Abstract
BackgroundThe histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; however, the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxy-terminal SET domain. To study effects of KMT2C expression in CRC cells, we restored one allele to wild type KMT2C in the two CRC cell lines RKO and HCT116, which both are homozygous c.8390delA mutant.ResultsGene editing resulted in increased KMT2C expression, increased H3K4me1 levels, altered gene expression profiles, and subtle negative effects on cell growth, where higher dependence and stronger effects of KMT2C expression were observed in RKO compared to HCT116 cells. Surprisingly, we found that the two RKO and HCT116 CRC cell lines have distinct baseline H3K4me1 epigenomic profiles. In RKO cells, a flatter genome-wide H3K4me1 profile was associated with more increased H3K4me1 deposition at enhancers, reduced cell growth, and more differential gene expression relative to HCT116 cells when KMT2C was restored. Profiling of H3K4me1 did not indicate a highly specific regulation of gene expression as KMT2C-induced H3K4me1 deposition was found globally and not at a specific enhancer sub-set in the engineered cells. Although we observed variation in differentially regulated gene sets between cell lines and individual clones, differentially expressed genes in both cell lines included genes linked to known cancer signaling pathways, estrogen response, hypoxia response, and aspects of immune system regulation.ConclusionsHere, KMT2C restoration reduced CRC cell growth and reinforced genome-wide H3K4me1 deposition at enhancers; however, the effects varied depending upon the H3K4me1 status of KMT2C deficient cells. Results indicate that KMT2C inactivation may promote colorectal cancer development through transcriptional dysregulation in several pathways with known cancer relevance.
Highlights
The histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; the effects of mutations on epigenome organization, gene expression, and cell growth are not clear
Restoration of KMT2C expression in microsatellite instability (MSI) colorectal cancer (CRC) cells by gene targeting A characteristic of MSI cancers is the accumulation of frameshift mutations in microsatellite repeats
This indicates that wild type KMT2C expression was restored in RKO and HCT116 KMT2CinsG cell lines and that the expression level was upregulated to the same extent in both cell lines
Summary
The histone 3 lysine 4 (H3K4) monomethylase KMT2C is mutated across several cancer types; the effects of mutations on epigenome organization, gene expression, and cell growth are not clear. A frequently recurring mutation in colorectal cancer (CRC) with microsatellite instability is a single nucleotide deletion within the exon 38 poly-A(9) repeat (c.8390delA) which results in frameshift preceding the functional carboxyterminal SET domain. The first family member to be linked to cancer development was KMT2A (MLL1), for which translocations resulting in oncogenic fusion proteins were first discovered in leukemias, and non-synonymous mutations, predominantly frameshift and nonsense, are frequently found in several solid tumor types [4, 5]. The genes encoding KMT2C (MLL3) and KMT2D (MLL4) are among the most frequently mutated in cancer [1]. These two proteins function in maintenance of histone 3 lysine 4 monomethylation (H3K4me1) levels at enhancer elements (reviewed in [6]). The mutational pattern suggests a tumor suppressor function of KMT2C which may be disrupted by differently localized mutations
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