letters represent the 35-bp random sequence from each clone, whereas lowercase letters represent the flanking sequence. Gray residues indicate greater than 60% identity, and a derived consensus KBE is shown.

FIG. 2

Electromobility shift assay of KS1 with ROB-purified sequences. An electromobility shift assay was performed using either the GST protein alone or the GST-KS1 fusion protein. Lanes 1 to 5, ROB1 probe; lane 6, GC box probe; lane 7, GCmut probe; lane 8, GT box probe; lane 1, no proteins; lane 2, 200 ng of GST; lanes 3 to 8, 200 ng of GST-KS1; lane 4, 5 μg of anti-GST antibody; lane 5, 10 mM EDTA.

FIG. 3

KS1 represses the transcription of promoters containing the KS1 binding sites. CHO cells were cotransfected with various reporter vectors and a KS1 or Kid-1 expression vector as indicated. Firefly luciferase activity was measured and normalized to Renilla luciferase activity by a dual luciferase reporter system, and the mean and standard deviation were determined for each experimental condition. Histogram of relative luciferase activity shows that KS1 represses both basal (A) and activated (B) transcription of reporters containing three copies of the ROB1 sequence. Note that KS1 has no effect on a reporter vector lacking these sites. SV40, simian virus 40. (C) Increasing amounts of KS1 expression vector (0 to 500 ng) were cotransfected into CHO cells with the 3× ROB1 reporter vector as indicated. As shown in the histogram, KS1 is able to repress the expression of this reporter in a dose-dependent manner. (D) The 3× ROB1 reporter vector was cotransfected into CHO cells with either a KS1 or Kid-1 expression vector. The histogram shows that KS1 and not the Kid-1 KRAB-ZFP represses the expression of this reporter.

FIG. 4

Defining the KBE. (A) Electromobility shift assay was performed using the GST-KS1 fusion protein with deletion and site-directed mutated KBE probes. Lane 1, no proteins; lane 2, 200 ng of GST; lanes 3 to 8, 200 ng of GST-KS1 with the various probes as indicated. The relative binding affinity of KS1 for the different probes is shown. (B) Luciferase assays using a reporter vector containing three copies of the ROB1, KBE, or Del1 sequence were performed as described for Fig. 3. The histogram of luciferase activity shows that KS1 is able to repress expression of a reporter containing three copies of the KBE. Note that KS1 is unable to repress a reporter containing three copies of the Del1 mutant sequence.

FIG. 5

Mapping of the zinc finger motifs required for KS1-mediated transcriptional repression. (A) Physical maps of the KS1 expression vectors transfected into CHO cells and tested in transcriptional regulatory assays. (B) Cells were transfected with either the 0× ROB1 or the 3× ROB1 reporter vector and the various deletion and site-directed mutants as indicated. Luciferase activities were determined as described for Fig. 3, and the 3×/0× values with standard deviations were graphed in the histogram. Note that the construct containing the nine clustered zinc fingers of KS1 was able to repress transcription [KS1(ZF2-10)], whereas all other zinc finger mutations abolished KS1-mediated transcriptional repression. (C) The various KS1 deletion proteins were detected by Western blot analysis as described in Materials and Methods. Note that all of the KS1 deletion constructs are expressed at similar or higher levels than the full-length KS1 protein.

FIG. 6

Mapping of the transcriptional repressor activity within KS1. (A) Physical maps of the KS1 expression vectors transfected into CHO cells and tested in transcriptional regulatory assays. (B) Cells were transfected with either the 0× ROB1 or the 3× ROB1 reporter vector and the various deletion and site-directed mutants as indicated. Luciferase activities were determined as described for Fig. 3, and the 3×/0× values were graphed in the histogram. Note that both constructs containing KRAB wt were able to repress transcription, whereas deletion or mutation of this region abolished KS1-mediated transcriptional repression. (C) The KS1 deletion proteins were detected by Western blot analysis as described in Materials and Methods. Note that all of the KS1 deletion constructs are expressed at similar or higher levels compared with the full-length KS1 protein.

FIG. 7

The KRAB domains of KS1 interact with KAP-1. (A) Comparison of the KS1 KRAB-A motif with the KRAB-A domain from other KRAB-ZFPs. Identical residues are shaded. The KRAB-A consensus sequence (Cons) is derived from the work of Bellefroid et al.; uppercase letters represent highly conserved residues, lowercase letters represent moderately conserved residues, and dots represent unconserved residues (2). The KRAB mutation that abolishes KS1-mediated transcriptional repression is indicated. (B) Coomassie blue gel analysis of the GST, GST-KRAB, and GST-KRAB mt fusion proteins used for the GST pulldown assays. (C) To determine the proteins with which the KRAB domain interacts, CHO cells were labeled with [³⁵S]methionine, lysed in RIPA buffer, and incubated with either the GST, GST-KRAB, or GST-KRAB mt protein. GST pulldown assays were performed as described in Materials and Methods. Note that KRAB wt interacts with a protein of approximately 100 kDa, whereas the mutant KRAB domain does not. The doublet of ≈30 kDa corresponds to endogenous GST. (D) To determine whether the 100-kDa KRAB-interacting protein was KAP-1, CHO cells were transfected with an HA-tagged form of KAP-1. Western blot analysis using an anti-HA antibody demonstrates that the KRAB wt of KS1 interacts with the KAP-1 protein.

FIG. 8

KS1 colocalizes with KAP-1. For KS1 and KAP-1 colocalization, CHO cells were cotransfected with an Xpress-tagged KS1 construct and an HA-tagged KAP-1 vector (A through H). KS1 expression was detected with an anti-Xpress mouse monoclonal antibody and a rhodamine-conjugated anti-mouse secondary antibody (A and E), whereas the KAP-1 protein was detected with an anti-HA-umouse monoclonal antibody directly conjugated to fluorescein (B and F). Superimposing these images demonstrates that KS1 and KAP-1 are localized to the same regions of the nucleus, as shown in yellow (C and G). For Kid-1 and KAP-1 colocalization, CHO cells were transfected with an Xpress-tagged KAP-1 construct and an HA-tagged Kid-1 expression vector (I through L). KAP-1 was detected with an anti-Xpress mouse monoclonal antibody and a fluorescein-conjugated anti-mouse secondary antibody (J), Kid-1 was detected with an anti-HA-umouse monoclonal antibody directly conjugated to rhodamine (I), and the images were subsequently superimposed (K). Hoechst staining was used to stain cellular DNA (D, H, and L). Note that an untransfected cell shown in panel H shows no specific staining.

FIG. 9

KAP-1 enhances KS1-mediated transcriptional repression. CHO cells were transfected with various constructs as indicated, and luciferase assays were performed as described for Fig. 3. (A) Histogram of luciferase activity from an experiment using either KRAB wt or KRAB mt, both of which lack the zinc finger motifs of the full-length KS1 protein. Note that expression of the KRAB domain alleviates the ability of KS1 to repress luciferase expression, whereas expression of the mutant KRAB domain has no effect on KS1-mediated repression. (B) Western blot analysis was used to detect the Xpress-tagged KS1 KRAB proteins (wt or mt) from CHO cells cotransfected with the Xpress-tagged full-length KS1 protein as indicated. (C) Histogram of luciferase activity from CHO cells transfected with increasing amounts of a KAP-1 expression vector. Note that addition of KAP-1 increases the transcriptional repression activity of KS1. (D) Western blot analysis was used to detect the Xpress-tagged full-length KS1 protein and the HA-tagged KAP-1 protein from transfected CHO cells as indicated.