Kaiso (ZBTB33) engages with non-canonical binding motifs to regulate DNA damage responses in breast cancer cells

Kaiso (ZBTB33) engages with non-canonical binding motifs to regulate DNA damage responses in breast cancer cells | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Kaiso (ZBTB33) engages with non-canonical binding motifs to regulate DNA damage responses in breast cancer cells Maximillian Rätze, Milou Tenhagen, Juul Vollaers, Christopher Benner, and 9 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7846387/v1 This work is licensed under a CC BY 4.0 License Status: Under Revision Version 1 posted 11 You are reading this latest preprint version Abstract In breast cancer, nuclear localization of Kaiso (ZBTB33), a dual specificity transcription factor, is a hallmark of high grade ductal-type carcinomas, especially in estrogen receptor negative disease. Regulation of gene expression by Kaiso is orchestrated via its engagement to distinct Kaiso binding sequence (KBS) motifs defined as canonical (cKBS; TCCTGCNA ) or CG-containing palindromic KBS (CG-KBS; TCTCGCGAGA ), depending on context. While there exists a clinical connection between localization of Kaiso expression and breast cancer, it remains unclear how Kaiso controls bi-modal canonical versus noncanonical transcriptional modulation. Here, we have combined Kaiso-specific chromatin immunoprecipitation (ChIP) and Kaiso-Dam methyltransferase identification (Dam-ID) approaches to map genomic Kaiso binding sites in breast cancer cells. We find that Kaiso mostly occupies the non-canonical CG-KBS sites in transcriptionally active and hypo-methylated (H3K4me3 and H3K27Ac enriched) promoter regions. Noncanonical CG-KBS targets are actively transcribed and linked to fast biological processes such as metabolism, cell cycle regulation and DNA damage repair. Functionally, we show that Kaiso expression is essential to prevent DNA damage in breast cancer cells. Loss of Kaiso leads to reduced expression of DNA damage response gene expression and treatment with the chemotherapeutic agent cisplatin leads to overt accumulation of DNA damage in cells devoid of Kaiso. Our data thus favor a model in which Kaiso promotes fast transcriptional activation of key cellular processes in cancer cells through binding of its non-canonical consensus site. Biological sciences/Biochemistry Biological sciences/Cancer Biological sciences/Cell biology Biological sciences/Computational biology and bioinformatics Biological sciences/Molecular biology Health sciences/Oncology Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Introduction Kaiso (ZBTB33) is a member of the zinc finger and broad-complex, tramtrack and bric-à-brac or poxvirus and zinc finger (BTB/POZ-ZF) transcription factor family that was initially identified in a yeast-two-hybrid screen as a p120-catenin (p120) binding partner 1 . In that setting, Kaiso functions as a transcriptional repressor through binding to a specific DNA sequence, TCCTGCNA , defined as the canonical Kaiso Binding Sequence (cKBS) 2 . The interaction between Kaiso and p120 results in repression relief of Kaiso target genes like Wnt11 3 , Cyclin D1 ( CCND1 ) 4 , Matrix metalloproteinase 7 ( MMP7 ) 2 and ID2 5 . Relief of canonical Kaiso target gene suppression is dependent on nuclear translocation of p120 through a conserved NLS signal, an event exacerbated in anchorage-independent conditions 6 , 7 . More recently, the palindromic CG-containing consensus binding site TCTCGCGAGA (CG-KBS) has been shown to be required for promoter-proximal noncanonical Kaiso-dependent recruitment of SMRT in terminal adipogenesis. Following this, the CG-KBS was also identified based on Kaiso chromatin immunoprecipitation (ChIP) data and subsequent in silico analyses 8 , 9 . Interestingly, while it was initially shown in vitro that methylation of this site is required for binding, analyses performed in the ENCODE dataset revealed that binding to the CG-KBS overlaps with highly active promoters and high levels of acetylated histones but without methylation enrichment of these sites, suggesting a noncanonical role for this particular Kaiso binding site in transcriptional activation. Binding of Kaiso to either the cKBS or CG-KBS occurs through three zinc finger domains that recognize specific bases in the major groove of the cKBS and CpG-KBS sites through both classical and methyl hydrogen bonds 10 , 11 , or residues in the Kaiso C-terminus 12 . Kaiso can directly affect transcription by binding to CG-KBS sites in the promotor of Metastasin ( S100A4 ) 10 , Metastasis associated 1 family member 2 ( MTA2 ) 13 and Cyclin-dependent kinase inhibitor 2A ( CDKN2A ) 14 . Loss of Kaiso results in major developmental defects at the gastrulation stage in Xenopus and zebrafish embryos, which has been coupled to aberrant regulation of both canonical and non-canonical WNT target genes 3 , 4 , 15 , 16 . Given the assumed convergence of parallel p120/Kaiso and canonical TCF/LEF target genes in vertebrate development, these studies suggested a possible role for Kaiso in cancer (reviewed in: 17 ). Histological assessments have indicated that Kaiso localization is enriched in the cytoplasm in thymic carcinoma, non-small cell lung cancer, lobular breast cancer and colon cancer cells when compared to healthy tissues, a finding that associates with decreased overall survival and/or high-grade tumors 18 – 22 . In contrast, high grade prostate and breast cancers are characterized by elevated levels of nuclear Kaiso, a feature that is also associated with a poor prognosis 23 , 24 . While Kaiso binding to its canonical site has been extensive studied, the functional consequences of Kaiso binding to the CG-KBS remain unclear. Because of this gap in knowledge and the suggested involvement of nuclear Kaiso in breast cancer, we set out to perform a comprehensive analysis to identify the genome-wide Kaiso binding sites in breast cancer using both chromatin immunoprecipitation (ChIP) and DNA adenine methyltransferase identification (Dam-ID). We found that Kaiso binding is enriched at the noncanonical CG-containing KBS in active promotor regions. Our data identify non-canonical KBS genes that are implicated in fast processes important in high grade cancer, such as metabolism, cell cycle control and DNA damage repair. Materials and Methods Cell culture Trp53 Δ/Δ -3 mouse mammary carcinoma cells ( aka KP6) and human MCF7 cells were cultured as described 5 . Ecotropic Phoenix (293T) cells were grown in DMEM-F12 (Sigma-Aldrich, Amsterdam, The Netherlands) containing 6% fetal bovine serum (Sigma-Aldrich), 100 IU/mL penicillin and 100 µg/mL streptomycin (Lonza, Geleen, The Netherlands). Immunofluorescence Cells were grown on glass coverslips to 70% confluence and washed with Ca 2+ /Mg 2+ containing PBS. Cells were fixed with 4% PFA in PBS for 10 minutes at room temperature. Permeabilization was performed using 0.3% Triton X-100 in PBS for 3 minutes, followed by blocking in 4% BSA/PBS for 15 minutes. Samples were incubated with the primary antibody mouse anti-V5 (1:1000, 46–0705, Invitrogen, Carlsbad, CA) overnight at 4°C and rabbit anti-GFP (SC-8334, Santa Cruz, Dallas TX) for 2 hours at room temperature. Secondary antibody incubation with goat anti-mouse-Alexa Fluor 568 (1:600, A11031, Invitrogen) and goat anti-rabbit-Alexa Fluor 488 (1:600, A11034, Invitrogen) took place at room temperature for 1 hour. Samples were stained with DAPI for 5 minutes and mounted with Immu-Mount (Thermo Fisher Scientific, Carlsbad, CA). Slides were imaged using a Zeiss LSM 700 (Carl Zeiss, Oberkochen, Germany) and processed using ImageJ and Photoshop CS6 (Adobe, San Jose, CA). Cloning and retroviral transduction for DAM-ID To generate the pMSCV-EcoDam-V5-Kaiso-puro expression vector, Gateway Cloning was used to recombine mouse Kaiso cDNA (provided by P. McCrea, M.D. Anderson Cancer Center, Houston, Texas 58 ) into pMSCV-EcoDam-V5-puro 59 by LR reaction. Next, Ecotropic Phoenix cells were transfected with pMSCV-EcoDam-V5-GFP-puro 59 and pMSCV-EcoDam-V5-Kaiso-puro using XtremeGene 9 (Roche). After 48 hours, supernatant containing the virus was collected and filtered through a 45 µm filter before transducing Trp53 Δ/Δ -3 cells in the presence of 4 µg/mL polybrene (Sigma-Aldrich). After 72 hours, transduced cells were selected using puromycin (10 µg/mL). Chromatin immunoprecipitation ChIP was performed as described previously 60 . In short, cells were cross-linked with 2 mM DSG (Thermo Fisher Scientific) and 1% formaldehyde. Incubation with nuclear extraction buffer (20 mM Tris, 10 mM NaCl, 2 mM EDTA, 0.5% NP-40) was performed to lyse the cells and enrich for nuclei. Lysates were collected in sonication buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% NP-40 and 0.3% SDS) and chromatin was sheared to 200 to 600 bp fragments by ultrasonication at maximum power for 8 minutes (M220, Covaris, Brighton, UK). Immunoprecipitation was performed using antibodies directed against the following proteins: Kaiso (10 µg, 6F, 12723, Abcam, Cambridge, UK), RBP1 (5µg, PB-7C2, Euromedex, Souffelweyersheim, France), H3K4me3 (5µg, ab8580, Abcam) and H3K27Ac (5µg, ab4729, Abcam). DNA was purified using standard phenol/chloroform extraction and used for library preparation and sequencing on a HiSeq2000 (Illumina). mRNA sequencing Cells were seeded into a 6-well plate and grown to 80% confluence in serum-containing medium. After washing in Ca 2+ /Mg 2+ -containing PBS, RNA was isolated and purified using the RNeasy kit (Qiagen), followed by DNase treatment (Qiagen, Venlo, The Netherlands). RNA library preparation was performed as previously described 61 and used for cluster generation on a HiSeq 2000 (Illumina, Cambridge, UK). Kaiso CRISPR/Cas9-mediated targeting and Knockout Kaiso (ZBTB33) Knockout was performed using the previously described Baculoviral delivery of CRISPR/Cas9 62 . A guide RNA targeting ZBTB33 was designed (5’- GAG GCT TAT CGA CTG GTT GCA -3’) using the online CRISPR design tool ( http://crispr.mit.edu/ ). Guide RNAs were cloned into the BbsI site of the baculovirus donor plasmid pAceBAC1-GFP (a kind gift from Dr. Michael Hadders, Center for Molecular Medicine, Utrecht, Netherlands), which was subsequently recombined with bacmid DNA in EmBacY competent cells. Two partly overlapping gDNA arms (1kb each) for the CRISPR/Cas9 mediated incorporation of the E535A mutation were amplified through PCR with gDNA isolated from mouse KP6 cells using primers 5’ – TTC TGA AGC GGC CGC TTT CAG ATG TTG CAC CTA GTG CT − 3’ (F1mut) and 5’ – GCT TTG TGC GAT AT G CTG CAA GAG GAA ATA CCT TCT CAC AGT AAC GAC ACG GAT ACT TCT TCT CCC AAG − 3’ (REV1)(arm 1) and primers 5’ – CCT CTT GCA G C A TAT CGC ACA AAG CAT GAA ATT CAT CAC ACA GGA GAG CGA AGG TAT CAG TGT TTG G − 3’ (F2EA)and 5’ – TTC GAA GCG GCC GCC TTG GAC TAG CAG ATT ACA CAA CC − 3’(REV2) (arm 2). The E535A mutation is indicated in bold, the PAM site mutations are underlined. Next, a classic heteroduplex protocol with subsequent amplification using with primers F1mut and REV2 The homologous arm was cloned into the pAceBAC-GFP vector containing the guideRNA using the NotI site. Resulting Bacmids were transfected into Sf9 insect cells for baculovirus production. After transduction, GFP + cells were identified using a fluorescence microscope (Leica DMIL801, Leica, Amsterdam, The Netherlands), seeded as single cell clones, for subsequent verification of Kaiso knockout by TIDE analysis 63 and Western Blot. DNA damage analysis Cells were seeded at a density of 50,000 cells per 24-well on glass cover slip 24 hours prior to treatment, after which cells were incubated with 0.1 µM cisplatin for 24 hours, washed and fixed with 4% paraformaldehyde for 5 minutes at room temperature. Cells were permeabilized using 0.3% Triton X-100 for 3 minutes, blocked with 4% BSA and incubated overnight at 4°C with mouse anti phospho-γH2AX (1:1.000; #05-636, Millipore, Darmstadt, Germany). Samples were imaged in triplicate using a Zeiss LSM 700 (Carl Zeiss). Images were processed using ImageJ, to adjust brightness and count foci per nucleus. A minimum of 20 high power fields containing on average 20 nuclei were quantified. Computational analysis of ChIP and DAM-ID For mRNA-sequencing, reads were uniquely mapped to the mouse (mm9) reference genome using the ELAND or BWA program, allowing 1 mismatch, and subsequently used for bioinformatic analysis. RPKM (reads per kilobase of gene length per million reads) values for RefSeq genes were computed using tag counting scripts and used to analyze the expression level of genes. ChIP sequencing reads were processed and mapped to the mouse (mm9) reference genome using BWA programming allowing 1 mismatch. ChIP-peaks were identified using Model based Analysis of ChIP-seq (MACS1.3.3) with a cutoff p-value of 10 − 7 for peak detection 64 , 65 . Dam-ID sequencing reads were aligned to the mouse genome (mm9) using Bowtie. Only reads aligning uniquely to a single genomic location and aligning just downstream from a GATC sequence were used for downstream analysis. Putative Dam-ID-peaks were identified using HOMER 66 using an initial peak-size of 2,500 bp and a 3-fold enrichment of normalized Dam-ID-Kaiso reads over Dam-ID-GFP control reads. In addition, peaks were required to contain at least 100 normalized reads per peak (per 10 million reads sequenced) to remove low magnitude sites. For comparison between ChIP-peaks and Dam-ID-peaks, the ‘mergepeaks’ function of HOMER was used, applying a maximal distance between the peak centers of 2000 bp. HOMER was also used to determine the location of Kaiso binding sites in the genome, the overlap of Kaiso binding sites with CpG islands, and (de novo) motif analysis. Additionally, using the ‘annotatepeaks’ function, tag enrichment histograms of Kaiso ChIP, Dam-ID, RNA polymerase and histone marks were generated. DAVID gene ontology database 67 was used to identify enriched functional annotations. In addition to statistical analysis included in the aforementioned software packages, R (2.15.3, R Core team 2011) and Excel (Microsoft, Redmond, WA) were used to analyze RNA-seq data and perform Pearson’s correlation testing. Reverse transcriptase quantitative PCR Total mRNA was extracted from cell or organoid pellets using Trizol reagent (Thermo Fisher Scientific). Poly-T primers and a cDNA transcription kit (iScript Synthesis kit, Biorad, Veenendaal, The Netherlands) were used to generate cDNA. PCR primer sets used to evaluate expression values for the gene set are listed in Supplementary Table 3. Primer efficiency was assessed by serial dilution. Expression values were generated using ∆∆Ct values normalized to GAPDH and/or ACTB . Experiments were performed in triplicate over three independent biological and technical settings, using the CFX96 Real-Time System and CFX manager software (both Biorad). For each comparison, unpaired two-tailed Student’s t-tests were used to determine statistical significance. Results Identification of the genome-wide Kaiso binding sites by ChIP and Dam-ID Although clinical evidence implicates Kaiso in breast cancer, its genome-wide role has yet to be clarified. To shed light on the genomic regions at which Kaiso engages chromatin, we performed chromatin immunoprecipitation (ChIP) and Dam methyltransferase identification (Dam-ID) of Kaiso, followed by next generation sequencing (NGS) (Fig. 1 A). Nuclear translocation of p120 and its possible confounding impacts on Kaiso binding to DNA were circumvented by using E-cadherin expressing mammary ductal-type breast cancer cells that were derived from a conditional p53 knockout mouse model of breast cancer 25 . Using this setup, we performed genome wide ChIP analysis and identified 6,713 peaks (Fig. 1 B). In parallel, we performed Kaiso-specific Dam-ID to complement findings in the ChIP experiments. To minimize a-specific methylation by the fused DAM methylase and Kaiso proteins (Dam::Kaiso), we used retroviral transduction to achieve stable but low expression of Dam::Kaiso (SFig. 1). Methylated sequences were PCR-amplified for detection by NGS from cells expressing Dam::Kaiso or (control) Dam::GFP to identify Kaiso-specific Dam-ID-peaks. This was done by calculating the ratio of normalized Dam::Kaiso reads over Dam::GFP reads, setting a 3-fold increase cut-off Dam::Kaiso reads as specific. In total, we identified 16,288 Dam-ID-peaks (Fig. 1 B and 1 C). Comparison of Kaiso ChIP-peaks with Dam-ID-peaks revealed a positive correlation between Kaiso ChIP tags and the ratio of normalized Dam::Kaiso over Dam::FP tags and an approximate 10-fold enrichment (r = 0.342, Fig. 1 C). Moreover, we observed a positive correlation between the number of Kaiso ChIP and Dam::Kaiso tags (r = 0.310, Fig. S2A), while there is no correlation between Kaiso ChIP tags and Dam::GFP tags (r = 0.155, Fig. S2B). We mapped 1,321 overlapping regions between the identified ChIP and Dam-ID peaks, which comprise roughly 20% of all ChIP-peaks identified (Fig. 1 C and Fig. S2C-F). Overall, ChIP-seq identified 5,392 Kaiso binding sites that do not overlap with those mapped using Dam-ID. Conversely, Dam-ID yielded 14,967 potential Kaiso binding sites that were not identified using ChIP. As expected, DAM-ID peaks are distributed in a typical biphasic pattern normalized at positions juxtaposed upstream and downstream of the Kaiso ChIP peak (Fig. 1 D). Kaiso binds to a CG-containing KBS sequence in promotor-regions Analysis of Kaiso binding sites identified by ChIP-seq revealed a high occupancy of Kaiso in promotors and 5-prime UTR regions, representing a 34- and 48-fold enrichment respectively (Fig. 2 A). Kaiso binding sites identified by Dam-ID-seq are also enriched at these genomic locations surrounding the transcription start site (TSS; 3.8 and 3.6-fold respectively), although less pronounced as seen for ChIP-seq (Fig. 2 A). Kaiso-binding was enriched at regions within 1 kb flanking the TSS for both the ChIP and DAM-ID experiments (Fig. 2 B). Interestingly, Kaiso binding to promotors based on our ChIP experiments primarily occurs in CG-rich regions (Fig. 2 C). Although this can be explained by abundant Kaiso binding in promotor regions, which are generally rich in CG islands, we observed that 90.6% of all ChIP-peaks in promotors overlap with a CG island, while 53% of all mouse promotors contain a CG island. Subsequent motif analysis revealed a palindromic CG-containing motif that was previously mapped as a Kaiso binding sequence based on publicly available (ENCODE) ChIP data in myeloid (K562) and lymphoid (GM12878) cells 8 , 9 . The identified palindromic motif consists of 10 core nucleotides ( TCTCGCGAGA ; ZBTB33) that we refer to as the non-canonical (CG-containing) Kaiso Binding Sequence (CG-KBS)(Fig. 3 A). Of note, 234 of the total 6,713 Kaiso ChIP peaks contained the canonical KBS ( TCCTGCNA ), which is not a significant enrichment over the genome-wide occurrence of this motif. De novo discovery did not identify additional motifs aside from a truncated version of the CG-KBS (Supplemental Fig. 3). However, in addition to the CG-KBS, we identified CTCF and its paralogue BORIS as significantly enriched motifs (Fig. 3 A). Although CTCF and BORIS can act as insulators that facilitate chromatin loops and occupy promoter-proximal regions 26 , we did not observe enrichment of their motifs in promoters in our dataset (Fig. 3 B), suggesting a context-specific binding profile. The CG-KBS is a common promotor element occurring in 5% of all human TATA-less promotors and linked to expression of genes that control fast processes 27 . Indeed, we find that approximately 90% of the identified CG-KBS peaks occur within a region 1 kb up- or downstream of the transcriptional start site (TSS) in the mouse genome (Fig. 3 C). Detailed positional examination of the CTCF and CG-KBS motifs relative to the site of Kaiso-binding revealed an interesting pattern. While the CG-KBS displayed an expected sharp enrichment at the ChIP-peak centers, the CTCF motif was more broadly positioned around the peak signal (Fig. 3 D). These findings suggest that Kaiso and CTCF could function as co-regulators of a common gene, as has been observed for β-globin 28 . Finally, motif analysis solely on the Dam-ID data also identified several members of the AP1 transcription factor family in close vicinity of Kaiso binding sites (Fig. 3 A). However, inspection of the regions flanking the Kaiso binding sites from the ChIP assays did not show an enrichment of AP1 family member or any other known or novel motifs. In conclusion, we have identified two subsets of Kaiso binding sites using combined ChIP and Dam-ID; promotor-proximal CG-KBS-containing sites and sites in close vicinity of the CTCF/Boris motif distal to the TSS. Kaiso binding to the CG-KBS occurs in transcriptionally active promotors involved in processes linked to cancer progression. To assess which pathways could be affected by Kaiso repression through the CG-KBS, we first characterized the chromatin landscape of Kaiso-bound regions. We performed ChIP-sequencing for histone 3-lysine 4-tri-methylation (H3K4me3), an epigenetic active histone mark that is enriched at promotor regions with an open chromatin state. Additionally, we identified active enhancers and promotors of transcribed genes by performing ChIP-seq for histone 3-lysine 27 acetylation (H3K27Ac) and regions of active transcription by RNA polymerase II (Pol2) ChIP-seq 29,30 . Kaiso binding sites were divided into two groups; sites present within a promotor (defined as peaks occurring within 1 kb up- or downstream from the TSS) containing the CG-KBS, and sites without an apparent consensus site within a promoter. Although both Kaiso targets sites were accompanied by a high occupancy of all three markers of active transcription, this was particularly evident at the CG-KBS (Fig. 4 A). The presence of Pol2 marks at Kaiso target gene promoters did not correlate with a significant increase in mRNA expression levels (Fig. 4 B). To gain insight into the pathways and biological processes linked to CG-KBS target genes, we performed Gene Ontology Enrichment analysis, which revealed a significant enrichment of genes controlling processes such as cellular metabolism, RNA transcription, cell cycle regulation and DNA damage repair (DDR)(Fig. 4 C). We did not find a correlation between CG-KBS Kaiso targets and the specific stages of cell cycle regulation or types of DNA damage responses (data not shown). Taken together, our analyses show that Kaiso binding is enriched at the non-canonical Kaiso motif in promotor regions of actively transcribed genes that control distinct cellular processes important for cancer progression. Kaiso prevents cisplatin induced DNA damage in breast cancer cells. To specify the functional impact of the CG-KBS in cancer, we devised a strategy to use CRISPR-Cas9 to perform homozygous mutation of the methyl-specific recognizing residues Glu-535 and Tyr-536 in the second zinc finger of Kaiso. These residues were identified based on amide chemical shifts comparing the nuclear magnetic resonance spectra of Kaiso in complex with either the canonical KBS or the CG-KBS 31 . We hypothesized, also based on published electromobility shift data 32 , that mutation of the E535 residue would impact the CG-KBS interaction. Upon targeting of this mutation in human MCF7 breast cancer cells we screened approximately 150 clones for genetic mutations (Supplemental Fig. 4). Surprisingly, we did not obtain viable clones that contained homozygous E535A alleles. We did however, obtain clones that showed heterozygous knock-in of the E535 mutation, but this was uniformly accompanied by the presence of either a wild type or a knockout allele (Supplemental Table 1). Subsequent biochemical experiments revealed that these heterozygous knock-in clones did not express Kaiso (Supplemental Table 1). Based on these results, we conclude that homozygous mutation of the sites necessary for KBS binding is not compatible with cellular viability in MCF7. Nonetheless, using this approach, we generated Kaiso knock-out clones (Fig. 5 A), of which two were used for further functional analysis. For this, we focused on the possible cancer progression impact of Kaiso in the modulation of DDR responses. We selected DRR genes that were identified using the Kaiso ChIP experiments (Supplemental Table 2) and compared transcript expression levels by quantitative rt-PCR (qPCR) in MCF7 control versus MCF7 Kaiso knockout (MCF7::∆Kaiso) cells. We started by selecting a list of 26 DDR genes with a proximal site of Kaiso, as identified by ChIP-seq in mouse cells. Next, we cross-referenced these 26 genes to the 1,000 base pair promoter regions of the human gene orthologs from the MCF7 Encode ChIP-seq database to verify Kaiso binding 33 – 35 . After confirmation of specificity, we designed qPCR primers for a set of 11 genes based on expression levels in MCF7 (bold genes, Supplemental Table 2). Based on these transcriptional analyses, we detected a reduction of all 11 DDR genes assessed within this cohort (Fig. 5 B). Next, we assessed the impact of Kaiso knock-out on DNA damage induced by cisplatin, a DNA intercalating agent that causes DNA double strand breaks. DNA damage was assessed by immunofluorescence and quantification of phosphorylated (p-) γH2A.X (Ser139) DNA damage foci, an established marker for double strand break repair 36 . We observed that a low concentration (0.1 µM), that does not induce overt DNA damage in control MCF cells, causes a stark significant increase in p-γH2A.X DNA damage foci in MCF7::∆Kaiso cells (7.5 foci/nucleus versus 26.5 and 34.2 foci/nucleus respectively, p = 0.001, Fig. 5 C and 5 D). These results indicate that Kaiso protects breast cancer cells from DNA damage and suggest that this protection is established through positive transcriptional regulation of CG-KBS dependent DDR target genes. Discussion Kaiso is a versatile and context-dependent transcription factor, with a propensity to bind the canonical cKBS and/or the non-canonical CG-KBS 1 , 8 . Growing evidence indicates that Kaiso may have a dual function as both a repressive and activating transcription factor, depending on the DNA sequence occupied 5 , 7 , 8 , 10 , 37 . Kaiso functions as a repressor of genes that play a role in breast cancer such as CCND1 , WNT11 , MMP7, ID2 and ERBB3 , a function presumably through a p120-dependent mechanism upon loss of E-cadherin 38 , 39 . In cancer, Kaiso cytosolic localization and expression carries prognostic value in colorectal, breast, prostate and lung cancer (Reviewed in: 17,40 ). However, Kaiso is found enriched in the nucleus in high grade breast cancer, a prognostic association that implies roles in transcriptional activation 22 . To study Kaiso in this context, we employed E-cadherin expressing breast cancer cells and performed genome-wide Kaiso-specific ChIP-seq and Dam-ID-seq in proliferating cells to identify Kaiso binding sites and the resulting transcriptional programs. This setup yielded a 20% overlap between ChIP and Dam-ID peaks, which is comparable to previous studies 41 , 42 . Interestingly, our data reveal that many of the identified Kaiso binding sites contain the CG-KBS, a finding that is in line with the publicly available ENCODE data 9 . Surprisingly, the cKBS was not significantly enriched on a genome-wide scale in our dataset. Furthermore, we did not find enrichment of binding sites that have dual-interaction with both the cKBS and CG-KBS such as in the CCND1 promoter 43 , suggesting that this specific dual specificity DNA binding mechanism might be unique to CCND1 . Taken together, our data suggest that Kaiso-dependent transcriptional regulation in high grade breast cancer functions as an activating transcription factor in hypomethylated areas via the CG-KBS. Consistent with the above, Kaiso binding sites are enriched with histone marks related to open chromatin conformation (H3K4me3 and H3K27Ac) and RNA polymerase II, suggesting active transcription. These data strengthened our hypothesis that CG-KBS-dependent gene regulation by Kaiso promotes active gene transcription, either by direct transcriptional activation, or by promoting an open chromatin formation of the bound promoter. In support of this, it was shown previously that SUMOylation of K42 in Kaiso allows a switch from transcriptional repression to activation 37 . Since this post-translational event occurs in the BTB/POZ domain, it could prevent dimerization, or interaction and subsequent repression with DNA methylation complexes such as N-CoR and SMRT 1 , 13 , 44 . Moreover, Kaiso deficiency in mouse embryonic fibroblasts induces hypomethylation of binding sites for Oct4 and Nanog 45 , suggesting possible recruitment of methylation machinery to CG-KBS. We find that most of the Kaiso binding sites are enriched for the CG-KBS consensus in hypomethylated, actively transcribed promotor regions. Our findings agree with previous analyses by Blatter et al. , who have used ENCODE to show that binding of Kaiso to the CG-KBS occurs to mostly unmethylated CpGCpG-nucleotides, supportive of Kaiso being able to bind the CG-KBS independently of methylation status 9 . In contrast, others have shown that Kaiso might preferentially bind methylated CG-KBS sites. However, these data have been produced in vitro and therefore not conclusively exclude binding of Kaiso to an unmethylated CG-KBS in vivo 8 . How methylation of the CG-KBS affects Kaiso binding and/or downstream biological functions in vivo is currently largely unknown, especially since methylation of CGs is highly dependent on a multitude of factors such as cell type and culture conditions. In the context of cancer, hypermethylation of CG islands in promoters of hormone receptor target genes frequently occurs, with subsequent reduced expression 46 – 48 . However, most CG islands in vivo are hypomethylated and the associated genes show increased expression 49 . Supporting this is our observation that a coincidence of CTCF / BORIS motifs with Kaiso binding sites, suggesting that Kaiso may additionally be involved in transcriptional regulation more distal from promoters, similarly to the transcriptional regulation of β-globin 28 . Although Kaiso and CTCT were identified independently as protein-protein interacting partners via yeast 2-hybrid assays 50 , there appears to be no direct binding affinity between the 2 factors 51 .Therefore, because we mostly find CTCF motifs independently from CG-KBS sites, we propose that Kaiso may interact with CTCF indirectly. Our data point to an enrichment of Kaiso binding in promoters involved with cell cycle processes, cellular metabolism and DDR responses. Functionally, we show that Kaiso is essential for a proper DDR response; we observe a marked increase in DNA damage foci when comparing Kaiso-null versus Kaiso-expressing MCF7 cells after low dosage cisplatin treatment. Given these findings, we conclude that Kaiso may drive processes that require fast transcriptional activation of genes through the CG-KBS. In contrast, cKBS-dependent transcriptional regulation by Kaiso causes repression of transcription and appears to mediate highly specialized differentiation functions through canonical and non-canonical Wnt signaling 2 – 4 , 52 . These signals are essential for vertebrate development (for detailed reviews see 53 , 54 ) and require oncogenic activation to drive progression in colon and breast cancer 5556 . Although the function of subcellular Kaiso localization is still unclear, phosphorylation at Threonine 606 leads to Kaiso accumulation in the cytoplasm 57 . These alternative mechanisms reveal yet another level of regulation, possibly modulating Kaiso binding towards the cKBS, allowing for an optional regulation of fast responses versus slow transcriptional differentiation programs, respectively. The target genes identified here suggest that indeed, Kaiso binding to the CG-KBS is mostly responsible for the modulation of pro-oncogenic processes that require acute upregulation such as DDR, whereas binding of Kaiso to the cKBS is required for the regulation genes that control development, inhibit proliferation and foster anchorage independence of breast cancer cells, such as WNT11 , ERBB3 and ID2 3,5,7 . In short, we conclude that nuclear Kaiso acts as a transcriptional activator of oncogenic processes that underpin tumor progression in high-risk, high grade breast carcinomas. Declarations All authors declare that they have no competing financial interests in relation to the work described. Acknowledgements Members of former Hetzer lab are acknowledged for their help and suggestions. We thank Corlinda ten Brink and the UMC Utrecht Cell Microscopy Center for imaging support. We acknowledge Michael Hadders for assisting us with generating CRISPR/Cas9 Baculoviruses to perform knockout of Kaiso. Ethical Declaration Ethics approval was not required for this study. Funding Declaration Research was supported by grants from the UICC Yamagiwa-Yoshida Memorial International Cancer Study Grant (YY1/13/001), The Netherlands Organization for Scientific Research (NWO/ZonMW-VIDI 016.096.318) and the Dutch Cancer Society grants KWF-UU-2011-5230 and KWF-10245. Author Contributions: MAKR and MT were responsible for designing and executing experiments, performing data analyses and writing of the manuscript. JV and MIAN performed data analyses. AM, AAS supported and performed ChIP experiments and subsequent data analysis. MH, JHM and HGS provided reagents and feedback on the report. JMD provided conceptual input, reagents and contributed to writing the report. SP performed data analyses for the genome wide ChIP and DAM-ID data and contributed to writing the manuscript. PWBD designed the study, performed and analyzed DNA damage repair experiments, provided supervision and wrote the report. Data availability Genomic data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE309273. References Daniel JM, Reynolds a B. The catenin p120(ctn) interacts with Kaiso, a novel BTB/POZ domain zinc finger transcription factor. Molecular and cellular biology 1999; 19: 3614–3623. Daniel JM, Spring CM, Crawford HC, Reynolds AB, Baig A. The p120(ctn)-binding partner Kaiso is a bi-modal DNA-binding protein that recognizes both a sequence-specific consensus and methylated CpG dinucleotides. Nucleic acids research 2002; 30: 2911–2919. Kim SW, Park J-I, Spring CM, Sater AK, Ji H, Otchere AA et al. 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11:39:52","extension":"png","order_by":11,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":101072,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/24f6e99763906b8f74ccd093.png"},{"id":95379857,"identity":"ad850f09-5e44-456d-a8f8-4dad615c0f83","added_by":"auto","created_at":"2025-11-07 11:39:52","extension":"png","order_by":12,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":78825,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/25d183971257ff7176a86f3a.png"},{"id":95379858,"identity":"0dcc2a91-3528-4d22-b227-6d08d789948e","added_by":"auto","created_at":"2025-11-07 11:39:52","extension":"png","order_by":13,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":95551,"visible":true,"origin":"","legend":"","description":"","filename":"Onlinefloatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/fb378cbcb925b6b0ffc53511.png"},{"id":95526344,"identity":"ea85b53b-8cfb-4e50-8964-b504da794e71","added_by":"auto","created_at":"2025-11-10 10:06:49","extension":"xml","order_by":14,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":160123,"visible":true,"origin":"","legend":"","description":"","filename":"cada15e36a364cfb9036a12e8241c1541structuring.xml","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/909a7dff121c0848720de24e.xml"},{"id":95379862,"identity":"c664dd3d-2cec-44a2-bd8e-a833552221e3","added_by":"auto","created_at":"2025-11-07 11:39:53","extension":"html","order_by":15,"title":"","display":"","copyAsset":false,"role":"acdc-reference","size":176459,"visible":true,"origin":"","legend":"","description":"","filename":"earlyproof.html","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/124f6ed28ad31aafee3c200b.html"},{"id":95525406,"identity":"1ec87290-48a6-41f3-99d5-a42e1792b305","added_by":"auto","created_at":"2025-11-10 10:04:58","extension":"jpeg","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":277594,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaiso binding site identification by ChIP- and Dam-ID-sequencing.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003e Schematic representation of Chromatin Immuno Precipitation (ChIP, top) and DNA adenine methyltransferase Identification (Dam-ID, bottom) procedures. For ChIP-sequencing, protein-DNA interactions were immunoprecipitated, followed by next-generation sequencing (NGS). DNA-adenine-methyltransferase identification (Dam-ID) analyses were done after stable low-level expression of Dam-Kaiso or Dam-GFP. The ratio of Dam-V5-Kaiso (Dam::Kaiso) over Dam-V5-GFP (Dam::GFP) was used to map specific Kaiso binding sites. \u003cstrong\u003eB.\u003c/strong\u003e Overlap (green, n=1,321) between Kaiso ChIP-peaks (grey, n=6,713) and Dam-ID-peaks (purple, 16,288) at a maximum distance of 2 kb between peak-centers display a 10.7-fold enrichment over random. \u003cstrong\u003eC.\u003c/strong\u003e Kaiso Dam-ID ratios correlate with binding sites identified by ChIP-seq. Bivariate scatterplot showing the normalized tag count of Kaiso ChIP-seq (x-axis, grey dots) within a 5 kb window surrounding ChIP-peak-centers versus the ratio of Dam-Kaiso over Dam-GFP (y-axis) in those regions. ChIP-peaks that overlap with Dam-ID-peaks are highlighted in green, r = Pearson’s correlation coefficient.\u003cstrong\u003e D.\u003c/strong\u003e Sequencing read density of Dam-ID- and ChIP-seq surrounding ChIP-peak-centers. Depicted is the tag coverage in 500 bp bins. Note the specific increase in intensity of the Dam-ID signal (pink/purple) juxtaposed to the ChIP signal (grey).\u003c/p\u003e","description":"","filename":"image1.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/d7085a49d8485ba9f593ab70.jpeg"},{"id":95379843,"identity":"a8416217-fa7c-4443-9c87-6d8216393a21","added_by":"auto","created_at":"2025-11-07 11:39:52","extension":"jpeg","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":236451,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaiso binds to the non-canonical Kaiso Binding Sequence (CG-KBS) consensus site in promotors.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eMotif analysis of the identified Kaiso binding sites. Top 5 enriched motifs for Dam-ID, ChIP and overlapping peaks are depicted. \u003cstrong\u003eB.\u003c/strong\u003e CG-KBS sites are enriched within 1 kb up- and downstream of the TSS (promotor-proximal), while CTCF and BORIS recognition sites are enriched in promotor distal elements. Based on ChIP-seq data. \u003cstrong\u003eC.\u003c/strong\u003e CG-KBS motifs are primarily located surrounding the transcription start site (TSS). Presence of the CG-KBS motif was assessed in ChIP-peaks (grey) and in the mouse genome (black) located 1500 bp up- and downstream of the TSS in 30 bp bins.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eD.\u003c/strong\u003eHistogram of motif density showing the enrichment of the CG-KBS at the center of ChIP-peaks, while CTCF recognition sites are more evenly distributed within 100 bp up- and downstream of the ChIP-peak-center.\u003c/p\u003e","description":"","filename":"image2.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/af489ffd88aad0cbbd309105.jpeg"},{"id":95525107,"identity":"654418a0-a6ca-4fac-b8fc-2524e34f68f6","added_by":"auto","created_at":"2025-11-10 10:04:14","extension":"jpeg","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":316502,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding of Kaiso occurs in transcriptionally active promotor regions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eKaiso binding sites are located in active gene promoter regions. ChIP-seq for RNA polymerase II (Pol2, green line), H3K27Ac (purple line) and H3K4me3 (grey line) revealed an enrichment of the active transcription marks around the Kaiso binding sites within the promoters (ChIP-peak-center) without a CG-KBS consensus site (dashed lines) or CG-KBS Kaiso targets (solid line). Depicted is the fragment coverage in 5 bp bins. \u003cstrong\u003eB.\u003c/strong\u003e Expression of Kaiso target genes. mRNA-sequencing analysis revealed a significant increase in mRNA expression levels of genes containing a CG-KBS ChIP-peak in the promoter compared to all expressed mRNAs, and genes not containing a CG-KBS in their promoters. RPKM: reads per kilobase of gene length per million reads. **** p_adj\u0026lt;0.001. \u003cstrong\u003eC.\u003c/strong\u003eGene Ontology Enrichment analysis revealing pathways and processes upregulated by Kaiso. Selection of the top enriched GO-terms linked to active genes harboring a CG-KBS in ChIP-seq peaks. The numbers behind each bar represent the number of genes annotated to each category. Dashed line marks p = 0.05.\u003c/p\u003e","description":"","filename":"image3.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/970923c3c89146967b9ac442.jpeg"},{"id":95526430,"identity":"95db1367-ec2c-4245-a284-181c8fe8a08a","added_by":"auto","created_at":"2025-11-10 10:06:58","extension":"jpeg","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":286985,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eBinding of Kaiso occurs in transcriptionally active promotor regions.\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eKaiso binding sites are located in active gene promoter regions. ChIP-seq for RNA polymerase II (Pol2, green line), H3K27Ac (purple line) and H3K4me3 (grey line) revealed an enrichment of the active transcription marks around the Kaiso binding sites within the promoters (ChIP-peak-center) without a CG-KBS consensus site (dashed lines) or CG-KBS Kaiso targets (solid line). Depicted is the fragment coverage in 5 bp bins. \u003cstrong\u003eB.\u003c/strong\u003e Expression of Kaiso target genes. mRNA-sequencing analysis revealed a significant increase in mRNA expression levels of genes containing a CG-KBS ChIP-peak in the promoter compared to all expressed mRNAs, and genes not containing a CG-KBS in their promoters. RPKM: reads per kilobase of gene length per million reads. **** p_adj\u0026lt;0.001. \u003cstrong\u003eC.\u003c/strong\u003eGene Ontology Enrichment analysis revealing pathways and processes upregulated by Kaiso. Selection of the top enriched GO-terms linked to active genes harboring a CG-KBS in ChIP-seq peaks. The numbers behind each bar represent the number of genes annotated to each category. Dashed line marks p = 0.05.\u003c/p\u003e","description":"","filename":"image4.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/f13e2b067249c0054bc2881a.jpeg"},{"id":95379847,"identity":"cfb61135-91e5-47f4-85f6-3b4213ce1d2f","added_by":"auto","created_at":"2025-11-07 11:39:52","extension":"jpeg","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":315842,"visible":true,"origin":"","legend":"\u003cp\u003e\u003cstrong\u003eKaiso protects breast cancer cells from cisplatin induced DNA damage\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eA.\u003c/strong\u003eKaiso knockout MCF7 clones. Western Blot revealing Kaiso knockout in MCF7 cells. AKT was used as loading control. \u003cstrong\u003eB.\u003c/strong\u003e Kaiso loss results in a reduction of DDR genes. Heatmap depicting relative mRNA expression of DDR genes normalized to wild type MCF7 cells. \u003cem\u003eGAPDH\u003c/em\u003e and \u003cem\u003eACTIN\u003c/em\u003e were used as controls. Bold numbers indicate significant differences (p\u0026lt;0.05). \u003cstrong\u003eC.\u003c/strong\u003eKaiso protects breast cancer cells against DNA damage. Shown are immunofluorescence images using antibodies targeting the phosphorylated (p) γH2A.X (Ser139) (green signals) in wild type MCF7 (left panels and the two Kaiso knockout MCF7 cells (∆\u003cem\u003eZBTB33\u003c/em\u003e#1 and ∆\u003cem\u003eZBTB33\u003c/em\u003e#2) after treatment with 0.1 µM cisplatin. Lower panels depict cell marked in the upper panel (inset). Size bars = 5 µm. \u003cstrong\u003eD.\u003c/strong\u003e Quantification of the experiment shown in (C). Depicted are the number of DNA damage foci per nucleus. A minimum of 50 nuclei were quantified per condition in three repeated experiments and averaged.\u003c/p\u003e","description":"","filename":"image5.jpeg","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/1ef8eb12086f9cfc89aa5d25.jpeg"},{"id":95530987,"identity":"56994f31-d6d9-4a1b-a171-255af90fec5c","added_by":"auto","created_at":"2025-11-10 10:22:25","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":2464025,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/72a7de28-aa03-4dee-903f-9dc9dadc4ae1.pdf"},{"id":95379846,"identity":"3c64f52d-7528-4811-94de-a3382b1c2d48","added_by":"auto","created_at":"2025-11-07 11:39:52","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"supplement","size":797974,"visible":true,"origin":"","legend":"","description":"","filename":"KGWV6supplementaldatanpjbreastcancer.pdf","url":"https://assets-eu.researchsquare.com/files/rs-7846387/v1/227663b3a6cfc73623f3ef4a.pdf"}],"financialInterests":"No competing interests reported.","formattedTitle":"Kaiso (ZBTB33) engages with non-canonical binding motifs to regulate DNA damage responses in breast cancer cells","fulltext":[{"header":"Introduction","content":"\u003cp\u003eKaiso (ZBTB33) is a member of the zinc finger and broad-complex, tramtrack and bric-\u0026agrave;-brac or poxvirus and zinc finger (BTB/POZ-ZF) transcription factor family that was initially identified in a yeast-two-hybrid screen as a p120-catenin (p120) binding partner\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e\u003c/sup\u003e. In that setting, Kaiso functions as a transcriptional repressor through binding to a specific DNA sequence, \u003cem\u003eTCCTGCNA\u003c/em\u003e, defined as the canonical Kaiso Binding Sequence (cKBS)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e. The interaction between Kaiso and p120 results in repression relief of Kaiso target genes like Wnt11\u003csup\u003e3\u003c/sup\u003e, Cyclin D1 (\u003cem\u003eCCND1\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e, Matrix metalloproteinase 7 (\u003cem\u003eMMP7\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e and ID2\u003csup\u003e5\u003c/sup\u003e. Relief of canonical Kaiso target gene suppression is dependent on nuclear translocation of p120 through a conserved NLS signal, an event exacerbated in anchorage-independent conditions\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e. More recently, the palindromic CG-containing consensus binding site \u003cem\u003eTCTCGCGAGA\u003c/em\u003e (CG-KBS) has been shown to be required for promoter-proximal noncanonical Kaiso-dependent recruitment of SMRT in terminal adipogenesis. Following this, the CG-KBS was also identified based on Kaiso chromatin immunoprecipitation (ChIP) data and subsequent \u003cem\u003ein silico\u003c/em\u003e analyses\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Interestingly, while it was initially shown \u003cem\u003ein vitro\u003c/em\u003e that methylation of this site is required for binding, analyses performed in the ENCODE dataset revealed that binding to the CG-KBS overlaps with highly active promoters and high levels of acetylated histones but without methylation enrichment of these sites, suggesting a noncanonical role for this particular Kaiso binding site in transcriptional activation.\u003c/p\u003e\u003cp\u003eBinding of Kaiso to either the cKBS or CG-KBS occurs through three zinc finger domains that recognize specific bases in the major groove of the cKBS and CpG-KBS sites through both classical and methyl hydrogen bonds\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e, or residues in the Kaiso C-terminus\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e\u003c/sup\u003e. Kaiso can directly affect transcription by binding to CG-KBS sites in the promotor of Metastasin (\u003cem\u003eS100A4\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e\u003c/sup\u003e, Metastasis associated 1 family member 2 (\u003cem\u003eMTA2\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e and Cyclin-dependent kinase inhibitor 2A (\u003cem\u003eCDKN2A\u003c/em\u003e)\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eLoss of Kaiso results in major developmental defects at the gastrulation stage in Xenopus and zebrafish embryos, which has been coupled to aberrant regulation of both canonical and non-canonical WNT target genes\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e,\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e,\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e. Given the assumed convergence of parallel p120/Kaiso and canonical TCF/LEF target genes in vertebrate development, these studies suggested a possible role for Kaiso in cancer (reviewed in:\u003csup\u003e17\u003c/sup\u003e). Histological assessments have indicated that Kaiso localization is enriched in the cytoplasm in thymic carcinoma, non-small cell lung cancer, lobular breast cancer and colon cancer cells when compared to healthy tissues, a finding that associates with decreased overall survival and/or high-grade tumors\u003csup\u003e\u003cspan additionalcitationids=\"CR19 CR20 CR21\" citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. In contrast, high grade prostate and breast cancers are characterized by elevated levels of nuclear Kaiso, a feature that is also associated with a poor prognosis\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e,\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eWhile Kaiso binding to its canonical site has been extensive studied, the functional consequences of Kaiso binding to the CG-KBS remain unclear. Because of this gap in knowledge and the suggested involvement of nuclear Kaiso in breast cancer, we set out to perform a comprehensive analysis to identify the genome-wide Kaiso binding sites in breast cancer using both chromatin immunoprecipitation (ChIP) and DNA adenine methyltransferase identification (Dam-ID). We found that Kaiso binding is enriched at the noncanonical CG-containing KBS in active promotor regions. Our data identify non-canonical KBS genes that are implicated in fast processes important in high grade cancer, such as metabolism, cell cycle control and DNA damage repair.\u003c/p\u003e"},{"header":"Materials and Methods","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\u003ch2\u003eCell culture\u003c/h2\u003e\u003cp\u003eTrp53\u003csup\u003eΔ/Δ\u003c/sup\u003e-3 mouse mammary carcinoma cells (\u003cem\u003eaka\u003c/em\u003e KP6) and human MCF7 cells were cultured as described \u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e. Ecotropic Phoenix (293T) cells were grown in DMEM-F12 (Sigma-Aldrich, Amsterdam, The Netherlands) containing 6% fetal bovine serum (Sigma-Aldrich), 100 IU/mL penicillin and 100 \u0026micro;g/mL streptomycin (Lonza, Geleen, The Netherlands).\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eImmunofluorescence\u003c/h3\u003e\n\u003cp\u003eCells were grown on glass coverslips to 70% confluence and washed with Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e containing PBS. Cells were fixed with 4% PFA in PBS for 10 minutes at room temperature. Permeabilization was performed using 0.3% Triton X-100 in PBS for 3 minutes, followed by blocking in 4% BSA/PBS for 15 minutes. Samples were incubated with the primary antibody mouse anti-V5 (1:1000, 46\u0026ndash;0705, Invitrogen, Carlsbad, CA) overnight at 4\u0026deg;C and rabbit anti-GFP (SC-8334, Santa Cruz, Dallas TX) for 2 hours at room temperature. Secondary antibody incubation with goat anti-mouse-Alexa Fluor 568 (1:600, A11031, Invitrogen) and goat anti-rabbit-Alexa Fluor 488 (1:600, A11034, Invitrogen) took place at room temperature for 1 hour. Samples were stained with DAPI for 5 minutes and mounted with Immu-Mount (Thermo Fisher Scientific, Carlsbad, CA). Slides were imaged using a Zeiss LSM 700 (Carl Zeiss, Oberkochen, Germany) and processed using ImageJ and Photoshop CS6 (Adobe, San Jose, CA).\u003c/p\u003e\n\u003ch3\u003eCloning and retroviral transduction for DAM-ID\u003c/h3\u003e\n\u003cp\u003eTo generate the pMSCV-EcoDam-V5-Kaiso-puro expression vector, Gateway Cloning was used to recombine mouse Kaiso cDNA (provided by P. McCrea, M.D. Anderson Cancer Center, Houston, Texas\u003csup\u003e\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e\u003c/sup\u003e) into pMSCV-EcoDam-V5-puro\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e by LR reaction. Next, Ecotropic Phoenix cells were transfected with pMSCV-EcoDam-V5-GFP-puro\u003csup\u003e\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e\u003c/sup\u003e and pMSCV-EcoDam-V5-Kaiso-puro using XtremeGene 9 (Roche). After 48 hours, supernatant containing the virus was collected and filtered through a 45 \u0026micro;m filter before transducing Trp53\u003csup\u003eΔ/Δ\u003c/sup\u003e -3 cells in the presence of 4 \u0026micro;g/mL polybrene (Sigma-Aldrich). After 72 hours, transduced cells were selected using puromycin (10 \u0026micro;g/mL).\u003c/p\u003e\n\u003ch3\u003eChromatin immunoprecipitation\u003c/h3\u003e\n\u003cp\u003eChIP was performed as described previously\u003csup\u003e\u003cspan citationid=\"CR60\" class=\"CitationRef\"\u003e60\u003c/span\u003e\u003c/sup\u003e. In short, cells were cross-linked with 2 mM DSG (Thermo Fisher Scientific) and 1% formaldehyde. Incubation with nuclear extraction buffer (20 mM Tris, 10 mM NaCl, 2 mM EDTA, 0.5% NP-40) was performed to lyse the cells and enrich for nuclei. Lysates were collected in sonication buffer (20 mM Tris, 150 mM NaCl, 2 mM EDTA, 1% NP-40 and 0.3% SDS) and chromatin was sheared to 200 to 600 bp fragments by ultrasonication at maximum power for 8 minutes (M220, Covaris, Brighton, UK). Immunoprecipitation was performed using antibodies directed against the following proteins: Kaiso (10 \u0026micro;g, 6F, 12723, Abcam, Cambridge, UK), RBP1 (5\u0026micro;g, PB-7C2, Euromedex, Souffelweyersheim, France), H3K4me3 (5\u0026micro;g, ab8580, Abcam) and H3K27Ac (5\u0026micro;g, ab4729, Abcam). DNA was purified using standard phenol/chloroform extraction and used for library preparation and sequencing on a HiSeq2000 (Illumina).\u003c/p\u003e\n\u003ch3\u003emRNA sequencing\u003c/h3\u003e\n\u003cp\u003eCells were seeded into a 6-well plate and grown to 80% confluence in serum-containing medium. After washing in Ca\u003csup\u003e2+\u003c/sup\u003e/Mg\u003csup\u003e2+\u003c/sup\u003e-containing PBS, RNA was isolated and purified using the RNeasy kit (Qiagen), followed by DNase treatment (Qiagen, Venlo, The Netherlands). RNA library preparation was performed as previously described \u003csup\u003e\u003cspan citationid=\"CR61\" class=\"CitationRef\"\u003e61\u003c/span\u003e\u003c/sup\u003e and used for cluster generation on a HiSeq 2000 (Illumina, Cambridge, UK).\u003c/p\u003e\u003cdiv id=\"Sec8\" class=\"Section2\"\u003e\u003ch2\u003eKaiso CRISPR/Cas9-mediated targeting and Knockout\u003c/h2\u003e\u003cp\u003eKaiso (ZBTB33) Knockout was performed using the previously described Baculoviral delivery of CRISPR/Cas9 \u003csup\u003e62\u003c/sup\u003e. A guide RNA targeting ZBTB33 was designed (5\u0026rsquo;-\u003cem\u003eGAG GCT TAT CGA CTG GTT GCA\u003c/em\u003e-3\u0026rsquo;) using the online CRISPR design tool (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003ehttp://crispr.mit.edu/\u003c/span\u003e\u003cspan address=\"http://crispr.mit.edu/\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). Guide RNAs were cloned into the \u003cem\u003eBbsI\u003c/em\u003e site of the baculovirus donor plasmid pAceBAC1-GFP (a kind gift from Dr. Michael Hadders, Center for Molecular Medicine, Utrecht, Netherlands), which was subsequently recombined with bacmid DNA in EmBacY competent cells.\u003c/p\u003e\u003cp\u003eTwo partly overlapping gDNA arms (1kb each) for the CRISPR/Cas9 mediated incorporation of the E535A mutation were amplified through PCR with gDNA isolated from mouse KP6 cells using primers 5\u0026rsquo; \u0026ndash; TTC TGA AGC GGC CGC TTT CAG ATG TTG CAC CTA GTG CT \u0026minus;\u0026thinsp;3\u0026rsquo; (F1mut) and 5\u0026rsquo; \u0026ndash; GCT TTG TGC GAT AT\u003cb\u003eG\u003c/b\u003e CTG CAA GAG GAA ATA CCT TCT CAC AGT AAC GAC ACG GAT ACT TCT TCT CCC AAG \u0026minus;\u0026thinsp;3\u0026rsquo; (REV1)(arm 1) and primers 5\u0026rsquo; \u0026ndash; CCT CTT GCA G\u003cb\u003eC\u003c/b\u003eA TAT CGC ACA AAG CAT GAA ATT CAT CAC ACA GGA GAG CGA AGG TAT CAG TGT TTG G \u0026minus;\u0026thinsp;3\u0026rsquo; (F2EA)and 5\u0026rsquo; \u0026ndash; TTC GAA GCG GCC GCC TTG GAC TAG CAG ATT ACA CAA CC \u0026minus;\u0026thinsp;3\u0026rsquo;(REV2) (arm 2). The E535A mutation is indicated in bold, the PAM site mutations are underlined. Next, a classic heteroduplex protocol with subsequent amplification using with primers F1mut and REV2\u003c/p\u003e\u003cp\u003eThe homologous arm was cloned into the pAceBAC-GFP vector containing the guideRNA using the \u003cem\u003eNotI\u003c/em\u003e site. Resulting Bacmids were transfected into Sf9 insect cells for baculovirus production. After transduction, GFP\u0026thinsp;+\u0026thinsp;cells were identified using a fluorescence microscope (Leica DMIL801, Leica, Amsterdam, The Netherlands), seeded as single cell clones, for subsequent verification of Kaiso knockout by TIDE analysis\u003csup\u003e\u003cspan citationid=\"CR63\" class=\"CitationRef\"\u003e63\u003c/span\u003e\u003c/sup\u003e and Western Blot.\u003c/p\u003e\u003c/div\u003e\n\u003ch3\u003eDNA damage analysis\u003c/h3\u003e\n\u003cp\u003eCells were seeded at a density of 50,000 cells per 24-well on glass cover slip 24 hours prior to treatment, after which cells were incubated with 0.1 \u0026micro;M cisplatin for 24 hours, washed and fixed with 4% paraformaldehyde for 5 minutes at room temperature. Cells were permeabilized using 0.3% Triton X-100 for 3 minutes, blocked with 4% BSA and incubated overnight at 4\u0026deg;C with mouse anti phospho-γH2AX (1:1.000; #05-636, Millipore, Darmstadt, Germany). Samples were imaged in triplicate using a Zeiss LSM 700 (Carl Zeiss). Images were processed using ImageJ, to adjust brightness and count foci per nucleus. A minimum of 20 high power fields containing on average 20 nuclei were quantified.\u003c/p\u003e\n\u003ch3\u003eComputational analysis of ChIP and DAM-ID\u003c/h3\u003e\n\u003cp\u003eFor mRNA-sequencing, reads were uniquely mapped to the mouse (mm9) reference genome using the ELAND or BWA program, allowing 1 mismatch, and subsequently used for bioinformatic analysis. RPKM (reads per kilobase of gene length per million reads) values for RefSeq genes were computed using tag counting scripts and used to analyze the expression level of genes. ChIP sequencing reads were processed and mapped to the mouse (mm9) reference genome using BWA programming allowing 1 mismatch. ChIP-peaks were identified using Model based Analysis of ChIP-seq (MACS1.3.3) with a cutoff p-value of 10\u003csup\u003e\u0026minus;\u0026thinsp;7\u003c/sup\u003e for peak detection\u003csup\u003e\u003cspan citationid=\"CR64\" class=\"CitationRef\"\u003e64\u003c/span\u003e,\u003cspan citationid=\"CR65\" class=\"CitationRef\"\u003e65\u003c/span\u003e\u003c/sup\u003e. Dam-ID sequencing reads were aligned to the mouse genome (mm9) using Bowtie. Only reads aligning uniquely to a single genomic location and aligning just downstream from a GATC sequence were used for downstream analysis. Putative Dam-ID-peaks were identified using HOMER\u003csup\u003e\u003cspan citationid=\"CR66\" class=\"CitationRef\"\u003e66\u003c/span\u003e\u003c/sup\u003e using an initial peak-size of 2,500 bp and a 3-fold enrichment of normalized Dam-ID-Kaiso reads over Dam-ID-GFP control reads. In addition, peaks were required to contain at least 100 normalized reads per peak (per 10\u0026nbsp;million reads sequenced) to remove low magnitude sites. For comparison between ChIP-peaks and Dam-ID-peaks, the \u0026lsquo;mergepeaks\u0026rsquo; function of HOMER was used, applying a maximal distance between the peak centers of 2000 bp. HOMER was also used to determine the location of Kaiso binding sites in the genome, the overlap of Kaiso binding sites with CpG islands, and (de novo) motif analysis. Additionally, using the \u0026lsquo;annotatepeaks\u0026rsquo; function, tag enrichment histograms of Kaiso ChIP, Dam-ID, RNA polymerase and histone marks were generated. DAVID gene ontology database\u003csup\u003e\u003cspan citationid=\"CR67\" class=\"CitationRef\"\u003e67\u003c/span\u003e\u003c/sup\u003e was used to identify enriched functional annotations. In addition to statistical analysis included in the aforementioned software packages, R (2.15.3, R Core team 2011) and Excel (Microsoft, Redmond, WA) were used to analyze RNA-seq data and perform Pearson\u0026rsquo;s correlation testing.\u003c/p\u003e\u003cdiv id=\"Sec11\" class=\"Section2\"\u003e\u003ch2\u003eReverse transcriptase quantitative PCR\u003c/h2\u003e\u003cp\u003eTotal mRNA was extracted from cell or organoid pellets using Trizol reagent (Thermo Fisher Scientific). Poly-T primers and a cDNA transcription kit (iScript Synthesis kit, Biorad, Veenendaal, The Netherlands) were used to generate cDNA. PCR primer sets used to evaluate expression values for the gene set are listed in Supplementary Table\u0026nbsp;3. Primer efficiency was assessed by serial dilution. Expression values were generated using ∆∆Ct values normalized to \u003cem\u003eGAPDH\u003c/em\u003e and/or \u003cem\u003eACTB\u003c/em\u003e. Experiments were performed in triplicate over three independent biological and technical settings, using the CFX96 Real-Time System and CFX manager software (both Biorad). For each comparison, unpaired two-tailed Student\u0026rsquo;s t-tests were used to determine statistical significance.\u003c/p\u003e\u003c/div\u003e"},{"header":"Results","content":"\u003cdiv id=\"Sec13\" class=\"Section2\"\u003e\u003ch2\u003eIdentification of the genome-wide Kaiso binding sites by ChIP and Dam-ID\u003c/h2\u003e\u003cp\u003eAlthough clinical evidence implicates Kaiso in breast cancer, its genome-wide role has yet to be clarified. To shed light on the genomic regions at which Kaiso engages chromatin, we performed chromatin immunoprecipitation (ChIP) and Dam methyltransferase identification (Dam-ID) of Kaiso, followed by next generation sequencing (NGS) (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eA). Nuclear translocation of p120 and its possible confounding impacts on Kaiso binding to DNA were circumvented by using E-cadherin expressing mammary ductal-type breast cancer cells that were derived from a conditional p53 knockout mouse model of breast cancer\u003csup\u003e\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e\u003c/sup\u003e. Using this setup, we performed genome wide ChIP analysis and identified 6,713 peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB). In parallel, we performed Kaiso-specific Dam-ID to complement findings in the ChIP experiments. To minimize a-specific methylation by the fused DAM methylase and Kaiso proteins (Dam::Kaiso), we used retroviral transduction to achieve stable but low expression of Dam::Kaiso (SFig. 1). Methylated sequences were PCR-amplified for detection by NGS from cells expressing Dam::Kaiso or (control) Dam::GFP to identify Kaiso-specific Dam-ID-peaks. This was done by calculating the ratio of normalized Dam::Kaiso reads over Dam::GFP reads, setting a 3-fold increase cut-off Dam::Kaiso reads as specific. In total, we identified 16,288 Dam-ID-peaks (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eB and \u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Comparison of Kaiso ChIP-peaks with Dam-ID-peaks revealed a positive correlation between Kaiso ChIP tags and the ratio of normalized Dam::Kaiso over Dam::FP tags and an approximate 10-fold enrichment (r\u0026thinsp;=\u0026thinsp;0.342, Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC). Moreover, we observed a positive correlation between the number of Kaiso ChIP and Dam::Kaiso tags (r\u0026thinsp;=\u0026thinsp;0.310, Fig. S2A), while there is no correlation between Kaiso ChIP tags and Dam::GFP tags (r\u0026thinsp;=\u0026thinsp;0.155, Fig. S2B). We mapped 1,321 overlapping regions between the identified ChIP and Dam-ID peaks, which comprise roughly 20% of all ChIP-peaks identified (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eC and Fig. S2C-F). Overall, ChIP-seq identified 5,392 Kaiso binding sites that do not overlap with those mapped using Dam-ID. Conversely, Dam-ID yielded 14,967 potential Kaiso binding sites that were not identified using ChIP. As expected, DAM-ID peaks are distributed in a typical biphasic pattern normalized at positions juxtaposed upstream and downstream of the Kaiso ChIP peak (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003eD).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003c/div\u003e\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e\u003ch2\u003eKaiso binds to a CG-containing KBS sequence in promotor-regions\u003c/h2\u003e\u003cp\u003eAnalysis of Kaiso binding sites identified by ChIP-seq revealed a high occupancy of Kaiso in promotors and 5-prime UTR regions, representing a 34- and 48-fold enrichment respectively (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Kaiso binding sites identified by Dam-ID-seq are also enriched at these genomic locations surrounding the transcription start site (TSS; 3.8 and 3.6-fold respectively), although less pronounced as seen for ChIP-seq (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eA). Kaiso-binding was enriched at regions within 1 kb flanking the TSS for both the ChIP and DAM-ID experiments (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eB). Interestingly, Kaiso binding to promotors based on our ChIP experiments primarily occurs in CG-rich regions (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003eC). Although this can be explained by abundant Kaiso binding in promotor regions, which are generally rich in CG islands, we observed that 90.6% of all ChIP-peaks in promotors overlap with a CG island, while 53% of all mouse promotors contain a CG island.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eSubsequent motif analysis revealed a palindromic CG-containing motif that was previously mapped as a Kaiso binding sequence based on publicly available (ENCODE) ChIP data in myeloid (K562) and lymphoid (GM12878) cells \u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. The identified palindromic motif consists of 10 core nucleotides (\u003cem\u003eTCTCGCGAGA\u003c/em\u003e; ZBTB33) that we refer to as the non-canonical (CG-containing) Kaiso Binding Sequence (CG-KBS)(Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Of note, 234 of the total 6,713 Kaiso ChIP peaks contained the canonical KBS (\u003cem\u003eTCCTGCNA\u003c/em\u003e), which is not a significant enrichment over the genome-wide occurrence of this motif. \u003cem\u003eDe novo\u003c/em\u003e discovery did not identify additional motifs aside from a truncated version of the CG-KBS (Supplemental Fig.\u0026nbsp;3). However, in addition to the CG-KBS, we identified CTCF and its paralogue BORIS as significantly enriched motifs (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). Although CTCF and BORIS can act as insulators that facilitate chromatin loops and occupy promoter-proximal regions\u003csup\u003e\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e\u003c/sup\u003e, we did not observe enrichment of their motifs in promoters in our dataset (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eB), suggesting a context-specific binding profile. The CG-KBS is a common promotor element occurring in 5% of all human TATA-less promotors and linked to expression of genes that control fast processes\u003csup\u003e\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e\u003c/sup\u003e. Indeed, we find that approximately 90% of the identified CG-KBS peaks occur within a region 1 kb up- or downstream of the transcriptional start site (TSS) in the mouse genome (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eC). Detailed positional examination of the CTCF and CG-KBS motifs relative to the site of Kaiso-binding revealed an interesting pattern. While the CG-KBS displayed an expected sharp enrichment at the ChIP-peak centers, the CTCF motif was more broadly positioned around the peak signal (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eD). These findings suggest that Kaiso and CTCF could function as co-regulators of a common gene, as has been observed for β-globin\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Finally, motif analysis solely on the Dam-ID data also identified several members of the AP1 transcription factor family in close vicinity of Kaiso binding sites (Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003eA). However, inspection of the regions flanking the Kaiso binding sites from the ChIP assays did not show an enrichment of AP1 family member or any other known or novel motifs.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eIn conclusion, we have identified two subsets of Kaiso binding sites using combined ChIP and Dam-ID; promotor-proximal CG-KBS-containing sites and sites in close vicinity of the CTCF/Boris motif distal to the TSS.\u003c/p\u003e\u003cp\u003e\u003cb\u003eKaiso binding to the CG-KBS occurs in transcriptionally active promotors involved in processes linked to cancer progression.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo assess which pathways could be affected by Kaiso repression through the CG-KBS, we first characterized the chromatin landscape of Kaiso-bound regions. We performed ChIP-sequencing for histone 3-lysine 4-tri-methylation (H3K4me3), an epigenetic active histone mark that is enriched at promotor regions with an open chromatin state. Additionally, we identified active enhancers and promotors of transcribed genes by performing ChIP-seq for histone 3-lysine 27 acetylation (H3K27Ac) and regions of active transcription by RNA polymerase II (Pol2) ChIP-seq \u003csup\u003e29,30\u003c/sup\u003e. Kaiso binding sites were divided into two groups; sites present within a promotor (defined as peaks occurring within 1 kb up- or downstream from the TSS) containing the CG-KBS, and sites without an apparent consensus site within a promoter. Although both Kaiso targets sites were accompanied by a high occupancy of all three markers of active transcription, this was particularly evident at the CG-KBS (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eA). The presence of Pol2 marks at Kaiso target gene promoters did not correlate with a significant increase in mRNA expression levels (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eB).\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eTo gain insight into the pathways and biological processes linked to CG-KBS target genes, we performed Gene Ontology Enrichment analysis, which revealed a significant enrichment of genes controlling processes such as cellular metabolism, RNA transcription, cell cycle regulation and DNA damage repair (DDR)(Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003eC). We did not find a correlation between CG-KBS Kaiso targets and the specific stages of cell cycle regulation or types of DNA damage responses (data not shown). Taken together, our analyses show that Kaiso binding is enriched at the non-canonical Kaiso motif in promotor regions of actively transcribed genes that control distinct cellular processes important for cancer progression.\u003c/p\u003e\u003cp\u003e\u003cb\u003eKaiso prevents cisplatin induced DNA damage in breast cancer cells.\u003c/b\u003e\u003c/p\u003e\u003cp\u003eTo specify the functional impact of the CG-KBS in cancer, we devised a strategy to use CRISPR-Cas9 to perform homozygous mutation of the methyl-specific recognizing residues Glu-535 and Tyr-536 in the second zinc finger of Kaiso. These residues were identified based on amide chemical shifts comparing the nuclear magnetic resonance spectra of Kaiso in complex with either the canonical KBS or the CG-KBS\u003csup\u003e\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e\u003c/sup\u003e. We hypothesized, also based on published electromobility shift data \u003csup\u003e\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e\u003c/sup\u003e, that mutation of the E535 residue would impact the CG-KBS interaction. Upon targeting of this mutation in human MCF7 breast cancer cells we screened approximately 150 clones for genetic mutations (Supplemental Fig.\u0026nbsp;4). Surprisingly, we did not obtain viable clones that contained homozygous E535A alleles. We did however, obtain clones that showed heterozygous knock-in of the E535 mutation, but this was uniformly accompanied by the presence of either a wild type or a knockout allele (Supplemental Table\u0026nbsp;1). Subsequent biochemical experiments revealed that these heterozygous knock-in clones did not express Kaiso (Supplemental Table\u0026nbsp;1). Based on these results, we conclude that homozygous mutation of the sites necessary for KBS binding is not compatible with cellular viability in MCF7. Nonetheless, using this approach, we generated Kaiso knock-out clones (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eA), of which two were used for further functional analysis.\u003c/p\u003e\u003cp\u003e\u003c/p\u003e\u003cp\u003eFor this, we focused on the possible cancer progression impact of Kaiso in the modulation of DDR responses. We selected DRR genes that were identified using the Kaiso ChIP experiments (Supplemental Table\u0026nbsp;2) and compared transcript expression levels by quantitative rt-PCR (qPCR) in MCF7 control \u003cem\u003eversus\u003c/em\u003e MCF7 Kaiso knockout (MCF7::∆Kaiso) cells. We started by selecting a list of 26 DDR genes with a proximal site of Kaiso, as identified by ChIP-seq in mouse cells. Next, we cross-referenced these 26 genes to the 1,000 base pair promoter regions of the human gene orthologs from the MCF7 Encode ChIP-seq database to verify Kaiso binding\u003csup\u003e\u003cspan additionalcitationids=\"CR34\" citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e\u003c/sup\u003e. After confirmation of specificity, we designed qPCR primers for a set of 11 genes based on expression levels in MCF7 (bold genes, Supplemental Table\u0026nbsp;2). Based on these transcriptional analyses, we detected a reduction of all 11 DDR genes assessed within this cohort (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eB). Next, we assessed the impact of Kaiso knock-out on DNA damage induced by cisplatin, a DNA intercalating agent that causes DNA double strand breaks. DNA damage was assessed by immunofluorescence and quantification of phosphorylated (p-) γH2A.X (Ser139) DNA damage foci, an established marker for double strand break repair \u003csup\u003e\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e\u003c/sup\u003e. We observed that a low concentration (0.1 \u0026micro;M), that does not induce overt DNA damage in control MCF cells, causes a stark significant increase in p-γH2A.X DNA damage foci in MCF7::∆Kaiso cells (7.5 foci/nucleus \u003cem\u003eversus\u003c/em\u003e 26.5 and 34.2 foci/nucleus respectively, p\u0026thinsp;=\u0026thinsp;0.001, Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eC and \u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003eD). These results indicate that Kaiso protects breast cancer cells from DNA damage and suggest that this protection is established through positive transcriptional regulation of CG-KBS dependent DDR target genes.\u003c/p\u003e\u003c/div\u003e"},{"header":"Discussion","content":"\u003cp\u003eKaiso is a versatile and context-dependent transcription factor, with a propensity to bind the canonical cKBS and/or the non-canonical CG-KBS\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. Growing evidence indicates that Kaiso may have a dual function as both a repressive and activating transcription factor, depending on the DNA sequence occupied\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e,\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e,\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Kaiso functions as a repressor of genes that play a role in breast cancer such as \u003cem\u003eCCND1\u003c/em\u003e, \u003cem\u003eWNT11\u003c/em\u003e, \u003cem\u003eMMP7, ID2\u003c/em\u003e and \u003cem\u003eERBB3\u003c/em\u003e, a function presumably through a p120-dependent mechanism upon loss of E-cadherin\u003csup\u003e\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e. In cancer, Kaiso cytosolic localization and expression carries prognostic value in colorectal, breast, prostate and lung cancer (Reviewed in: \u003csup\u003e17,40\u003c/sup\u003e). However, Kaiso is found enriched in the nucleus in high grade breast cancer, a prognostic association that implies roles in transcriptional activation\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e. To study Kaiso in this context, we employed E-cadherin expressing breast cancer cells and performed genome-wide Kaiso-specific ChIP-seq and Dam-ID-seq in proliferating cells to identify Kaiso binding sites and the resulting transcriptional programs. This setup yielded a 20% overlap between ChIP and Dam-ID peaks, which is comparable to previous studies\u003csup\u003e\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e,\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e\u003c/sup\u003e. Interestingly, our data reveal that many of the identified Kaiso binding sites contain the CG-KBS, a finding that is in line with the publicly available ENCODE data\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. Surprisingly, the cKBS was not significantly enriched on a genome-wide scale in our dataset. Furthermore, we did not find enrichment of binding sites that have dual-interaction with both the cKBS and CG-KBS such as in the \u003cem\u003eCCND1\u003c/em\u003e promoter\u003csup\u003e\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e\u003c/sup\u003e, suggesting that this specific dual specificity DNA binding mechanism might be unique to \u003cem\u003eCCND1\u003c/em\u003e. Taken together, our data suggest that Kaiso-dependent transcriptional regulation in high grade breast cancer functions as an activating transcription factor in hypomethylated areas via the CG-KBS.\u003c/p\u003e\u003cp\u003eConsistent with the above, Kaiso binding sites are enriched with histone marks related to open chromatin conformation (H3K4me3 and H3K27Ac) and RNA polymerase II, suggesting active transcription. These data strengthened our hypothesis that CG-KBS-dependent gene regulation by Kaiso promotes active gene transcription, either by direct transcriptional activation, or by promoting an open chromatin formation of the bound promoter. In support of this, it was shown previously that SUMOylation of K42 in Kaiso allows a switch from transcriptional repression to activation\u003csup\u003e\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e. Since this post-translational event occurs in the BTB/POZ domain, it could prevent dimerization, or interaction and subsequent repression with DNA methylation complexes such as N-CoR and SMRT\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e,\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e. Moreover, Kaiso deficiency in mouse embryonic fibroblasts induces hypomethylation of binding sites for Oct4 and Nanog\u003csup\u003e\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e, suggesting possible recruitment of methylation machinery to CG-KBS. We find that most of the Kaiso binding sites are enriched for the CG-KBS consensus in hypomethylated, actively transcribed promotor regions. Our findings agree with previous analyses by Blatter \u003cem\u003eet al.\u003c/em\u003e, who have used ENCODE to show that binding of Kaiso to the CG-KBS occurs to mostly unmethylated CpGCpG-nucleotides, supportive of Kaiso being able to bind the CG-KBS independently of methylation status\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e. In contrast, others have shown that Kaiso might preferentially bind methylated CG-KBS sites. However, these data have been produced \u003cem\u003ein vitro\u003c/em\u003e and therefore not conclusively exclude binding of Kaiso to an unmethylated CG-KBS \u003cem\u003ein vivo\u003c/em\u003e\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e. How methylation of the CG-KBS affects Kaiso binding and/or downstream biological functions \u003cem\u003ein vivo\u003c/em\u003e is currently largely unknown, especially since methylation of CGs is highly dependent on a multitude of factors such as cell type and culture conditions. In the context of cancer, hypermethylation of CG islands in promoters of hormone receptor target genes frequently occurs, with subsequent reduced expression\u003csup\u003e\u003cspan additionalcitationids=\"CR47\" citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e. However, most CG islands \u003cem\u003ein vivo\u003c/em\u003e are hypomethylated and the associated genes show increased expression\u003csup\u003e\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e. Supporting this is our observation that a coincidence of \u003cem\u003eCTCF\u003c/em\u003e/\u003cem\u003eBORIS\u003c/em\u003e motifs with Kaiso binding sites, suggesting that Kaiso may additionally be involved in transcriptional regulation more distal from promoters, similarly to the transcriptional regulation of β-globin\u003csup\u003e\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e. Although Kaiso and CTCT were identified independently as protein-protein interacting partners via yeast 2-hybrid assays\u003csup\u003e\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e\u003c/sup\u003e, there appears to be no direct binding affinity between the 2 factors\u003csup\u003e\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e\u003c/sup\u003e.Therefore, because we mostly find \u003cem\u003eCTCF\u003c/em\u003e motifs independently from CG-KBS sites, we propose that Kaiso may interact with CTCF indirectly.\u003c/p\u003e\u003cp\u003eOur data point to an enrichment of Kaiso binding in promoters involved with cell cycle processes, cellular metabolism and DDR responses. Functionally, we show that Kaiso is essential for a proper DDR response; we observe a marked increase in DNA damage foci when comparing Kaiso-null \u003cem\u003eversus\u003c/em\u003e Kaiso-expressing MCF7 cells after low dosage cisplatin treatment. Given these findings, we conclude that Kaiso may drive processes that require fast transcriptional activation of genes through the CG-KBS. In contrast, cKBS-dependent transcriptional regulation by Kaiso causes repression of transcription and appears to mediate highly specialized differentiation functions through canonical and non-canonical Wnt signaling\u003csup\u003e\u003cspan additionalcitationids=\"CR3\" citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e,\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e\u003c/sup\u003e. These signals are essential for vertebrate development (for detailed reviews see\u003csup\u003e\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e,\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e\u003c/sup\u003e) and require oncogenic activation to drive progression in colon and breast cancer\u003csup\u003e5556\u003c/sup\u003e. Although the function of subcellular Kaiso localization is still unclear, phosphorylation at Threonine 606 leads to Kaiso accumulation in the cytoplasm\u003csup\u003e\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e\u003c/sup\u003e. These alternative mechanisms reveal yet another level of regulation, possibly modulating Kaiso binding towards the cKBS, allowing for an optional regulation of fast responses \u003cem\u003eversus\u003c/em\u003e slow transcriptional differentiation programs, respectively. The target genes identified here suggest that indeed, Kaiso binding to the CG-KBS is mostly responsible for the modulation of pro-oncogenic processes that require acute upregulation such as DDR, whereas binding of Kaiso to the cKBS is required for the regulation genes that control development, inhibit proliferation and foster anchorage independence of breast cancer cells, such as \u003cem\u003eWNT11\u003c/em\u003e, \u003cem\u003eERBB3\u003c/em\u003e and \u003cem\u003eID2\u003c/em\u003e\u003csup\u003e3,5,7\u003c/sup\u003e.\u003c/p\u003e\u003cp\u003eIn short, we conclude that nuclear Kaiso acts as a transcriptional activator of oncogenic processes that underpin tumor progression in high-risk, high grade breast carcinomas.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003eAll authors declare that they have no competing financial interests in relation to the work described.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMembers of former Hetzer lab are acknowledged for their help and suggestions. We thank Corlinda ten Brink and the UMC Utrecht Cell Microscopy Center for imaging support. We acknowledge Michael Hadders for assisting us with generating CRISPR/Cas9 Baculoviruses to perform knockout of Kaiso.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthical Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eEthics approval was not required for this study.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding Declaration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eResearch was supported by grants from the UICC Yamagiwa-Yoshida Memorial International Cancer Study Grant (YY1/13/001), The Netherlands Organization for Scientific Research (NWO/ZonMW-VIDI 016.096.318) and the Dutch Cancer Society grants KWF-UU-2011-5230 and KWF-10245.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthor Contributions:\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eMAKR and MT were responsible for designing and executing experiments, performing data analyses and writing of the manuscript. JV and MIAN performed data analyses. AM, AAS supported and performed ChIP experiments and subsequent data analysis. MH, JHM and HGS provided reagents and feedback on the report. JMD provided conceptual input, reagents and contributed to writing the report. SP performed data analyses for the genome wide ChIP and DAM-ID data and contributed to writing the manuscript. PWBD designed the study, performed and analyzed DNA damage repair experiments, provided supervision and wrote the report.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eData availability\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGenomic data generated in this study have been deposited in the Gene Expression Omnibus (GEO) under accession number GSE309273.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eDaniel JM, Reynolds a B. 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A Conformational Switch in the Zinc Finger Protein Kaiso Mediates Differential Readout of Specific and Methylated DNA Sequences. \u003cem\u003eBiochemistry\u003c/em\u003e 2020; 59: 1909\u0026ndash;1926.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":false,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"npj-breast-cancer","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjbcancer","sideBox":"Learn more about [npj Breast Cancer](http://www.nature.com/npjbcancer/)","snPcode":"41523","submissionUrl":"https://mts-npjbcancer.nature.com/","title":"npj Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true},"keywords":"","lastPublishedDoi":"10.21203/rs.3.rs-7846387/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-7846387/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003cp\u003eIn breast cancer, nuclear localization of Kaiso (ZBTB33), a dual specificity transcription factor, is a hallmark of high grade ductal-type carcinomas, especially in estrogen receptor negative disease. Regulation of gene expression by Kaiso is orchestrated via its engagement to distinct Kaiso binding sequence (KBS) motifs defined as canonical (cKBS; \u003cem\u003eTCCTGCNA\u003c/em\u003e) or CG-containing palindromic KBS (CG-KBS; \u003cem\u003eTCTCGCGAGA\u003c/em\u003e), depending on context. While there exists a clinical connection between localization of Kaiso expression and breast cancer, it remains unclear how Kaiso controls bi-modal canonical \u003cem\u003eversus\u003c/em\u003e noncanonical transcriptional modulation. Here, we have combined Kaiso-specific chromatin immunoprecipitation (ChIP) and Kaiso-Dam methyltransferase identification (Dam-ID) approaches to map genomic Kaiso binding sites in breast cancer cells. We find that Kaiso mostly occupies the non-canonical CG-KBS sites in transcriptionally active and hypo-methylated (H3K4me3 and H3K27Ac enriched) promoter regions. Noncanonical CG-KBS targets are actively transcribed and linked to fast biological processes such as metabolism, cell cycle regulation and DNA damage repair. Functionally, we show that Kaiso expression is essential to prevent DNA damage in breast cancer cells. Loss of Kaiso leads to reduced expression of DNA damage response gene expression and treatment with the chemotherapeutic agent cisplatin leads to overt accumulation of DNA damage in cells devoid of Kaiso. Our data thus favor a model in which Kaiso promotes fast transcriptional activation of key cellular processes in cancer cells through binding of its non-canonical consensus site.\u003c/p\u003e","manuscriptTitle":"Kaiso (ZBTB33) engages with non-canonical binding motifs to regulate DNA damage responses in breast cancer cells","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2025-11-07 11:39:47","doi":"10.21203/rs.3.rs-7846387/v1","editorialEvents":[{"type":"communityComments","content":0},{"type":"decision","content":"Revision requested","date":"2025-12-01T20:42:36+00:00","index":"","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-12-01T01:43:57+00:00","index":"hide","fulltext":""},{"type":"editorInvitedReview","content":"","date":"2025-11-26T21:03:20+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"281019531543159407715604410532247158945","date":"2025-11-10T16:07:16+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"23882890700467327358489142319374109417","date":"2025-11-10T15:23:43+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"148430257675682518871858513349732974852","date":"2025-11-10T02:44:52+00:00","index":"hide","fulltext":""},{"type":"reviewerAgreed","content":"315130611968649519984830198459018456143","date":"2025-10-28T18:09:26+00:00","index":"hide","fulltext":""},{"type":"reviewersInvited","content":"","date":"2025-10-28T15:58:22+00:00","index":"","fulltext":""},{"type":"editorAssigned","content":"","date":"2025-10-26T15:18:07+00:00","index":"","fulltext":""},{"type":"checksComplete","content":"","date":"2025-10-24T09:16:28+00:00","index":"","fulltext":""},{"type":"submitted","content":"npj Breast Cancer","date":"2025-10-13T08:17:50+00:00","index":"","fulltext":""}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"npj-breast-cancer","isNatureJournal":false,"hasQc":false,"allowDirectSubmit":false,"externalIdentity":"npjbcancer","sideBox":"Learn more about [npj Breast Cancer](http://www.nature.com/npjbcancer/)","snPcode":"41523","submissionUrl":"https://mts-npjbcancer.nature.com/","title":"npj Breast Cancer","twitterHandle":"","acdcEnabled":true,"dfaEnabled":true,"editorialSystem":"ejp","reportingPortfolio":"NPJ","inReviewEnabled":true,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"08b9de31-ad23-41e4-92fb-45a53cd0fbe2","owner":[],"postedDate":"November 7th, 2025","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"in-revision","subjectAreas":[{"id":57304015,"name":"Biological sciences/Biochemistry"},{"id":57304016,"name":"Biological sciences/Cancer"},{"id":57304017,"name":"Biological sciences/Cell biology"},{"id":57304020,"name":"Biological sciences/Computational biology and bioinformatics"},{"id":57304024,"name":"Biological sciences/Molecular biology"},{"id":57304027,"name":"Health sciences/Oncology"}],"tags":[],"updatedAt":"2025-12-01T20:53:22+00:00","versionOfRecord":[],"versionCreatedAt":"2025-11-07 11:39:47","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-7846387","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-7846387","identity":"rs-7846387","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}