Sequence-specific recruitment of heterochromatin protein 1 via interaction with Krüppel-like factor 11, a human transcription factor involved in tumor suppression and metabolic diseases

Abstract

Heterochromatin protein 1 (HP1) proteins are "gatekeepers" of epigenetic gene silencing that is mediated by lysine 9 of histone H3 methylation (H3K9me). Current knowledge supports a paradigm whereby HP1 proteins achieve repression by binding to H3K9me marks and interacting to H3K9 histone methyltransferases (HMTs), such as SUV39H1, which methylate this residue on adjacent nucleosomes thereby compacting chromatin and silencing gene expression. However, the mechanism underlying the recruitment of this epigenetic regulator to target gene promoters remains poorly characterized. In the current study, we reveal for the first time a mechanism whereby HP1 is recruited to promoters by a well characterized Krüppel-like transcription factor (KLF), in a sequence-specific manner, to mediate complex biological phenomena. A PXVXL HP1-interacting domain identified at position 487-491 of KLF11 mediates the binding of HP1α and KLF11 in vitro and in cultured cells. KLF11 also recruits HP1α and its histone methyltransferase, SUV39H1, to promoters to limit KLF11-mediated gene activation. Indeed, a KLF11ΔHP1 mutant derepresses KLF11-regulated cancer genes, by inhibiting HP1-SUV39H1 recruitment, decreasing H3K9me3, while increasing activation-associated marks. Biologically, impairment of KLF11-mediated HP1-HMT recruitment abolishes tumor suppression, providing direct evidence that HP1-HMTs act in a sequence-specific manner to achieve this function rather than its well characterized binding to methylated chromatin without intermediary. Collectively, these studies reveal a novel role for HP1 as a cofactor in tumor suppression, expand our mechanistic understanding of a KLF associated to human disease, and outline cellular and biochemical mechanisms underlying this phenomenon, increasing the specificity of targeting HP1-HMT complexes to gene promoters.

FIGURE 1

HP1 interacts with the sequence-specific tumor suppressor, KLF11 in vitro.A, KLF11 contains a PXVX(L/M) domain. HP1-interacting protein sequences containing the pentameric consensus (top, gray-shaded regions) align with the C terminus of KLF11. Amino acid positions are denoted to the right. Dm, D. melanogaster; Mm, Mus muculus; Hs, Homo sapiens. B, HP1 interacts with KLF11 in vitro. KLF11 interacts with all 3 HP1 isoforms with HP1α displaying the strongest binding. Phosphorimage results of ³⁵S-KLF11 binding (upper) and Coomassie staining of GST-HP1 proteins as a loading control (lower) are shown. GST alone was included as a negative control. Input (10%) of the in vitro translated (IVT) protein is shown to the right. C, HP1 does not bind within the N-terminal domains of KLF11. GST-HP1α still interacted with KLF11 upon deletion of the R1 (42–495) or both, the R1 and R2 together along with the intervening sequence (162–495). GST-HP1α interacted with KLF11 (379–495), indicating that HP1 interacts with the C terminus of KLF11. Phosphorimage results of binding (upper) and loading control Coomassie staining of GST proteins (lower) are shown. GST alone was included as a negative control for each. Inputs (10%) of in vitro translated proteins are shown to the right. The schematic (top) depicts domains of KLF11, including the R1, R2, R3, and zinc finger (ZFs) domains. Various deletion mutants of KLF11, utilized for mapping, are shown. D, HP1 interacts with residues 487–495 of KLF11. Deletion KLF11 (486), or KLF11ΔHP1, abolished binding of HP1α, indicating that this PXVX(L/M) domain is the site of HP1 interaction. Phosphorimage results of binding (upper) and Coomassie staining of GST proteins for a loading control (lower) are shown. Inputs of in vitro translated proteins are shown to the right.

FIGURE 2

HP1 binds to KLF11 in vivo.A, endogenous HP1α and KLF11 proteins interact in cells. Endogenous HP1α immunoprecipitates endogenous KLF11 from BxPC3 nuclear extracts. Upon immunoprecipitation with an HP1α-specific antibody, a Western blot was performed using a KLF11-specific antibody. Mouse IgG was used as a negative control. B, HP1α-KLF11 interaction in cells requires its C-terminal PXVX(L/M) motif. Immunoprecipitation (IP) with an HP1α-specific antibody (lower) demonstrates binding of His-tagged KLF11 WT (upper), but is disrupted with His-tagged KLF11ΔHP1. EV was used as a negative control. C, KLF11ΔHP1 does not impair binding to other KLF11 co-factors. Although KLF11ΔHP1 does not interact with HP1α, upon immunoprecipitation of the His-tagged deletion mutant, interactions with other KLF11 corepressors/coactivators, namely Sin3a and p300, are not disrupted, supporting mutant specificity. D–H, BiFC of HP1 and KLF11 demonstrates nuclear interaction. Models of predicted results are shown (D). Cotransfection of N-terminal EYFP protein (EYFP(1)) fused to KLF11 with the C-terminal EYFP (EYFP(2)) fused to HP1α demonstrates interaction in the nucleus through fluorescence reconstitution (E) and nuclear DAPI co-stain (F). A negative control leucine zipper protein fused to EYFP with either KLF11 (G) or HP1 (not shown) fused to their respective EYFP halves did not reconstitute fluorescence. DAPI staining is shown to visualize the nucleus (H). Scale bar represents 5 μm.

FIGURE 4

Functional site in the CXCR4 promoter is defined by HP1 recruitment in a sequence-specific manner by KLF11.A, KLF11ΔHP1 increases CXCR4 protein levels. Protein levels of CXCR4 corroborated the expression pattern observed by array and RT-PCR, consistent with derepression by KLF11ΔHP1. Endogenous levels of KLF11 and HP1α protein remained unchanged. α-Tubulin was used as a loading control. OMNI D8 antibody was used to detect ectopic His-tagged KLF11 WT and KLF11ΔHP1 as an expression control. Densitometric analysis expressed as CXCR4/α-tubulin is shown on the right. Error bars represent S.D. * denotes p < 0.05 for EV versus KLF11ΔHP1, as well as KLF11WT versus KLF11ΔHP1. B, KLFΔHP1 derepresses the −800-bp CXCR4 promoter. A −800-bp fragment of the CXCR4 promoter was utilized for luciferase-based reporter assays, validating the expression pattern observed by microarray, RT-PCR, and Western blot. KLF11ΔHP1 significantly derepressed CXCR4 promoter activity, whereas KLF11 WT showed negligible changes. * denotes p < 0.05 for EV versus KLF11ΔHP1, as well as KLF11WT versus KLF11ΔHP1. C, the KLF-HP1 site is located within the −300-bp CXCR4 promoter. Similar results to the −800-bp promoter were observed with a −300-bp promoter, indicating that the KLF-HP1 site is located within this smaller fragment of the promoter. KLF11 WT with knockdown of HP1α by shRNA effectively reproduced the derepression observed with KLF11ΔHP1. Inset shows a control Western blot of HP1α knockdown. The mean ± S.E. from at least three independent experiments performed in triplicate are shown for both reporters. * denotes p < 0.05. D, knockdown of HP1α results in increased CXCR4 protein. Similar to KLF11ΔHP1, siRNA-mediated knockdown of HP1α (siHP1α) increases CXCR4 at the protein level compared with scrambled siRNA control (Scr). HP1α levels are shown as control of efficient knockdown. Endogenous KLF11 protein levels remain unchanged, and α-tubulin was used as a loading control. Densitometric analysis expressed as CXCR4/α-tubulin is shown on the right. Error bars represent S.D. * denotes p < 0.05.

FIGURE 3

Sequence-specific recruitment of HP1 by KLF11 regulates expression of cancer-associated genes.A, KLF11ΔHP1 displays a distinct pattern of gene expression. Heat map of microarray data is shown comparing the expression patterns from Panc1 cells infected with EV, KLF11 WT, or KLF11ΔHP1. Inset displays genes derepressed by KLF11ΔHP1. B, RT-PCR validates the expression pattern of select genes derepressed by KLF11ΔHP1. β2-Tubulin (TUBB2) was used as an internal control. C, KLF11ΔHP1 regulates a large number of cancer-associated genes. Upper chart depicts that 34% of genes derepressed by KLF11ΔHP1 are related to cancer-associated processes. A break-down of these cancer-associated processes is also shown (lower chart).

FIGURE 5

KLF11 and HP1α occupy the CXCR4 promoter in vivo.A, the CXCR4 proximal promoter contains KLF sites. Map of the −500-bp proximal promoter of CXCR4 shows the relative location of 4 putative KLF sites (solid lines labeled 1–4). Numbering of the promoter is relative to the transcription start site (+1, TSS). Half-arrows indicate positions of the primer pairs used in ChIP experiments. B, HP1α and its HMT, SUV39H1, are recruited to the CXCR4 promoter by KLF11. ChIP results demonstrate that in the presence of KLF11 WT, HP1α and SUV39H1 occupy the CXCR4 promoter (left). However, replacement for KLF11ΔHP1 abolishes their recruitment. Recruitment of other KLF11 co-factors, Sin3A, HDAC2, and p300 remains unchanged (right). Mouse IgG (mIgG) and rabbit IgG (rIgG) serve as negative controls. Both KLF11 WT and ΔHP1 proteins bind to this promoter, confirming that KLF11ΔHP1 does not disrupt KLF11 promoter recognition and DNA binding. C, KLF11 and HP1α co-occupy the CXCR4 promoter by sequential ChIP. Sequential ChIP experiments were performed in which chromatin-DNA complexes containing HP1α were immunoprecipitated in the first round and rIgG (HP1α:rIgG) or KLF11 (HP1α:KLF11) in the second round of immunoprecipitation. CXCR4 was detected after sequential HP1α:KLF11 ChIP from KLF11 WT, but not KLF11ΔHP1-transfected cells, confirming that these two proteins co-occupy this region of the promoter. D, histone marks at the KLF-HP1 site change in a manner congruent with different promoter states. ChIP experiments were performed on the CXCR4 promoter for acetyl-H3 (K9, K14), H3K4me3, and H3K9me3. KLF11 WT enriched the region with H3K9me3, but KLF11ΔHP1 lost its ability to support the writing of the H3K9me3 mark. The pattern of histone marks present at the CXCR4 promoter predicts that KLF11ΔHP1 triggers a derepression event due to high levels of acetyl-H3 (K9 and K14) and H3K4me3 with concomitant loss of the H3K9me3 mark. Positive amplification of PCR products is shown in the input DNA lanes demonstrating that the region of the CXCR4 promoter is present in all samples before immunoprecipitation.

FIGURE 6

The KLF site at −119 to −129 mediates HP1 recruitment to the CXCR4 promoter.A, the CXCR4 promoter has four putative KLF binding sites. The diagram represents a schematic of the human CXCR4 proximal promoter (−50 to −190 relative to TSS) containing four KLF elements (underlined, 1–4 relative to TSS). Site 4, highlighted with an asterisk (*), was previously identified as a KLF2 site (46). B, KLF11 binds to site 3 of the CXCR4 promoter in vitro. EMSA was performed using recombinant KLF11 protein (lanes 1, 3, 5, and 7) or control GST protein (lanes 2, 4, 6, and 8) with radiolabeled oligonucleotides for each of the four putative KLF binding sites, as indicated. Each probe was also loaded without protein (probe alone lanes, a–d). A KLF11/CXCR4 oligo complex was significant with site 3 (lane 5), as indicated by the arrow. C, KLF11 binding to site 3 demonstrates binding specificity in vitro. EMSA was performed on WT CXCR4 KLF site 3 (WT site 3; lanes 1 and 3–8) with GST protein (lane 3), recombinant KLF11 (KLF11; lanes 2 and 4–8), or probe alone (lane 1). Specific complexes between KLF11 and probe, as well as the free probe, are indicated by the arrows on the left. Although a GST antibody disrupted the KLF11/CXCR4 WT site 3 complex (lane 5), the same amount of anti-mIgG did not (lane 6), indicating specificity. Excess unlabeled WT site 3 probe robustly competed for binding (×125 and 250; lanes 7 and 8, respectively), whereas excess mutant probe did not (×125; lane d). Radiolabeled mutant probe failed to bind recombinant KLF11 (lane c). Probe alone (lane a) and probe with GST protein (lane b) are shown as controls. D, functionality of the CXCR4 KLF-HP1 site is provided by an intact site 3. Activity of the site 3 mutant CXCR4 promoter was no longer derepressed by KLF11ΔHP1, indicating disruption of the KLF-HP1 site, whereas KLF11 WT showed negligible changes. Graphical depiction of the results is the mean ± S.E. from two independent experiments performed in triplicate.

FIGURE 7

HP1 recruitment is necessary for KLF11-mediated apoptosis and senescence.A and B, HP1 plays a role in the ability of KLF11 to induce apoptosis., Apoptosis was measured by both, nuclear morphology via Hoechst 33342 staining (A) and caspase 3 cleavage assay (B). As observed by Hoescht staining (A), KLF11 WT increases apoptosis. KLF11ΔHP1 is defective in inducing this effect, with an apoptotic index similar to EV. The depicted results are the mean ± S.E. from three independent experiments. In caspase 3 cleavage assays (B), KLF11 WT increased the amount of caspase 3 cleavage, as shown by Western blot (upper), whereas KLF11ΔHP1 had reduced levels similar to control. Pro-caspase 3 was used as a control (lower). The levels of caspase 3 cleavage are shown for both, 48- and 72-h post-serum starvation. C, KLF11ΔHP1 demonstrates an increase in adherent cell number compared with KLF WT. Assessment of adherent cell count is shown after normalization to EV. Although KLF11 WT results in a significant decrease in cell numbers, this effect is blunted with KLF11ΔHP1. The graph represents the mean ± S.E. of results from three independent experiments. D and E, loss of HP1 recruitment eliminates the ability of KLF11 to increase senescence. To measure cells undergoing senescence, senescence-associated β-galactosidase staining was performed in primary fibroblasts. KLF11 increased the percentage of cells undergoing senescence. However, KLF11ΔHP1 lost this ability, behaving similar to control. The graph (D) depicts the mean ± S.E. from three independent experiments. A representative picture of senescence-associated β-galactosidase staining for each experimental group is shown (E). F, KLF11ΔHP1 is no longer able to repress the telomerase promoter. Activity of the hTERT promoter, as measured by the luciferase reporter assay, was significantly repressed by KLF11 WT, however, KLF11ΔHP1 loses this repression. Graphical depiction of the results is the mean ± S.E. from at least three independent experiments performed in triplicate. * denotes p < 0.05.

FIGURE 8

HP1 recruitment is required for KLF11-mediated suppression of neoplastic cell growth.A and B, KLF11ΔHP1 is no longer able to suppress KRAS-mediated foci formation in NIH/3T3 cells. A representative photo (A) is shown for each: KLF11ΔHP1 alone (no KRAS), EV control + KRAS, KLF11 WT + KRAS, and KLF11ΔHP1 + KRAS. Note that KLF11ΔHP1 does not form foci alone in the absence of KRAS. Graphical depiction of the results (B) as the mean ± S.E. from three independent experiments performed at least in triplicate demonstrates that KLF11 WT significantly suppressed KRAS-mediated foci formation by 48%, whereas KLF11ΔHP1 failed to suppress KRAS-mediated foci formation. C and D, recruitment of HP1 is necessary for KLF11-mediated suppression of colony growth in soft agar. Panc1 cells were infected with adenovirus containing EV control, KLF11 WT, or KLF11ΔHP1 and monitored for visible colonies in soft agar. A representative photo is shown for each condition, including agar plated with no cells (C). KLF11 WT suppressed soft agar colony formation by ∼60%; however, KLF11ΔHP1 was unable to suppress colony formation. The graph (D) represents the mean ± S.E. from three independent experiments performed in triplicate. E and F, HP1 is necessary for KLF11-mediated tumor suppression in vivo. Flanks of athymic nude mice were injected with L3.6 pancreatic cancer cells transfected with control (EV), KLF11 WT, or KLF11ΔHP1. E, representative images of flank tumor burden (upper) and excised tumors at time of sacrifice (middle) are shown. Western blot with the OMNI D8 antibody confirms ectopic expression of His-tagged KLF11 WT and mutant proteins in injected cells (lower). F, graph depicts the mean tumor volume and S.D. at each week post-injection until sacrifice. KLF11 WT significantly decreases tumor size compared with control. However, KLF11ΔHP1 significantly impairs KLF11 tumor suppression, as seen in tumor sizes comparable with controls rather than KLF11 WT. * denotes p < 0.05.

FIGURE 9

Model of HP1 recruitment in KLF11-mediated gene regulation. An extended KLF11 binding site mediates the sequence-specific recruitment of HP1-SUV39H1 to regulate gene promoters (upper). Our experimental data demonstrate that engagement of HP1-SUV39H1 recruitment by KLF11 binding leads to the deposition and/or removal of key histone marks that change the state of chromatin and impact on transcription. Disruption of this recruitment mechanism, as achieved by KLF11ΔHP1, leads to derepression of gene promoters (lower).