Arne H. Smits

Dynamic Readers for 5-(Hydroxy)Methylcytosine and Its Oxidized Derivatives

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.

Stoichiometry of chromatin-associated protein complexes revealed by label-free quantitative mass spectrometry-based proteomics

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.

H2A.Z demarcates intergenic regions of the plasmodium falciparum epigenome that are dynamically marked by H3K9ac and H3K4me3.

Epigenetic regulatory mechanisms and their enzymes are promising targets for malaria therapeutic intervention; however, the epigenetic component of gene expression in P. falciparum is poorly understood. Dynamic or stable association of epigenetic marks with genomic features provides important clues about their function and helps to understand how histone variants/modifications are used for indexing the Plasmodium epigenome. We describe a novel, linear amplification method for next-generation sequencing (NGS) that allows unbiased analysis of the extremely AT-rich Plasmodium genome. We used this method for high resolution, genome-wide analysis of a histone H2A variant, H2A.Z and two histone H3 marks throughout parasite intraerythrocytic development. Unlike in other organisms, H2A.Z is a constant, ubiquitous feature of euchromatic intergenic regions throughout the intraerythrocytic cycle. The almost perfect colocalisation of H2A.Z with H3K9ac and H3K4me3 suggests that these marks are preferentially deposited on H2A.Z-containing nucleosomes. By performing RNA-seq on 8 time-points, we show that acetylation of H3K9 at promoter regions correlates very well with the transcriptional status whereas H3K4me3 appears to have stage-specific regulation, being low at early stages, peaking at trophozoite stage, but does not closely follow changes in gene expression. Our improved NGS library preparation procedure provides a foundation to exploit the malaria epigenome in detail. Furthermore, our findings place H2A.Z at the cradle of P. falciparum epigenetic regulation by stably defining intergenic regions and providing a platform for dynamic assembly of epigenetic and other transcription related complexes.

Quantitative Dissection and Stoichiometry Determination of the Human SET1/MLL Histone Methyltransferase Complexes

ABSTRACT 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.

Active DNA demethylation at enhancers during the vertebrate phylotypic period

The vertebrate body plan and organs are shaped during a conserved embryonic phase called the phylotypic stage. However, the mechanisms that guide the epigenome through this transition and their evolutionary conservation remain elusive. Here we report widespread DNA demethylation of enhancers during the phylotypic period in zebrafish, Xenopus tropicalis and mouse. These enhancers are linked to developmental genes that display coordinated transcriptional and epigenomic changes in the diverse vertebrates during embryogenesis. Binding of Tet proteins to (hydroxy)methylated DNA and enrichment of 5-hydroxymethylcytosine in these regions implicated active DNA demethylation in this process. Furthermore, loss of function of Tet1, Tet2 and Tet3 in zebrafish reduced chromatin accessibility and increased methylation levels specifically at these enhancers, indicative of DNA methylation being an upstream regulator of phylotypic enhancer function. Overall, our study highlights a regulatory module associated with the most conserved phase of vertebrate embryogenesis and suggests an ancient developmental role for Tet dioxygenases.

Insight into the Architecture of the NuRD Complex STRUCTURE OF THE RbAp48-MTA1 SUBCOMPLEX

Background: The NuRD complex controls gene expression through altering chromatin structure. Results: The MTA1-RbAp48 structure shows how the RbAp46/p48 histone chaperones are recruited to NuRD. Conclusion: The MTA subunits act as scaffolds for NuRD complex assembly. Significance: The MTA/RbAp48 interaction prevents binding of histone H4, which is crucial for understanding the role of the RbAp46/p48 chaperones in the complex. The nucleosome remodeling and deacetylase (NuRD) complex is a widely conserved transcriptional co-regulator that harbors both nucleosome remodeling and histone deacetylase activities. It plays a critical role in the early stages of ES cell differentiation and the reprogramming of somatic to induced pluripotent stem cells. Abnormalities in several NuRD proteins are associated with cancer and aging. We have investigated the architecture of NuRD by determining the structure of a subcomplex comprising RbAp48 and MTA1. Surprisingly, RbAp48 recognizes MTA1 using the same site that it uses to bind histone H4, showing that assembly into NuRD modulates RbAp46/48 interactions with histones. Taken together with other results, our data show that the MTA proteins act as scaffolds for NuRD complex assembly. We further show that the RbAp48-MTA1 interaction is essential for the in vivo integration of RbAp46/48 into the NuRD complex.

Characterizing Protein–Protein Interactions Using Mass Spectrometry: Challenges and Opportunities

During the past decades, mass spectrometry (MS)-based proteomics has become an important technology to identify protein-protein interactions (PPIs). The application of a quantitative filter in protein enrichments from crude lysates to discriminate bona fide interactors from background proteins has proved to be particularly powerful. Recently, many different approaches to identify PPIs have been developed, including proximity-ligation technology and global interactome profiling based on the co-behavior of protein complexes in biochemical purification or perturbation experiments. Furthermore, methodologies have been introduced that provide information regarding the stoichiometry and topology of detected PPIs. We review these novel methodologies and emphasize the need to miniaturize workflows to analyze protein interactions in biological and pathological contexts where sample amounts are limited.

Cdyl, a new partner of the inactive X chromosome and potential reader of H3K27me3 and H3K9me2.

ABSTRACT X chromosome inactivation is a remarkable example of chromosome-wide gene silencing and facultative heterochromatin formation. Numerous histone posttranslational modifications, including H3K9me2 and H3K27me3, accompany this process, although our understanding of the enzymes that lay down these marks and the factors that bind to them is still incomplete. Here we identify Cdyl, a chromodomain-containing transcriptional corepressor, as a new chromatin-associated protein partner of the inactive X chromosome (Xi). Using mouse embryonic stem cell lines with mutated histone methyltransferase activities, we show that Cdyl relies on H3K9me2 for its general association with chromatin in vivo. For its association with Xi, Cdyl requires the process of differentiation and the presence of H3K9me2 and H3K27me3, which both become chromosomally enriched following Xist RNA coating. We further show that the removal of the PRC2 component Eed and subsequent loss of H3K27me3 lead to a reduction of both Cdyl and H3K9me2 enrichment on inactive Xi. Finally, we show that Cdyl associates with the H3K9 histone methyltransferase G9a and the MGA protein, both of which are also found on Xi. We propose that the combination of H3K9me2 and H3K27me3 recruits Cdyl to Xi, and this, in turn, may facilitate propagation of the H3K9me2 mark by anchoring G9a.

Plasmodium falciparum centromeres display a unique epigenetic makeup and cluster prior to and during schizogony

Centromeres are essential for the faithful transmission of chromosomes to the next generation, therefore being essential in all eukaryotic organisms. The centromeres of Plasmodium falciparum, the causative agent of the most severe form of malaria, have been broadly mapped on most chromosomes, but their epigenetic composition remained undefined. Here, we reveal that the centromeric histone variant PfCENH3 occupies a 4–4.5 kb region on each P. falciparum chromosome, which is devoid of pericentric heterochromatin but harbours another histone variant, PfH2A.Z. These CENH3 covered regions pinpoint the exact position of the centromere on all chromosomes and revealed that all centromeric regions have similar size and sequence composition. Immunofluorescence assay of PfCENH3 strongly suggests that P. falciparum centromerescluster to a single nuclear location prior to and during mitosis and cytokinesis but dissociate soon after invasion. In summary, we reveal a dynamic association of Plasmodium centromeres, which bear a unique epigenetic signature and conform to a strict structure. These findings suggest that DNA‐associated and epigenetic elements play an important role in centromere establishment in this important human pathogen.

Mbd5 and mbd6 interact with the human pr-dub complex through their methyl-cpg-binding domain

MBD5 and MBD6 are two members of the methyl‐CpG‐binding domain (MBD) family of proteins that are poorly characterized. Studies performed thus far have failed to show binding of the MBD5 and MBD6 MBD to methylated DNA. Here, we show that both MBD5 and MBD6 interact with the mammalian PR‐DUB Polycomb protein complex in a mutually exclusive manner. Strikingly, the MBD of MBD5 and MBD6 is both necessary and sufficient to mediate this interaction. Chromatin immunoprecipitation analyses reveal that MBD6 and FOXK2/PR‐DUB share a subset of genomic target genes, suggesting a functional interaction in vivo. Finally, we show that MBD6, but not MBD5, is recruited to sites of DNA damage in a PR‐DUB independent manner. Our study thus implies a shared function for MBD5 and MBD6 through an interaction with PR‐DUB, as well as an MBD6‐specific recruitment to sites of DNA damage.