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RRBS analysis of Braf-activated MEFs

ABSTRACT: We investigated DNA methylation status by RRBS method after oncogenic Braf activation using MEFs isolated from Braf knockin mice. Window-based analysis made it clear that the regions <500 bp from TSS were hypermethylated after Braf activation, whereas those >5000 bp from TSS were less methylated after Braf activation compared with wild-type MEFs. A hallmark of human cancer genomes is global DNA hypomethylation accompanied by focal DNA hypermethylation. Although factors and pathways that mediate DNA hypermethylation in cancer cells have been described, the basis of DNA hypomethylation remains to be determined. Here we show that expression of an activated BRAF oncoprotein, BRAFV600E, is sufficient to promote global DNA hypomethylation in mouse embryonic fibroblasts (MEFs). Conversion of 5-methylcytosine (5-mC) to its oxidation product 5-hydroxymethylcytosine (5-hmC) is thought to be the first step in a pathway leading to complete DNA demethylation. We find that BRAFV600E-directed global DNA hypomethylation occurs through conversion of 5-mC to 5-hmC by TET3, a member of the ten-eleven translocation (TET) family of proteins. In normal cells, TET3 is maintained at low levels resulting from ubiquitination by a SKP1–CUL1–F-box (SCF)-type ubiquitin ligase containing the F-box protein FBXW7, a known tumor suppressor, and subsequent proteasome-mediated degradation. BRAFV600E increases TET3 levels through inhibition of glycogen synthase kinase-3b (GSK3b), a protein kinase that phosphorylates TET3 and is required for SCFFBXW7-mediated ubiquitination. Consistent with these cell culture results, we find increased levels of TET3 and 5-hmC in experimentally induced mouse lung adenomas and patient-derived human colorectal adenomas that contain BRAFV600E. Moreover, we show that RNAi-mediated knockdown of Tet3 results in decreased BRAFV600E-induced tumorigenesis. Finally, to test the generality of our findings, we repeated several key experiments using a KRASG12D oncoprotein rather than BRAFV600E and obtained similar results. Collectively, our results elucidate a mechanism of global DNA hypomethylation promoted by oncogenic BRAF and KRAS, and establish an essential role for TET3 at an early stage of oncogenesis.

ORGANISM(S): Mus musculus

PROVIDER: GSE73770 | GEO | 2023/04/05

REPOSITORIES: GEO

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Project description:Tet proteins oxidize 5-methylcytosine (mC) to generate 5-hydroxymethyl (hmC), 5-formyl (fC) and 5-carboxylcytosine (caC). The exact function of these oxidative cytosine bases remains elusive. We applied quantitative mass spectrometry-based proteomics to identify readers for mC and hmC in mouse embryonic stem cells (mESC), neuronal progenitor cells (NPC) and adult mouse brain tissue. Readers for these modifications are only partially overlapping and some readers, such as Rfx proteins, display strong specificity. Interactions are dynamic during differentiation, as for example evidenced by the mESC-specific binding of Klf4 to mC and the NPC-specific binding of Uhrf2 to hmC, suggesting specific biological roles for mC and hmC. Oxidized derivatives of mC recruit distinct transcription regulators as well as a large number of DNA repair proteins in mouse ES cells, implicating the DNA damage response as a major player in active DNA demethylation. Peptides were separated on an EASY-nLC (Proxeon) connected online to an LTQ-Orbitrap-Velos mass spectrometer. Spectra were recorded in CID mode. A gradient of organic solvent (5-30% acetonitrile) was applied (120 minutes) and the top 15 most abundant peptides were fragmented for MS/MS, using an exclusion list of 500 proteins for 45 seconds. Raw data were analyzed using Maxquant version 1.2.2.5 and the integrated Andromeda search engine against protein database ipi.MOUSE.v3.68. Using Perseus, data was filtered for contaminants, reverse hits, number of peptides (>1) and unique peptides (>0). Ratios were logarithmitized (log2) and groups (consisting of forward and reverse) were defined. Proteins were filtered to have at least 2 valid values in one of the groups and missing values were imputed based on a normal distribution (width=0.2 and shift=0), after which Significance B was calculated (Benj.Hoch.FDR=0.05). Scatterplots were made using R. Proteins were defined to be significant when both forward and reverse significance p<0.05 and minimal ratios were >2 in both experiments. The H/L ratios shown in Figure2A-C were calculated using the formula (log(forward ratio) - log(reverse ratio))/2.

Project description:5-hydroxymethylcytosine (5-hmC), a derivative of 5-methylcytosine (5-mC), is abundant in the brain for unknown reasons. We mapped the genomic distribution of 5-hmC and 5-mC in human and mouse tissues using glucosylation of 5-hmC coupled with restriction enzyme digestion, and interrogation on microarrays. We detected 5-hmC enrichment in genes with synapse-related functions in the brain. We also identified significant, tissue-specific differential distributions of these DNA modifications at the exon-intron boundary, in both human and mouse. This boundary change was mainly due to 5-hmC in the brain, but due to 5-mC in non-neural contexts. This pattern was replicated in multiple independent datasets, and the brain-specific change in 5-hmC was validated using single-molecule sequencing. Moreover, in the brain, constitutive exons contained higher levels of 5-hmC, relative to alternatively-spliced exons. Our study suggests a novel role for 5-hmC in RNA splicing and synaptic function in the brain The M-NM-2-glucosyltransferase (BGT) enzyme transfers a glucose molecule specifically to the hydroxymethyl group of 5-hmC, thus rendering it resistant to digestion by the methylation insensitive MspI enzyme at the ChmCGG target site; 5-hmC is thus detected by differential resistance to MspI-digestion with and without glucosylation of genomic DNA (gDNA). HpaII (targets the same site, CCGG) cannot cut CmCGG or ChmCGG, and conceptually its difference with MspI digestion is a measure of both 5-mC and 5-hmC. Subtraction of 5-hmC from the HpaII-based estimate therefore measures 5-mC. These estimates were measured on respective Affymetrix whole-genome tiling arrays (2.0 R ) MspI - APRIL_fc1.ch02_coverage.bed Undigested DNA - APRIL_fc1.ch01_coverage.bed GluMspI - APRIL_fc1.ch04_coverage_NONTARGET.bed GluMspI - APRIL_fc1.ch04_coverage.bed MspI - APRIL_fc1.ch02_coverage_NONTARGET.bed Undigested DNA - APRIL_fc1.ch01_coverage_NONTARGET.bed MspI - MARCH_fc1.ch02_coverage_NONTARGET.bed Undigested DNA - MARCH_fc1.ch01_coverage_NONTARGET.bed GluMspI - MARCH_fc1.ch04_coverage_NONTARGET.bed GluMspI - MARCH_fc1.ch04_coverage.bed MspI - MARCH_fc1.ch02_coverage.bed Undigested DNA - MARCH_fc1.ch01_coverage.bed MspI - MAY_fc1.ch02_coverage.bed GluMspI - MAY_fc1.ch04_coverage_NONTARGET.bed GluMspI - MAY_fc1.ch04_coverage.bed Undigested DNA - MAY_fc1.ch01_coverage.bed MspI - MAY_fc1.ch02_coverage_NONTARGET.bed Undigested DNA - MAY_fc1.ch01_coverage_NONTARGET.bed

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RRBS analysis of Braf-activated MEFs

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