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.

RNA modifications are integral to the regulation of RNA metabolism. One abundant mRNA modification is N6-methyladenosine (m6A), which affects various aspects of RNA metabolism, including splicing, translation and degradation. Current knowledge about the proteins recruited to m6A to carry out these molecular processes is still limited. Here we describe comprehensive and systematic mass-spectrometry-based screening of m6A interactors in various cell types and sequence contexts. Among the main findings, we identified G3BP1 as a protein that is repelled by m6A and positively regulates mRNA stability in an m6A-regulated manner. Furthermore, we identified FMR1 as a sequence-context-dependent m6A reader, thus revealing a connection between an mRNA modification and an autism spectrum disorder. Collectively, our data represent a rich resource and shed further light on the complex interplay among m6A, m6A interactors and mRNA homeostasis.

Deregulation in lysine methylation signaling has emerged as a common etiologic factor in cancer pathogenesis, with inhibitors of several histone lysine methyltransferases (KMTs) being developed as chemotherapeutics1. The largely cytoplasmic KMT SMYD3 (SET and MYND domain containing protein 3) is overexpressed in numerous human tumors2-4. However, the molecular mechanism by which SMYD3 regulates cancer pathways and its relationship to tumorigenesis in vivo are largely unknown. Here we show that methylation of MAP3K2 by SMYD3 increases MAP Kinase signaling and promotes the formation of Ras-driven carcinomas. Using mouse models for pancreatic ductal adenocarcinoma (PDAC) and lung adenocarcinoma (LAC), we found that abrogating SMYD3 catalytic activity inhibits tumor development in response to oncogenic Ras. We employed protein array technology to identify the MAP3K2 kinase as a target of SMYD3. In cancer cell lines, SMYD3-mediated methylation of MAP3K2 at lysine 260 potentiates activation of the Ras/Raf/MEK/ERK signaling module. Finally, the PP2A phosphatase complex, a key negative regulator of the MAP Kinase pathway, binds to MAP3K2 and this interaction is blocked by methylation. Together, our results elucidate a new role for lysine methylation in integrating cytoplasmic kinase-signaling cascades and establish a pivotal role for SMYD3 in the regulation of oncogenic Ras signaling.

A key step in gene repression by Polycomb is trimethylation of histone H3 K27 by PCR2 to form H3K27me3. H3K27me3 provides a binding surface for PRC1. We show that monoubiquitination of histone H2A by PRC1-type complexes to form H2Aub creates a binding site for Jarid2-Aebp2-containing PRC2 and promotes H3K27 trimethylation on H2Aub nucleosomes. Jarid2, Aebp2 and H2Aub thus constitute components of a positive feedback loop establishing H3K27me3 chromatin domains.

Many cellular proteins assemble into macromolecular protein complexes. The identification of protein–protein interactions and quantification of their stoichiometry is therefore crucial to understand the molecular function of protein complexes. Determining the stoichiometry of protein complexes is usually achieved by mass spectrometry-based methods that rely on introducing stable isotope-labeled reference peptides into the sample of interest. However, these approaches are laborious and not suitable for high-throughput screenings. Here, we describe a robust and easy to implement label-free relative quantification approach that combines the detection of high-confidence protein–protein interactions with an accurate determination of the stoichiometry of the identified protein–protein interactions in a single experiment. We applied this method to two chromatin-associated protein complexes for which the stoichiometry thus far remained elusive: the MBD3/NuRD and PRC2 complex. For each of these complexes, we accurately determined the stoichiometry of the core subunits while at the same time identifying novel interactors and their stoichiometry.

While the core subunits of Polycomb group (PcG) complexes are well characterized, little is known about the dynamics of these protein complexes during cellular differentiation. We used quantitative interaction proteomics and genome-wide profiling to study PcG proteins in mouse embryonic stem cells (ESCs) and neural progenitor cells (NPCs). We found the stoichiometry and genome-wide binding of PRC1 and PRC2 to be highly dynamic during neural differentiation. Intriguingly, we observed a downregulation and loss of PRC2 from H3K27me3-marked chromatin during differentiation, whereas PRC1 was retained at these sites. Additionally, we found PRC1 at enhancer and promoter regions independent of PRC2 binding and H3K27me3. Finally, overexpression of NPC-specific PRC1 interactors in ESCs led to increased Ring1b binding to and decreased expression of NPC-enriched Ring1b target genes. In summary, our integrative analyses have uncovered dynamic PcG subcomplexes and widespread co-localization with active chromatin marks during differentiation.

Methylation of lysine 4 on histone H3 (H3K4) at promoters is tightly linked to transcriptional regulation in human cells. At least six different COMPASS-like multisubunit (SET1/MLL) complexes that contain methyltransferase activity for H3K4 have been described, but a comprehensive and quantitative analysis of these SET1/MLL complexes is lacking. We applied label-free quantitative mass spectrometry to determine the subunit composition and stoichiometry of the human SET1/MLL complexes. We identified both known and novel, unique and shared interactors and determined their distribution and stoichiometry over the different SET1/MLL complexes. In addition to being a core COMPASS subunit, the Dpy30 protein is a genuine subunit of the NURF chromatin remodeling complex. Furthermore, we identified the Bod1 protein as a discriminator between the SET1B and SET1A complexes, and we show that the H3K36me-interactor Psip1 preferentially binds to the MLL2 complex. Finally, absolute protein quantification in crude lysates mirrors many of the observed SET1/MLL complex stoichiometries. Our findings provide a molecular framework for understanding the diversity and abundance of the different SET1/MLL complexes, which together establish the H3K4 methylation landscape in human cells.

NuRD (nucleosome remodeling and histone deacetylase) is a versatile multi-protein complex with roles in transcription regulation and the DNA damage response. Here, we show that ZMYND8 bridges NuRD to a number of putative DNA-binding zinc finger proteins. The MYND domain of ZMYND8 directly interacts with PPPLΦ motifs in the NuRD subunit GATAD2A. Both GATAD2A and GATAD2B exclusively form homodimers and define mutually exclusive NuRD subcomplexes. ZMYND8 and NuRD share a large number of genome-wide binding sites, mostly active promoters and enhancers. Depletion of ZMYND8 does not affect NuRD occupancy genome-wide and only slightly affects expression of NuRD/ZMYND8 target genes. In contrast, the MYND domain in ZMYND8 facilitates the rapid, poly(ADP-ribose)-dependent recruitment of GATAD2A/NuRD to sites of DNA damage to promote repair by homologous recombination. Thus, these results show that a specific substoichiometric interaction with a NuRD subunit paralogue provides unique functionality to distinct NuRD subcomplexes.