Genome-wide investigation of the dynamic changes of epigenome modifications after global DNA methylation editing.

Chromatin properties are regulated by complex networks of epigenome modifications. Currently, it is unclear how these modifications interact and if they control downstream effects such as gene expression. We employed promiscuous chromatin binding of a zinc finger fused catalytic domain of DNMT3A to introduce DNA methylation in HEK293 cells at many CpG islands (CGIs) and systematically investigated the dynamics of the introduced DNA methylation and the consequent changes of the epigenome network. We observed efficient methylation at thousands of CGIs, but it was unstable at about 90% of them, highlighting the power of genome-wide molecular processes that protect CGIs against DNA methylation. Partially stable methylation was observed at about 1000 CGIs, which showed enrichment in H3K27me3. Globally, the introduced DNA methylation strongly correlated with a decrease in gene expression indicating a direct effect. Similarly, global but transient reductions in H3K4me3 and H3K27ac were observed after DNA methylation but no changes were found for H3K9me3 and H3K36me3. Our data provide a global and time-resolved view on the network of epigenome modifications, their connections with DNA methylation and the responses triggered by artificial DNA methylation revealing a direct repressive effect of DNA methylation in CGIs on H3K4me3, histone acetylation, and gene expression.

A functional LSD1 coregulator screen reveals a novel transcriptional regulatory cascade connecting R-loop homeostasis with epigenetic regulation.

The lysine specific demethylase 1 (LSD1) plays a pivotal role in cellular differentiation by regulating the expression of key developmental genes in concert with different coregulatory proteins. This process is impaired in different cancer types and incompletely understood. To comprehensively identify functional coregulators of LSD1, we established a novel tractable fluorescent reporter system to monitor LSD1 activity in living cells. Combining this reporter system with a state-of-the-art multiplexed RNAi screen, we identify the DEAD-box helicase 19A (DDX19A) as a novel coregulator and demonstrate that suppression of Ddx19a results in an increase of R-loops and reduced LSD1-mediated gene silencing. We further show that DDX19A binds to tri-methylated lysine 27 of histone 3 (H3K27me3) and it regulates gene expression through the removal of transcription promoting R-loops. Our results uncover a novel transcriptional regulatory cascade where the downregulation of genes is dependent on the LSD1 mediated demethylation of histone H3 lysine 4 (H3K4). This allows the polycomb repressive complex 2 (PRC2) to methylate H3K27, which serves as a binding site for DDX19A. Finally, the binding of DDX19A leads to the efficient removal of R-loops at active promoters, which further de-represses LSD1 and PRC2, establishing a positive feedback loop leading to a robust repression of the target gene.

Transcriptional plasticity promotes primary and acquired resistance to BET inhibition.

Following the discovery of BRD4 as a non-oncogene addiction target in acute myeloid leukaemia (AML), bromodomain and extra terminal protein (BET) inhibitors are being explored as a promising therapeutic avenue in numerous cancers. While clinical trials have reported single-agent activity in advanced haematological malignancies, mechanisms determining the response to BET inhibition remain poorly understood. To identify factors involved in primary and acquired BET resistance in leukaemia, here we perform a chromatin-focused RNAi screen in a sensitive MLL-AF9;Nras(G12D)-driven AML mouse model, and investigate dynamic transcriptional profiles in sensitive and resistant mouse and human leukaemias. Our screen shows that suppression of the PRC2 complex, contrary to effects in other contexts, promotes BET inhibitor resistance in AML. PRC2 suppression does not directly affect the regulation of Brd4-dependent transcripts, but facilitates the remodelling of regulatory pathways that restore the transcription of key targets such as Myc. Similarly, while BET inhibition triggers acute MYC repression in human leukaemias regardless of their sensitivity, resistant leukaemias are uniformly characterized by their ability to rapidly restore MYC transcription. This process involves the activation and recruitment of WNT signalling components, which compensate for the loss of BRD4 and drive resistance in various cancer models. Dynamic chromatin immunoprecipitation sequencing and self-transcribing active regulatory region sequencing of enhancer profiles reveal that BET-resistant states are characterized by remodelled regulatory landscapes, involving the activation of a focal MYC enhancer that recruits WNT machinery in response to BET inhibition. Together, our results identify and validate WNT signalling as a driver and candidate biomarker of primary and acquired BET resistance in leukaemia, and implicate the rewiring of transcriptional programs as an important mechanism promoting resistance to BET inhibitors and, potentially, other chromatin-targeted therapies.

The histone chaperone CAF-1 safeguards somatic cell identity.

Cellular differentiation involves profound remodelling of chromatic landscapes, yet the mechanisms by which somatic cell identity is subsequently maintained remain incompletely understood. To further elucidate regulatory pathways that safeguard the somatic state, we performed two comprehensive RNA interference (RNAi) screens targeting chromatin factors during transcription-factor-mediated reprogramming of mouse fibroblasts to induced pluripotent stem cells (iPS cells). Subunits of the chromatin assembly factor-1 (CAF-1) complex, including Chaf1a and Chaf1b, emerged as the most prominent hits from both screens, followed by modulators of lysine sumoylation and heterochromatin maintenance. Optimal modulation of both CAF-1 and transcription factor levels increased reprogramming efficiency by several orders of magnitude and facilitated iPS cell formation in as little as 4 days. Mechanistically, CAF-1 suppression led to a more accessible chromatin structure at enhancer elements early during reprogramming. These changes were accompanied by a decrease in somatic heterochromatin domains, increased binding of Sox2 to pluripotency-specific targets and activation of associated genes. Notably, suppression of CAF-1 also enhanced the direct conversion of B cells into macrophages and fibroblasts into neurons. Together, our findings reveal the histone chaperone CAF-1 to be a novel regulator of somatic cell identity during transcription-factor-induced cell-fate transitions and provide a potential strategy to modulate cellular plasticity in a regenerative setting.

Mutations of R882 change flanking sequence preferences of the DNA methyltransferase DNMT3A and cellular methylation patterns.

Somatic DNMT3A mutations at R882 are frequently observed in AML patients including the very abundant R882H, but also R882C, R882P and R882S. Using deep enzymology, we show here that DNMT3A-R882H has more than 70-fold altered flanking sequence preferences when compared with wildtype DNMT3A. The R882H flanking sequence preferences mainly differ on the 3' side of the CpG site, where they resemble DNMT3B, while 5' flanking sequence preferences resemble wildtype DNMT3A, indicating that R882H behaves like a DNMT3A/DNMT3B chimera. Investigation of the activity and flanking sequence preferences of other mutations of R882 revealed that they cause similar effects. Bioinformatic analyses of genomic methylation patterns focusing on flanking sequence effects after expression of wildtype DNMT3A and R882H in human cells revealed that genomic methylation patterns reflect the details of the altered flanking sequence preferences of R882H. Concordantly, R882H specific hypermethylation in AML patients was strongly correlated with the R882H flanking sequence preferences. R882H specific DNA hypermethylation events in AML patients were accompanied by R882H specific mis-regulation of several genes with strong cancer connection, which are potential downstream targets of R882H. In conclusion, our data provide novel and detailed mechanistic understanding of the pathogenic mechanism of the DNMT3A R882H somatic cancer mutation.

The GEF-H1/PKD3 signaling pathway promotes the maintenance of triple-negative breast cancer stem cells.

Protein kinase D3 (PKD3) is upregulated in triple-negative breast cancer (TNBC) and associated with cell proliferation and metastasis development but its precise pro-oncogenic function is unknown. Here we show that PKD3 is required for the maintenance of the TNBC stem cell population. The depletion of PKD3 in MDA-MB-231 cells reduced the cancer stem cell frequency in vitro and tumor initiation potential in vivo. We further provide evidence that the RhoGEF GEF-H1 is upstream of PKD3 activation in TNBC stem cells. Most importantly, pharmacological PKD inhibition in combination with paclitaxel synergistically decreased oncosphere and colony formation efficiency in vitro and tumor recurrence in vivo. Based on our results we propose that targeting the GEF-H1/PKD3 signaling pathway in combination with chemotherapy might provide an effective therapeutic option for TNBC.

Chromatin-dependent allosteric regulation of DNMT3A activity by MeCP2.

Despite their central importance in mammalian development, the mechanisms that regulate the DNA methylation machinery and thereby the generation of genomic methylation patterns are still poorly understood. Here, we identify the 5mC-binding protein MeCP2 as a direct and strong interactor of DNA methyltransferase 3 (DNMT3) proteins. We mapped the interaction interface to the transcriptional repression domain of MeCP2 and the ADD domain of DNMT3A and find that binding of MeCP2 strongly inhibits the activity of DNMT3A in vitro. This effect was reinforced by cellular studies where a global reduction of DNA methylation levels was observed after overexpression of MeCP2 in human cells. By engineering conformationally locked DNMT3A variants as novel tools to study the allosteric regulation of this enzyme, we show that MeCP2 stabilizes the closed, autoinhibitory conformation of DNMT3A. Interestingly, the interaction with MeCP2 and its resulting inhibition were relieved by the binding of K4 unmodified histone H3 N-terminal tail to the DNMT3A-ADD domain. Taken together, our data indicate that the localization and activity of DNMT3A are under the combined control of MeCP2 and H3 tail modifications where, depending on the modification status of the H3 tail at the binding sites, MeCP2 can act as either a repressor or activator of DNA methylation.

Mapping the chemical chromatin reactivation landscape identifies BRD4-TAF1 cross-talk.

Bromodomain-containing proteins of the BET family recognize histone lysine acetylation and mediate transcriptional activation of target genes such as the MYC oncogene. Pharmacological inhibitors of BET domains promise therapeutic benefits in a variety of cancers. We performed a high-diversity chemical compound screen for agents capable of modulating BRD4-dependent heterochromatization of a generic reporter in human cells. In addition to known and new compounds targeting BRD4, we identified small molecules that mimic BRD4 inhibition without direct engagement. One such compound was a potent inhibitor of the second bromodomain of TAF1. Using this inhibitor, we discovered that TAF1 synergizes with BRD4 to control proliferation of cancer cells, making TAF1 an attractive epigenetic target in cancers driven by MYC.

Fluorescent Reporter Systems to Investigate Chromatin Effector Proteins in Living Cells.

Epigenetic research faces the challenge of the high complexity and tight regulation in chromatin modification networks. Although many isolated mechanisms of chromatin-mediated gene regulation have been described, solid approaches for the comprehensive analysis of specific processes as parts of the bigger epigenome network are missing. In order to expand the toolbox of methods by a system that will help to capture and describe the complexity of transcriptional regulation, we describe here a robust protocol for the generation of stable reporter systems for transcriptional activity and summarize their applications. The system allows for the induced recruitment of a chromatin regulator to a fluorescent reporter gene, followed by the detection of transcriptional changes using flow cytometry. The reporter gene is integrated into an endogenous chromatin environment, thus enabling the detection of regulatory dependencies of the investigated chromatin regulator on endogenous cofactors. The system allows for an easy and dynamic readout at the single-cell level and the ability to compensate for cell-to-cell variances of transcription. The modular design of the system enables the simple adjustment of the method for the investigation of different chromatin regulators in a broad panel of cell lines. We also summarize applications of this technology to characterize the silencing velocity of different chromatin effectors, removal of activating histone modifications, analysis of stability and reversibility of epigenome modifications, the investigation of the effects of small molecule on chromatin effectors and of functional effector-coregulator relationships. The presented method allows to investigate the complexity of transcriptional regulation by epigenetic effector proteins in living cells.

Selenium nanoparticles modulate histone methylation via lysine methyltransferase activity and S-adenosylhomocysteine depletion.

At physiological levels, the trace element selenium plays a key role in redox reactions through the incorporation of selenocysteine in antioxidant enzymes. Selenium has also been evaluated as a potential anti-cancer agent, where selenium nanoparticles have proven effective, and are well tolerated in vivo at doses that are toxic as soluble Se. The use of such nanoparticles, coated with either serum albumin or the naturally occurring alkaline polysaccharide chitosan, also serves to enhance biocompatibility and bioavailability. Here we demonstrate a novel role for selenium in regulating histone methylation in ovarian cancer cell models treated with inorganic selenium nanoparticles coated with serum albumin or chitosan. As well as inducing thioredoxin reductase expression, ROS activity and cancer cell cytotoxicity, coated nanoparticles caused significant increases in histone methylation. Specifically, selenium nanoparticles triggered an increase in the methylation of histone 3 at lysines K9 and K27, histone marks involved in both the activation and repression of gene expression, thus suggesting a fundamental role for selenium in these epigenetic processes. This direct function was confirmed using chemical inhibitors of the histone lysine methyltransferases EZH2 (H3K27) and G9a/EHMT2 (H3K9), both of which blocked the effect of selenium on histone methylation. This novel role for selenium supports a distinct function in histone methylation that occurs due to a decrease in S-adenosylhomocysteine, an endogenous inhibitor of lysine methyltransferases, the metabolic product of methyl-group transfer from S-adenosylmethionine in the one-carbon metabolism pathway. These observations provide important new insights into the action of selenium nanoparticles. It is now important to consider both the classic antioxidant and novel histone methylation effects of this key redox element in its development in cancer therapy and other applications.

Chromatin methylation activity of Dnmt3a and Dnmt3a/3L is guided by interaction of the ADD domain with the histone H3 tail.

Using peptide arrays and binding to native histone proteins, we show that the ADD domain of Dnmt3a specifically interacts with the H3 histone 1-19 tail. Binding is disrupted by di- and trimethylation of K4, phosphorylation of T3, S10 or T11 and acetylation of K4. We did not observe binding to the H4 1-19 tail. The ADD domain of Dnmt3b shows the same binding specificity, suggesting that the distinct biological functions of both enzymes are not related to their ADD domains. To establish a functional role of the ADD domain binding to unmodified H3 tails, we analyzed the DNA methylation of in vitro reconstituted chromatin with Dnmt3a2, the Dnmt3a2/Dnmt3L complex, and the catalytic domain of Dnmt3a. All Dnmt3a complexes preferentially methylated linker DNA regions. Chromatin substrates with unmodified H3 tail or with H3K9me3 modification were methylated more efficiently by full-length Dnmt3a and full-length Dnmt3a/3L complexes than chromatin trimethylated at H3K4. In contrast, the catalytic domain of Dnmt3a was not affected by the H3K4me3 modification. These results demonstrate that the binding of the ADD domain to H3 tails unmethylated at K4 leads to the preferential methylation of DNA bound to chromatin with this modification state. Our in vitro results recapitulate DNA methylation patterns observed in genome-wide DNA methylation studies.

The Dnmt3a PWWP domain reads histone 3 lysine 36 trimethylation and guides DNA methylation.

The Dnmt3a DNA methyltransferase contains in its N-terminal part a PWWP domain that is involved in chromatin targeting. Here, we have investigated the interaction of the PWWP domain with modified histone tails using peptide arrays and show that it specifically recognizes the histone 3 lysine 36 trimethylation mark. H3K36me3 is known to be a repressive modification correlated with DNA methylation in mammals and heterochromatin in Schizosaccharomyces pombe. These results were confirmed by equilibrium peptide binding studies and pulldown experiments with native histones and purified native nucleosomes. The PWWP-H3K36me3 interaction is important for the subnuclear localization of enhanced yellow fluorescent protein-fused Dnmt3a. Furthermore, the PWWP-H3K36me3 interaction increases the activity of Dnmt3a for methylation of nucleosomal DNA as observed using native nucleosomes isolated from human cells after demethylation of the DNA with 5-aza-2'-deoxycytidine as substrate for methylation with Dnmt3a. These data suggest that the interaction of the PWWP domain with H3K36me3 is involved in targeting of Dnmt3a to chromatin carrying that mark, a model that is in agreement with several studies on the genome-wide distribution of DNA methylation and H3K36me3.

Structure, Activity and Function of the NSD3 Protein Lysine Methyltransferase.

NSD3 is one of six H3K36-specific lysine methyltransferases in metazoans, and the methylation of H3K36 is associated with active transcription. NSD3 is a member of the nuclear receptor-binding SET domain (NSD) family of histone methyltransferases together with NSD1 and NSD2, which generate mono- and dimethylated lysine on histone H3. NSD3 is mutated and hyperactive in some human cancers, but the biochemical mechanisms underlying such dysregulation are barely understood. In this review, the current knowledge of NSD3 is systematically reviewed. Finally, the molecular and functional characteristics of NSD3 in different tumor types according to the current research are summarized.

G Protein-Coupled Estrogen Receptor Correlates With Dkk2 Expression and Has Prognostic Impact in Ovarian Cancer Patients.

Purpose: Wnt pathway modulator Dickkopf 2 (Dkk2) and signaling of the G protein-coupled estrogen receptor (GPER) seem to have essential functions in numerous cancer types. For epithelial ovarian cancer (EOC), it has not been proven if either Dkk2 or the GPER on its own have an independent impact on overall survival (OS). So far, the correlation of both factors and their clinical significance has not systematically been investigated before. Methods: Expression levels of Dkk2 were immunohistochemically analyzed in 156 patient samples from different histologic subtypes of EOC applying the immune-reactivity score (IRS). Expression analyses were correlated with clinical and pathological parameters to assess for prognostic relevance. Data analysis was performed using Spearman's correlations, Kruskal-Wallis-test and Kaplan-Meier estimates. Results: Highest Dkk2 expression of all subtypes was observed in clear cell carcinoma. In addition, Dkk2 expression differed significantly (p<0.001) between low and high grade serous ovarian cancer. A significant correlation of Dkk2 with the cytoplasmic GPER expression was noted (p=0.001) but not for the nuclear estrogen receptor alpha (ERα) or beta (ERß). Patients exhibiting both, high expression Dkk2 (IRS>4) and GPER (IRS>8), had a significantly better overall survival compared to patients with low expression (61 months vs. 33 months; p=0.024). Conclusion: Dkk2 and GPER expression correlates in EOC and combined expression of both is associated with improved OS. These findings underline the clinical significance of both pathways and indicate a possible prognostic impact as well as a potential for treatment strategies addressing interactions between estrogen and Wnt signaling in ovarian cancer.

Functional Analysis of Non-Genetic Resistance to Platinum in Epithelial Ovarian Cancer Reveals a Role for the MBD3-NuRD Complex in Resistance Development.

Epithelial ovarian cancer (EOC) is the most lethal disease of the female reproductive tract, and although most patients respond to the initial treatment with platinum (cPt)-based compounds, relapse is very common. We investigated the role of epigenetic changes in cPt-sensitive and -resistant EOC cell lines and found distinct differences in their enhancer landscape. Clinical data revealed that two genes (JAK1 and FGF10), which gained large enhancer clusters in resistant EOC cell lines, could provide novel biomarkers for early patient stratification with statistical independence for JAK1. To modulate the enhancer remodeling process and prevent the acquisition of cPt resistance in EOC cells, we performed a chromatin-focused RNAi screen in the presence of cPt. We identified subunits of the Nucleosome Remodeling and Deacetylase (NuRD) complex as critical factors sensitizing the EOC cell line A2780 to platinum treatment. Suppression of the Methyl-CpG Binding Domain Protein 3 (MBD3) sensitized cells and prevented the establishment of resistance under prolonged cPt exposure through alterations of H3K27ac at enhancer regions, which are differentially regulated in cPt-resistant cells, leading to a less aggressive phenotype. Our work establishes JAK1 as an independent prognostic marker and the NuRD complex as a potential target for combinational therapy.

Inhibition of CBP synergizes with the RNA-dependent mechanisms of Azacitidine by limiting protein synthesis.

The nucleotide analogue azacitidine (AZA) is currently the best treatment option for patients with high-risk myelodysplastic syndromes (MDS). However, only half of treated patients respond and of these almost all eventually relapse. New treatment options are urgently needed to improve the clinical management of these patients. Here, we perform a loss-of-function shRNA screen and identify the histone acetyl transferase and transcriptional co-activator, CREB binding protein (CBP), as a major regulator of AZA sensitivity. Compounds inhibiting the activity of CBP and the closely related p300 synergistically reduce viability of MDS-derived AML cell lines when combined with AZA. Importantly, this effect is specific for the RNA-dependent functions of AZA and not observed with the related compound decitabine that is only incorporated into DNA. The identification of immediate target genes leads us to the unexpected finding that the effect of CBP/p300 inhibition is mediated by globally down regulating protein synthesis.

Protein lysine methyltransferase G9a acts on non-histone targets.

By methylation of peptide arrays, we determined the specificity profile of the protein methyltransferase G9a. We show that it mostly recognizes an Arg-Lys sequence and that its activity is inhibited by methylation of the arginine residue. Using the specificity profile, we identified new non-histone protein targets of G9a, including CDYL1, WIZ, ACINUS and G9a (automethylation), as well as peptides derived from CSB. We demonstrate potential downstream signaling pathways for methylation of non-histone proteins.

Analysis of the substrate specificity of the Dim-5 histone lysine methyltransferase using peptide arrays.

Histone methylation is an epigenetic mark essential for gene regulation and development. We introduce peptide SPOT synthesis to study sequence specificity of the Dim-5 histone-3 lysine-9 methyltransferase. Dim-5 recognizes R8-G12 of the H3 tail with T11 and G12 being the most important specificity determinants. Exchange of H3 tail residue S10 and T11 by E strongly reduced methylation by Dim-5, suggesting that phosphorylation of S10 or T11 may regulate the activity of Dim-5. In the Dim-5/peptide structure, E227 interacts with H3R8 and D209 with H3-S10. Mutations of E227 or D209 caused predictable changes in the substrate preference, illustrating that peptide recognition of histone methyltransferases can be altered by protein design. Comparative analyses of peptide arrays with wild-type and mutant enzymes, therefore, are well suited to investigate the target specificity of protein methyltransferases and study epigenetic crosstalk.

Somatic Cancer Mutations in the SUV420H1 Protein Lysine Methyltransferase Modulate Its Catalytic Activity.

SUV420H1 is a protein lysine methyltransferase that introduces di- and trimethylation of H4K20 and is frequently mutated in human cancers. We investigated the functional effects of eight somatic cancer mutations on SUV420H1 activity in vitro and in cells. One group of mutations (S255F, K258E, A269V) caused a reduction of the catalytic activity on peptide and nucleosome substrates. The mutated amino acids have putative roles in AdoMet binding and recognition of H4 residue D24. Group 2 mutations (E238V, D249N, E320K) caused a reduction of activity on peptide substrates, which was partially recovered when using nucleosomal substrates. The corresponding residues could have direct or indirect roles in peptide and AdoMet binding, but the effects of the mutations can be overcome by additional interactions between SUV420H1 and the nucleosome substrate. The third group of mutations (S283L, S304Y) showed enhanced activity with peptide substrates when compared with nucleosomal substrates, suggesting that these residues are involved in nucleosomal interaction or allosteric activation of SUV420H1 after nucleosome binding. Group 2 and 3 mutants highlight the role of nucleosomal contacts for SUV420H1 regulation in agreement with the high activity of this enzyme on nucleosomal substrates. Strikingly, seven of the somatic cancer mutations studied here led to a reduction of the catalytic activity of SUV420H1 in cells, suggesting that SUV420H1 activity might have a tumor suppressive function. This could be explained by the role of H4K20me2/3 in DNA repair, suggesting that loss or reduction of SUV420H1 activity could contribute to a mutator phenotype in cancer cells.

MTHFD1 interaction with BRD4 links folate metabolism to transcriptional regulation.

The histone acetyl reader bromodomain-containing protein 4 (BRD4) is an important regulator of chromatin structure and transcription, yet factors modulating its activity have remained elusive. Here we describe two complementary screens for genetic and physical interactors of BRD4, which converge on the folate pathway enzyme MTHFD1 (methylenetetrahydrofolate dehydrogenase, cyclohydrolase and formyltetrahydrofolate synthetase 1). We show that a fraction of MTHFD1 resides in the nucleus, where it is recruited to distinct genomic loci by direct interaction with BRD4. Inhibition of either BRD4 or MTHFD1 results in similar changes in nuclear metabolite composition and gene expression; pharmacological inhibitors of the two pathways synergize to impair cancer cell viability in vitro and in vivo. Our finding that MTHFD1 and other metabolic enzymes are chromatin associated suggests a direct role for nuclear metabolism in the control of gene expression.