The lysine‐specific methyltransferase KMT 2C/ MLL 3 regulates DNA repair components in cancer
2019; Springer Nature; Volume: 20; Issue: 3 Linguagem: Inglês
10.15252/embr.201846821
ISSN1469-3178
AutoresΘεόδωρος Ράμπιας, Dimitris Karagiannis, Margaritis Avgeris, Alexander Polyzos, Antonis Kokkalis, Zoi Kanaki, Evgenia Kousidou, Maria Tzetis, Emmanouil Kanavakis, Konstantinos Stravodimos, Kalliopi N. Manola, Gabriel E. Pantelias, Andreas Scorilas, Apostolos Klinakis,
Tópico(s)PARP inhibition in cancer therapy
ResumoArticle21 January 2019Open Access Source DataTransparent process The lysine-specific methyltransferase KMT2C/MLL3 regulates DNA repair components in cancer Theodoros Rampias Theodoros Rampias orcid.org/0000-0002-5460-5334 Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Dimitris Karagiannis Dimitris Karagiannis orcid.org/0000-0002-4394-1076 Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Margaritis Avgeris Margaritis Avgeris orcid.org/0000-0002-2135-9886 Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Alexander Polyzos Alexander Polyzos Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Antonis Kokkalis Antonis Kokkalis Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Zoi Kanaki Zoi Kanaki Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Evgenia Kousidou Evgenia Kousidou Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Maria Tzetis Maria Tzetis Department of Medical Genetics, Medical School, "Aghia Sophia" Children's Hospital, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Emmanouil Kanavakis Emmanouil Kanavakis Department of Medical Genetics, Medical School, "Aghia Sophia" Children's Hospital, National and Kapodistrian University of Athens, Athens, Greece University Research Institute for the Study and Treatment of Childhood Genetic and Malignant Diseases, "Aghia Sophia" Children's Hospital, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Konstantinos Stravodimos Konstantinos Stravodimos First Department of Urology, "Laiko" General Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Kalliopi N Manola Kalliopi N Manola Laboratory of Health Physics, Radiobiology & Cytogenetics, National Center for Scientific Research (NCSR) "Demokritos", Athens, Greece Search for more papers by this author Gabriel E Pantelias Gabriel E Pantelias Laboratory of Health Physics, Radiobiology & Cytogenetics, National Center for Scientific Research (NCSR) "Demokritos", Athens, Greece Search for more papers by this author Andreas Scorilas Andreas Scorilas Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Apostolos Klinakis Corresponding Author Apostolos Klinakis [email protected] orcid.org/0000-0002-3923-861X Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Theodoros Rampias Theodoros Rampias orcid.org/0000-0002-5460-5334 Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Dimitris Karagiannis Dimitris Karagiannis orcid.org/0000-0002-4394-1076 Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Margaritis Avgeris Margaritis Avgeris orcid.org/0000-0002-2135-9886 Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Alexander Polyzos Alexander Polyzos Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Antonis Kokkalis Antonis Kokkalis Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Zoi Kanaki Zoi Kanaki Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Evgenia Kousidou Evgenia Kousidou Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Maria Tzetis Maria Tzetis Department of Medical Genetics, Medical School, "Aghia Sophia" Children's Hospital, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Emmanouil Kanavakis Emmanouil Kanavakis Department of Medical Genetics, Medical School, "Aghia Sophia" Children's Hospital, National and Kapodistrian University of Athens, Athens, Greece University Research Institute for the Study and Treatment of Childhood Genetic and Malignant Diseases, "Aghia Sophia" Children's Hospital, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Konstantinos Stravodimos Konstantinos Stravodimos First Department of Urology, "Laiko" General Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Kalliopi N Manola Kalliopi N Manola Laboratory of Health Physics, Radiobiology & Cytogenetics, National Center for Scientific Research (NCSR) "Demokritos", Athens, Greece Search for more papers by this author Gabriel E Pantelias Gabriel E Pantelias Laboratory of Health Physics, Radiobiology & Cytogenetics, National Center for Scientific Research (NCSR) "Demokritos", Athens, Greece Search for more papers by this author Andreas Scorilas Andreas Scorilas Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece Search for more papers by this author Apostolos Klinakis Corresponding Author Apostolos Klinakis [email protected] orcid.org/0000-0002-3923-861X Biomedical Research Foundation Academy of Athens, Athens, Greece Search for more papers by this author Author Information Theodoros Rampias1, Dimitris Karagiannis1, Margaritis Avgeris2, Alexander Polyzos1, Antonis Kokkalis1, Zoi Kanaki1, Evgenia Kousidou1, Maria Tzetis3, Emmanouil Kanavakis3,4, Konstantinos Stravodimos5, Kalliopi N Manola6, Gabriel E Pantelias6, Andreas Scorilas2 and Apostolos Klinakis *,1 1Biomedical Research Foundation Academy of Athens, Athens, Greece 2Department of Biochemistry and Molecular Biology, Faculty of Biology, National and Kapodistrian University of Athens, Athens, Greece 3Department of Medical Genetics, Medical School, "Aghia Sophia" Children's Hospital, National and Kapodistrian University of Athens, Athens, Greece 4University Research Institute for the Study and Treatment of Childhood Genetic and Malignant Diseases, "Aghia Sophia" Children's Hospital, National and Kapodistrian University of Athens, Athens, Greece 5First Department of Urology, "Laiko" General Hospital, Medical School, National and Kapodistrian University of Athens, Athens, Greece 6Laboratory of Health Physics, Radiobiology & Cytogenetics, National Center for Scientific Research (NCSR) "Demokritos", Athens, Greece *Corresponding author. Tel: +30 2106597069; E-mail: [email protected] EMBO Reports (2019)20:e46821https://doi.org/10.15252/embr.201846821 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Genome-wide studies in tumor cells have indicated that chromatin-modifying proteins are commonly mutated in human cancers. The lysine-specific methyltransferase 2C (KMT2C/MLL3) is a putative tumor suppressor in several epithelia and in myeloid cells. Here, we show that downregulation of KMT2C in bladder cancer cells leads to extensive changes in the epigenetic status and the expression of DNA damage response and DNA repair genes. More specifically, cells with low KMT2C activity are deficient in homologous recombination-mediated double-strand break DNA repair. Consequently, these cells suffer from substantially higher endogenous DNA damage and genomic instability. Finally, these cells seem to rely heavily on PARP1/2 for DNA repair, and treatment with the PARP1/2 inhibitor olaparib leads to synthetic lethality, suggesting that cancer cells with low KMT2C expression are attractive targets for therapies with PARP1/2 inhibitors. Synopsis The histone methyltransferase KMT2C/MLL3 is commonly mutated in cancer and involved in the transcriptional regulation of several DNA repair genes. Its loss leads to increased DNA damage and dependence on PARP1/2. KMT2C loss in bladder cancer cells affects the expression of DNA repair genes. KMT2C loss leads to extensive chromosomal instability as well as elevated DNA damage. KMT2C-depleted cancer cells depend on PARP1/2 and are sensitive to PARP inhibitors. Introduction It is well established that epigenetic dysregulation is an integral component of cancer etiology and progression 1. Therefore, it is not surprising that numerous epigenetic modifiers, such as DNMT3A, EZH2, and the MLL proteins, are frequently found genetically altered in cancer 2, 3. Lysine (K)-specific methyltransferase 2C (KMT2C, also known as MLL3) belongs to the mixed-lineage leukemia (MLL) family of histone methyltransferases which methylate the histone 3 tail at lysine 4 (H3K4) 4 as part of the complex proteins associated with Set 1 (COMPASS) complex 5. Although originally identified as oncogenic fusions in leukemia 6, recent genome-wide mutation studies have revealed frequent, presumably loss-of-function, mutations in various members of the MLL family, including MLL2/KMT2B, MLL3/KMT2C, and MLL4/KMT2D in a variety of malignancies, particularly solid tumors 7-11. Mouse studies have also uncovered a tumor suppressor role for KMT2C in acute myeloid leukemia (AML) 12 and urothelial tumorigenesis 13. Mechanistic studies of KMT2C in normal cells have focused primarily on its role in enhancer regulation 14, 15 by deposition of H3K4me1 marks. Interestingly, recent reports also indicate roles for KMT2C in transcription regulation, which are independent of its H3K4 monomethylation activity on enhancers 16, 17. However, its role in tumorigenesis remains largely undefined. Bladder cancer is the fifth most common human malignancy and the second most frequently diagnosed genitourinary tumor after prostate cancer 18. The majority of malignant tumors arising in the urinary bladder are urothelial carcinomas. Superficial carcinoma accounts for approximately 75% of the newly diagnosed cases while the remaining 25% represents muscle-invasive bladder cancer 19. The latter, often originating from superficial carcinoma, is a life-threatening disease with high metastatic potential. Recent genome-wide studies on superficial and muscle-invasive urothelial carcinoma have indicated that epigenetic regulators, including KMT2C, are commonly mutated in both types 11, 20. Here, we show that KMT2C is downregulated in neoplastic tissue in several epithelial cancers including urothelial carcinoma. As expected, KMT2C knockdown leads to epigenetic and expression changes. Of interest, genes involved in DNA damage response (DDR) and DNA repair, particularly homologous recombination (HR)-mediated DNA repair, are downregulated. This leads to increased DNA damage and chromosomal instability, highlighted by generation of micronuclei and numerical/regional chromosome losses. In our experiments, cells with reduced KMT2C expression are highly dependent on the alternative end-joining (alt-EJ) pathway for repair of double-strand breaks (DSBs), while inhibition of PARP1/2 causes synthetic lethality. Results KMT2C is downregulated in human epithelial cancers Mutational data from published studies show that the majority of KMT2C mutations cluster within the plant homeodomain (PHD) fingers 1–3 located in the N-terminus of the protein (Catalogue of Somatic Mutations in Cancer—COSMIC). KMT2C PHD fingers 1–3 act as "readers" of the histone methylation status, recognizing monomethylated H3K4 (H3K4me1), while the catalytic Su(var)3-9, Enhancer of zeste, Trithorax (SET) domain, located in the C-terminus, is the "writer" that adds methyl- groups to complete the methylation process 21. KMT2C is commonly mutated in high-grade muscle-invasive urothelial carcinoma 7, in which mutations were recently found equally distributed within the two major subtypes, luminal papillary and basal squamous 11. Little is known, however, about low-grade/early-stage tumors, including superficial papillomas. To address this issue, we sequenced the N- and C-terminus of the KMT2C transcript in tumors and matching normal tissues from a cohort of 72 patients diagnosed with superficial or muscle-invasive urothelial cancer of variable grade 22. We identified mutations primarily within PHD fingers 1–3 (Fig 1A), which showed no statistical preference with respect to grade and stage (mutations were found in 12/43 high grade vs. 4/29 low grade, and 9/32 invasive vs. 7/40 superficial tumors). Interestingly, a recent study on non-invasive bladder cancer also identified a high frequency (15%) of KMT2C likely loss-of-function mutations in non-invasive bladder cancer 20, indicating that KMT2C inactivation might occur early in carcinogenesis. In our mutation detection study, both frameshift and missense mutations were identified, the vast majority of which are predicted to be damaging (Fig 1A and Table EV1). Recently identified missense mutations within the PHD fingers 1–3 have been shown to disrupt the interaction between KMT2C and BAP1 leading to reduced recruitment of KMT2C to gene enhancers 1. Our KMT2C expression analysis in 104 matched normal/cancer tissue pairs from an expanded bladder cancer patients cohort (n = 138; Appendix Tables S1 and S2) revealed that, in comparison with normal tissues, KMT2C expression is downregulated in the majority of tumors at both the RNA and protein levels (71/104, P < 0.001; Fig 1B and C). Figure 1. KMT2C downregulation in cancer tissue KMT2C mutations identified in our study cohort of human bladder cancers. Mutations in red are predicted to be damaging while those in black benign, according to the PolyPHEN-2 algorithm (D and B, respectively, in Table EV1) 95. Comparison of KMT2C expression in cancer/healthy matched tissue pairs (n = 104) of the study cohort. Expression is presented as log(ratio tumor/healthy) in the y-axis. Data obtained from qRT–PCR analysis. P value calculated by Wilcoxon signed-rank test. Immunofluorescence (top) and Western blot analysis (bottom) against KMT2C on representative human bladder cancers with variable KMT2C transcript levels: 11th, 4th, 93rd, and 79th percentiles for UCC30, 6, 7, and 29, respectively (Appendix Table S2), from the differential expression analysis of the study cohort. Antibodies against KRT5 or KRT20 were used to stain urothelial cells and DAPI as nuclear counterstain. β-Actin is used as loading control in Western blots. Scale bars indicate 50 μm. Comparison of KMT2C expression in human healthy and cancer tissues from bladder cancer (BC, n = 136), colorectal adenocarcinoma (COAD, n = 128), non-small-cell lung cancer (NSCLC, n = 341), and head and neck squamous cell carcinoma (HNSCC, n = 174) patients. For NSCLC analysis, separate cohorts from adenocarcinoma and squamous cell carcinoma were combined. Separate analysis of the two NSCLC subtypes (adenocarcinoma and squamous cell carcinoma) yielded the same results. For COAD, the y-axis is the log2(ratio tumor/normal) of KMT2C expression as assessed with Affymetrix microarray. All expression data were obtained from TCGA through cbioportal.org. P values calculated by Mann–Whitney U-test. The middle lines inside the boxes indicate the median (50th percentile). The lower and the upper box boundaries represent the 25th percentile and the 75th percentile, respectively. The lower and upper whiskers extend to the lowest and highest values, respectively, within the 1.5× interquartile range (box height) from the box boundaries. Source data are available online for this figure. Source Data for Figure 1 [embr201846821-sup-0005-SDataFig1.pdf] Download figure Download PowerPoint KMT2C is mutated in several epithelial cancers 8, implying a general role as a tumor suppressor. To investigate this hypothesis, we performed a meta-analysis on publicly available RNA-seq data from The Cancer Genome Atlas (TCGA) Consortium 23-26. We found that similarly to bladder cancer (BC), KMT2C is downregulated in comparison with normal tissue in colorectal adenocarcinoma (COAD), non-small-cell lung cancer (NSCLC), and head and neck squamous cell carcinoma (HNSCC; Fig 1D). These data indicate that KMT2C downregulation is a rather common event in tumorigenesis in several human epithelial tissues. On the other hand, a recent report 27 and our own meta-analysis of non-epithelial cancers with the use of the GEPIA web server 28 indicated that, in comparison with respective healthy tissue, KMT2C is expressed at higher levels in glioblastoma multiforme (GBM), brain lower grade glioma (LGG), diffuse large B-cell lymphomas (DLBL), acute myeloid leukemia (AML), and sarcomas (SARC; Appendix Fig S1). This is in agreement with the fact that KMT2C truncating mutations account for only 0.6% in these cancer types (2/397, 2/512, 0/41, 3/200, and 2/254 cases, respectively; not shown). Our meta-analysis of publicly available DNA methylation data 7 obtained from the MethHC database 29 indicates that two Illumina methylation detection probes (cg17322443 and cg19258062) located within a CpG island (chr7:152435133–152437025, assembly GRCh38/hg38, ENCODE) spanning the KMT2C proximal promoter are subject to DNA methylation in bladder tumor samples, while remaining methylation-free in normal tissue (Fig EV1A and B), confirming a previously published report 30. More importantly, the same CpG island within the KMT2C proximal promoter is also hypermethylated in tumor samples from COAD, NSCLC, and HNSCC (Fig EV1C). Collectively, these data indicate that both mutational inactivation and transcriptional downregulation via promoter methylation of KMT2C might contribute to reduced activity facilitating tumor development in several epithelial cancers. Click here to expand this figure. Figure EV1. KMT2C promoter methylation in human cancers Schematic of the upstream promoter region of the KMT2C locus indicating the position and sequence of methylation detection probes within the CpG island (located at chr7:152435133–152437025, assembly GRCh38/hg38) that encompasses the KMT2C promoter region. Comparison of the methylation levels of the above probes in tumor samples and normal bladder tissue. Methylation data were obtained from TCGA through the MethHC database for n = 21 healthy/tumor pairs. Wilcoxon matched-pairs signed-rank test was used. Tumor vs. normal paired comparison of the methylation levels in the KMT2C promoter in various cancer types; cg1: cg17322443; cg2: cg19258062. Methylation data were obtained from the MethHC database (Huang et al 29). BC: n = 21, COAD: n = 21, NSCLC: n = 70, HNSCC: n = 50. For NSCLC analysis, separate cohorts from adenocarcinoma and squamous cell carcinoma were combined. Separate analysis of the two NSCLC subtypes yielded the same results. Wilcoxon matched-pairs signed-rank test was used. * designates P-value < 0.05 and ****P-value < 0.0001. Download figure Download PowerPoint KMT2C loss affects enhancer activity and gene expression in a subset of genes To investigate its role in urothelial carcinoma cells, we used two independent shRNAs (KD1/KD2) to knock down KMT2C levels in human BC cell lines (Fig 2A). While the loss of KMT2C activity did not affect cell proliferation or apoptosis (Appendix Fig S2), RNA-seq experiments in HTB9 cells showed that, directly or indirectly, 3,324 genes were transcriptionally affected upon KMT2C silencing (1.4-fold and higher change in expression levels). Of those, 1,846 were downregulated while 1,478 were upregulated. Gene ontology (GO) analysis indicated that many of the affected genes are involved in DDR, DNA repair, DNA replication, cell cycle control, and apoptosis, all of which are considered hallmarks of cancer, and are associated with tumor aggressiveness 31 (Fig 2B). In order to study directly the role of KMT2C and to circumvent the lack of chromatin immunoprecipitation (ChIP)-grade anti-KMT2C antibodies, we exogenously expressed a Flag-tagged KMT2C protein (fKMT2C) in HTB9/KD1 cells (Fig 2C). Figure 2. KMT2C loss leads to extensive epigenetic changes in human bladder cancer cells KMT2C transcript (left) and protein (right) levels in human bladder cancer cell lines stably transduced with lentiviral vectors expressing shRNAs against KMT2C (KD1 and KD2) in comparison with Scr control cells expressing scrambled shRNAs (Scr). RBBP5, another COMPASS complex protein used as internal control and b-actin as loading control. Transcript levels were assessed by qRT–PCR in triplicates, and values shown represent mean ± SEM. Bar graph showing selected biological processes and signaling pathways obtained from Gene Ontology (GO) enrichment analysis for the 3,324 differentially expressed genes between Scr control and KMT2C/KD1 HTB9 cells. Expression values were obtained from RNA-seq data. Quantitative RT–PCR for KMT2C in HTB9/KD1 cells, and HTB9/KD1 cells stably transfected with a plasmid expressing a Flag-tagged full-length KMT2C protein (fKMT2C). Expression levels are shown in the y-axis as respective ratios over KMT2C expression in Scr control cells (Scr expression corresponds to 1). Experiments were performed in triplicates and analyzed with Mann–Whitney U-test. Values shown represent mean ± SEM. * designates P-value < 0.05. Genome distribution of KMT2C peaks in HTB9/KD1 cells complemented with fKMT2C. Data obtained from ChIP-seq experiments. Density plot indicating KMT2C binding and H3K27ac levels on active enhancers in Scr control and KD1 HTB9 cells. Bar graph showing selected biological processes and signaling pathways obtained from Gene Ontology (GO) enrichment analysis for 253 genes in proximity to active enhancers affected by KMT2C knockdown and heatmap of their expression (> 1.5-fold H3K27ac and mRNA downregulation). Data obtained from ChIP-seq and RNA-seq experiments. Bedgraph indicating KMT2C binding and H3K27ac at a putative enhancer of the ITGB1 locus before and after KMT2C knockdown in HTB9 cells. Transcription factor binding motif analysis on active enhancers affected by KMT2C knockdown. Data obtained from ChIP-seq experiments. Source data are available online for this figure. Source Data for Figure 2 [embr201846821-sup-0006-SDataFig2.pdf] Download figure Download PowerPoint To gain further insight into the function of KMT2C in gene transcription regulation, we used fKMT2C-complemented HTB9/KD1 cells to map KMT2C binding genome-wide through ChIP-sequencing (ChIP-seq). In addition, to measure the epigenetic effects of KMT2C loss we performed ChIP-seq experiments for histone 3 lysine 27 acetylation (H3K27ac), histone 3 lysine 4 trimethylation (H3K4me3), and histone 3 lysine 9 acetylation (H3K9ac) histone modifications on HTB9 KMT2C/KD1 and control Scr cells. ChIP-seq experiments performed with anti-Flag antibodies indicated that KMT2C binding sites are equally dispersed among promoter, gene body, and intergenic regions (12,417, 10,882, and 9,885 peaks, respectively; Fig 2D). In agreement with its role in enhancer regulation, KMT2C colocalizes with the active enhancer mark H3K27ac on intergenic sites likely representing active enhancers 32 (Fig 2E). Our ChIP-Seq analysis identified 2,808 genes proximally located to enhancers that are characterized by KMT2C binding and significant H3K27ac loss upon KMT2C silencing. GO analysis on genes of this group that are also downregulated upon KMT2C silencing (1.5-fold or higher reduction) revealed an enrichment in processes such as focal adhesion and integrin-mediated adhesion as well as ErbB and Wnt signaling pathways (Fig 2F). More specifically, we identified genes that encode proteins which are critical for cell adherence to the epithelial basement membrane: ITGB1, ITGB6, RHOB, a putative tumor suppressor also commonly mutated in BC 7, 20, MMP7; (Fig 2G); the extracellular matrix organization LOXL2, LOXL4 and TIMP4, an epigenetically silenced putative tumor suppressor in bladder carcinoma 33, and epithelial development and differentiation (SMAD6, SOX2, EREG, WNT11, BMP2). Interestingly, KMT2D/MLL4 was recently reported to regulate the enhancers of genes involved in cell–cell and cell–matrix adhesion as well as in differentiation of keratinocytes affecting the expression of ITGB2, ITGB4, LOXL1, LOXL2, SOX7, WNT10A genes by a similar way 34. An analysis of transcription factor binding motifs in KMT2C peaks, located at enhancers that are characterized by significant H3K27ac loss upon KMT2C silencing, identified JUNB, TEAD, RUNX1, and MAFA as the most enriched transcription factors (Fig 2H). KMT2C localizes at promoters and controls the expression of DNA damage response and repair genes Interestingly, our ChIP-seq experiments also revealed 12417 fKMT2C binding sites enriched at transcription start site proximal regions (TSS ± 1,500 bp) that contain large domains of H3K4me3 H3K9ac and H3K27ac marks (Fig 3A and B). KMT2C silencing was associated with transcriptional suppression of 1,368 genes, which are characterized by promoter-only KMT2C binding. This finding indicates that besides enhancer regulation, KMT2C is also involved in promoter activation in cancer cells. Transcription factor binding motif analysis of fKMT2C-bound regions yielded a totally different set of transcription factors from those identified in enhancers. The most prominent of these is ELK1 (Fig 3C), a prominent RAS/MAPK target controlling components of the basal transcriptional machinery, the spliceosome and the ribosome 35. Figure 3. KMT2C controls the expression of DDR and DNA repair genes in BC cells Density plot indicating KMT2C binding and H3K4me3, H3K27ac, and H3K9ac levels on transcription start sites (TSS) in HTB9 cells. Histogram indicating distribution of histone modifications around transcription start sites (TSS ± 5,000 bp). Data obtained from ChIP-seq with antibodies against the indicated histone modifications. Transcription factor binding motif analysis on TSS of genes transcriptionally affected by KMT2C knockdown. Data obtained from ChIP-seq experiments. Boxplot indicating expression (left) and H3K4me3 levels (right) of genes with KMT2C presence on their promoters in Scr control and KMT2C/KD1 cells. Median comparison of Expr/K4 m3 values was performed with two-tailed paired Wilcoxon rank sum test with continuity correction. The middle lines inside the boxes indicate the median (50th percentile). The lower and the upper box boundaries represent the 25th percentile and the 75th percentile, respectively. The lower and upper whiskers extend to the lowest and highest values, respectively, within the 1.5× interquartile range (box height) from the box boundaries. Heatmap comparison of the expression levels of genes implicated in DDR between control (Scr) and KMT2C-knockdown (KD1) HTB9 cells (left). Expression data were obtained from RNA-seq experiments. Western blot analysis of selected proteins in control (Scr) and KMT2C-knockdown (KD1 and KD2) HTB9 cells (right). Expression level restoration of selected genes in KMT2C/KD1 HTB9 cells complemented with exogenously expressed Flag-tagged KMT2C (fKMT2C). Data obtained by qRT–PCR. Experiments were performed in triplicates, and values shown represent mean ± SEM. Source data are available online for this figure. Source Data for Figure 3 [embr201846821-sup-0007-SDataFig3.pdf] Download figure Download PowerPoint Our ChIP-seq and RNA-seq data indicated that upon KMT2C silencing, the subgroup of genes showing reduced expression levels also show reduced H3K4me3 levels at the respective TSSs (Fig 3D). GO analysis on this group of the 1,368 downregulated genes revealed several processes such as DDR and DSB repair by HR, which interestingly presented the highest score (see also Fig 2B). More specifically, KMT2C silencing was associated with decreased expression of key components of DDR (ATM, ATR) and the HR DNA repair pathway (BRCA1, BRCA2, RAD50, RAD51; Fig 3E). Interestingly, restoration of KMT2C activity by means of exogenous expression of fKMT2C also restored the expression levels of these genes (Fig 3F). Our own ChIP-seq data as well as ENCODE data indicate that KMT2C and the COMPASS complex component RBBP5 colocalize together with ELK1 upon the TSS of ATM, ATR, BRCA1, and BRCA2 genes (Fig 4A). Moreover, KMT2C levels modulate positively the H3K4me3 enrichment on TSS of these genes, indicating an important role for this histone methyltransferase on their transcriptional activation. More specifically, upon KMT2C silencing, H3K4me3 levels were significantly reduced, whereas restoration of KMT2C activity also restored H3K4me3 levels. Promoter region immunoprecipitation either as direct binding or through long-range enhancer interactions has previously been reported for both KMT2C and KMT2D 36, 37. KMT2C binding upon the promoter region of the ATM, ATR, BRCA1, and BRCA2 genes is independently corroborated in a recently published analysis 38 (Appendix Fig S3). Interestingly in the same study, a 32% of KMT2C is located within promoter regions, indicating roles for KMT2C besides enhancer H3K4 monomethylation. Figure 4. KMT2C controls the expression of DDR and DNA repair genes in various cancers Bedgraphs indicating KMT2C and H3K4me3 binding at the TSS of indicated loci in HTB9 cel
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