Development of a sensitive microplate assay for characterizing RNA methyltransferase activity: Implications for epitranscriptomics and drug development.

RNA methylation is a ubiquitous post-transcriptional modification found in diverse RNA classes and is a critical regulator of gene expression. In this study, we used Zika virus RNA methyltransferase (MTase) to develop a highly sensitive microplate assay that uses a biotinylated RNA substrate and radiolabeled AdoMet coenzyme. The assay is fast, highly reproducible, exhibits linear progress-curve kinetics under multiple turnover conditions, has high sensitivity in competitive inhibition assays, and significantly lower background levels compared with the currently used method. Using our newly developed microplate assay, we observed no significant difference in the catalytic constants of the full-length nonstructural protein 5 enzyme and the truncated MTase domain. These data suggest that, unlike the Zika virus RNA-dependent RNA polymerase activity, the MTase activity is unaffected by RNA-dependent RNA polymerase-MTase interdomain interaction. Given its quantitative nature and accuracy, this method can be used to characterize various RNA MTases, and, therefore, significantly contribute to the field of epitranscriptomics and drug development against infectious diseases.

Integrative genomic analysis of pediatric T-cell lymphoblastic lymphoma reveals candidates of clinical significance.

T-cell lymphoblastic lymphoma (T-LBL) is a heterogeneous malignancy of lymphoblasts committed to T-cell lineage. The dismal outcomes (15%-30%) after T-LBL relapse warrant establishing risk-based treatment. To our knowledge, this study presents the first comprehensive, systematic, integrated, genome-wide analysis including relapsed cases that identifies molecular markers of prognostic relevance for T-LBL. NOTCH1 was identified as the putative driver for T-LBL. An activated NOTCH/PI3K-AKT signaling axis and alterations in cell cycle regulators constitute the core oncogenic program for T-LBL. Mutated KMT2D was identified as a prognostic marker. The cumulative incidence of relapse was 47% ± 17% in patients with KMT2D mutations, compared with 14% ± 3% in wild-type KMT2D. Structural analysis of the mutated domains of KMT2D revealed a plausible impact on structure and functional consequences. These findings provide new insights into the pathogenesis of T-LBL, including high translational potential. The ongoing LBL 2018 trial (www.clinicaltrials.gov #NCT04043494) allows for prospective validation and subsequent fine tuning of the stratification criteria for T-LBL risk groups to improve survival of pediatric patients.

The acute myeloid leukemia variant DNMT3A Arg882His is a DNMT3B-like enzyme.

We have previously shown that the highly prevalent acute myeloid leukemia (AML) mutation, Arg882His, in DNMT3A disrupts its cooperative mechanism and leads to reduced enzymatic activity, thus explaining the genomic hypomethylation in AML cells. However, the underlying cause of the oncogenic effect of Arg882His in DNMT3A is not fully understood. Here, we discovered that DNMT3A WT enzyme under conditions that favor non-cooperative kinetic mechanism as well as DNMT3A Arg882His variant acquire CpG flanking sequence preference akin to that of DNMT3B, which is non-cooperative. We tested if DNMT3A Arg882His could preferably methylate DNMT3B-specific target sites in vivo. Rescue experiments in Dnmt3a/3b double knockout mouse embryonic stem cells show that the corresponding Arg878His mutation in mouse DNMT3A severely impairs its ability to methylate major satellite DNA, a DNMT3A-preferred target, but has no overt effect on the ability to methylate minor satellite DNA, a DNMT3B-preferred target. We also observed a previously unappreciated CpG flanking sequence bias in major and minor satellite repeats that is consistent with DNMT3A and DNMT3B specificity suggesting that DNA methylation patterns are guided by the sequence preference of these enzymes. We speculate that aberrant methylation of DNMT3B target sites could contribute to the oncogenic potential of DNMT3A AML variant.

DNMT3L facilitates DNA methylation partly by maintaining DNMT3A stability in mouse embryonic stem cells.

DNMT3L (DNMT3-like), a member of the DNMT3 family, has no DNA methyltransferase activity but regulates de novo DNA methylation. While biochemical studies show that DNMT3L is capable of interacting with both DNMT3A and DNMT3B and stimulating their enzymatic activities, genetic evidence suggests that DNMT3L is essential for DNMT3A-mediated de novo methylation in germ cells but is dispensable for de novo methylation during embryogenesis, which is mainly mediated by DNMT3B. How DNMT3L regulates DNA methylation and what determines its functional specificity are not well understood. Here we show that DNMT3L-deficient mouse embryonic stem cells (mESCs) exhibit downregulation of DNMT3A, especially DNMT3A2, the predominant DNMT3A isoform in mESCs. DNA methylation analysis of DNMT3L-deficient mESCs reveals hypomethylation at many DNMT3A target regions. These results confirm that DNMT3L is a positive regulator of DNA methylation, contrary to a previous report that, in mESCs, DNMT3L regulates DNA methylation positively or negatively, depending on genomic regions. Mechanistically, DNMT3L forms a complex with DNMT3A2 and prevents DNMT3A2 from being degraded. Restoring the DNMT3A protein level in DNMT3L-deficient mESCs partially recovers DNA methylation. Thus, our work uncovers a role for DNMT3L in maintaining DNMT3A stability, which contributes to the effect of DNMT3L on DNMT3A-dependent DNA methylation.

The transcription factor Vezf1 represses the expression of the antiangiogenic factor Cited2 in endothelial cells.

Formation of the vasculature by angiogenesis is critical for proper development, but angiogenesis also contributes to the pathogenesis of various disorders, including cancer and cardiovascular diseases. Vascular endothelial zinc finger 1 (Vezf1), is a Krüppel-like zinc finger protein that plays a vital role in vascular development. However, the mechanism by which Vezf1 regulates this process is not fully understood. Here, we show that Vezf1-/- mouse embryonic stem cells (ESC) have significantly increased expression of a stem cell factor, Cbp/p300-interacting transactivator 2 (Cited2). Compared with WT ESCs, Vezf1-/- ESCs inefficiently differentiated into endothelial cells (ECs), which exhibited defects in the tube-formation assay. These defects were due to reduced activation of EC-specific genes concomitant with lower enrichment of histone 3 acetylation at Lys27 (H3K27) at their promoters. We hypothesized that overexpression of Cited2 in Vezf1-/- cells sequesters P300/CBP away from the promoters of proangiogenic genes and thereby contributes to defective angiogenesis in these cells. This idea was supported by the observation that shRNA-mediated depletion of Cited2 significantly reduces the angiogenic defects in the Vezf1-/- ECs. In contrast to previous studies that have focused on the role of Vezf1 as a transcriptional activator of proangiogenic genes, our findings have revealed a role for Vezf1 in modulating the expression of the antiangiogenic factor Cited2. Vezf1 previously has been characterized as an insulator protein, and our results now provide insights into the mechanism, indicating that Vezf1 can block inappropriate, nonspecific interactions of promoters with cis-located enhancers, preventing aberrant promoter activation.

Dnmt3b Methylates DNA by a Noncooperative Mechanism, and Its Activity Is Unaffected by Manipulations at the Predicted Dimer Interface.

The catalytic domains of the de novo DNA methyltransferases Dnmt3a-C and Dnmt3b-C are highly homologous. However, their unique biochemical properties could potentially contribute to differences in the substrate preferences or biological functions of these enzymes. Dnmt3a-C forms tetramers through interactions at the dimer interface, which also promote multimerization on DNA and cooperativity. Similar to the case for processive enzymes, cooperativity allows Dnmt3a-C to methylate multiple sites on the same DNA molecule; however, it is unclear whether Dnmt3b-C methylates DNA by a cooperative or processive mechanism. The importance of the tetramer structure and cooperative mechanism is emphasized by the observation that the R882H mutation in the dimer interface of DNMT3A is highly prevalent in acute myeloid leukemia and leads to a substantial loss of its activity. Under conditions that distinguish between cooperativity and processivity, we show that in contrast to that of Dnmt3a-C, the activity of Dnmt3b-C is not cooperative and confirm the processivity of Dnmt3b-C and the full length Dnmt3b enzyme. Whereas the R878H mutation (mouse homologue of R882H) led to the loss of cooperativity of Dnmt3a-C, the activity and processivity of the analogous Dnmt3b-C R829H variant were comparable to those of the wild-type enzyme. Additionally, buffer acidification that attenuates the dimer interface interactions of Dnmt3a-C had no effect on Dnmt3b-C activity. Taken together, these results demonstrate an important mechanistic difference between Dnmt3b and Dnmt3a and suggest that interactions at the dimer interface may play a limited role in regulating Dnmt3b-C activity. These new insights have potential implications for the distinct biological roles of Dnmt3a and Dnmt3b.

Mammalian DNA methyltransferases: new discoveries and open questions.

As part of the epigenetic network, DNA methylation is a major regulator of chromatin structure and function. In mammals, it mainly occurs at palindromic CpG sites, but asymmetric methylation at non-CpG sites is also observed. Three enzymes are involved in the generation and maintenance of DNA methylation patterns. DNMT1 has high preference for hemimethylated CpG sites, and DNMT3A and DNMT3B equally methylate unmethylated and hemimethylated DNA, and also introduce non-CpG methylation. Here, we review recent observations and novel insights into the structure and function of mammalian DNMTs (DNA methyltransferases), including new structures of DNMT1 and DNMT3A, data on their mechanism, regulation by post-translational modifications and on the function of DNMTs in cells. In addition, we present news findings regarding the allosteric regulation and targeting of DNMTs by chromatin modifications and chromatin proteins. In combination, the recent publications summarized here impressively illustrate the intensity of ongoing research in this field. They provide a deeper understanding of key mechanistic properties of DNMTs, but they also document still unsolved issues, which need to be addressed in future research.

An epigenetic switch regulates de novo DNA methylation at a subset of pluripotency gene enhancers during embryonic stem cell differentiation.

Coordinated regulation of gene expression that involves activation of lineage specific genes and repression of pluripotency genes drives differentiation of embryonic stem cells (ESC). For complete repression of pluripotency genes during ESC differentiation, chromatin at their enhancers is silenced by the activity of the Lsd1-Mi2/NuRD complex. The mechanism/s that regulate DNA methylation at these enhancers are largely unknown. Here, we investigated the affect of the Lsd1-Mi2/NuRD complex on the dynamic regulatory switch that induces the local interaction of histone tails with the Dnmt3 ATRX-DNMT3-DNMT3L (ADD) domain, thus promoting DNA methylation at the enhancers of a subset of pluripotency genes. This is supported by previous structural studies showing a specific interaction between Dnmt3-ADD domain with H3K4 unmethylated histone tails that is disrupted by histone H3K4 methylation and histone acetylation. Our data suggest that Dnmt3a activity is triggered by Lsd1-Mi2/NuRD-mediated histone deacetylation and demethylation at these pluripotency gene enhancers when they are inactivated during mouse ESC differentiation. Using Dnmt3 knockout ESCs and the inhibitors of Lsd1 and p300 histone modifying enzymes during differentiation of E14Tg2A and ZHBTc4 ESCs, our study systematically reveals this mechanism and establishes that Dnmt3a is both reader and effector of the epigenetic state at these target sites.

Characterization of Small Molecules Inhibiting the Pro-Angiogenic Activity of the Zinc Finger Transcription Factor Vezf1.

Discovery of inhibitors for endothelial-related transcription factors can contribute to the development of anti-angiogenic therapies that treat various diseases, including cancer. The role of transcription factor Vezf1 in vascular development and regulation of angiogenesis has been defined by several earlier studies. Through construction of a computational model for Vezf1, work here has identified a novel small molecule drug capable of inhibiting Vezf1 from binding to its cognate DNA binding site. Using structure-based design and virtual screening of the NCI Diversity Compound Library, 12 shortlisted compounds were tested for their ability to interfere with the binding of Vezf1 to DNA using electrophoretic gel mobility shift assays. We identified one compound, T4, which has an IC50 of 20 µM. Using murine endothelial cells, MSS31, we tested the effect of T4 on endothelial cell viability and angiogenesis by using tube formation assay. Our data show that addition of T4 in cell culture medium does not affect cell viability at concentrations lower or equal to its IC 50 but strongly inhibits the network formation by MSS31 in the tube formation assays. Given its potential efficacy, this inhibitor has significant therapeutic potential in several human diseases.

A refined DNA methylation detection method using MspJI coupled quantitative PCR.

DNA methylation is a highly conserved epigenetic modification with critical roles ranging from protection against phage infection in bacteria to the regulation of gene expression in mammals. DNA methylation at specific sequences can be measured by using methylation dependent or sensitive restriction enzymes coupled to semi- or quantitative PCR (MD-qPCR). This study reports a refined MD-qPCR method for detecting gain or loss of DNA methylation at specific sites through the specific use of MspJI or HpaII, respectively. By employing varying concentrations of DNA with methylation ranging from 0 to 100%, our data provide evidence that compared to HpaII, MspJI increases the sensitivity and accuracy of detecting relative DNA methylation gains by MD-qPCR. We also show that the MspJI-coupled MD-qPCR can accurately determine the percent gain in DNA methylation at the Sall4 enhancer and is more sensitive than HpaII in detecting relative gains in DNA methylation at the Oct4 proximal enhancer during embryonic stem cell (ESC) differentiation. The high specificity and sensitivity of this targeted approach increases its potential as a diagnostic tool to detect relatively smaller gains in DNA methylation at specific sites from limited amounts of sample.

Vezf1 protein binding sites genome-wide are associated with pausing of elongating RNA polymerase II.

The protein Vezf1 plays multiple roles important for embryonic development. In Vezf1(-/-) mouse embryonic stem (mES) cells, our earlier data showed widespread changes in gene-expression profiles, including decreased expression of the full-length active isoform of Dnmt3b methyltransferase and concomitant genome-wide reduction in DNA methylation. Here we show that in HeLaS3 cells there is a strong genome-wide correlation between Vezf1 binding and peaks of elongating Ser2-P RNA polymerase (Pol) ll, reflecting Vezf1-dependent slowing of elongation. In WT mES cells, the elongating form of RNA pol II accumulates near Vezf1 binding sites within the dnmt3b gene and at several other Vezf1 sites, and this accumulation is significantly reduced at these sites in Vezf1(-/-) mES cells. Depending upon genomic location, Vezf1-mediated Pol II pausing can have different regulatory roles in transcription and splicing. We find examples of genes in which Vezf1 binding sites are located near cassette exons, and in which loss of Vezf1 leads to a change in the relative abundance of alternatively spliced messages. We further show that Vezf1 interacts with Mrg15/Mrgbp, a protein that recognizes H3K36 trimethylation, consistent with the role of histone modifications at alternatively spliced sites.

Signaling Pathways Governing Cardiomyocyte Differentiation.

Cardiomyocytes are the largest cell type that make up the heart and confer beating activity to the heart. The proper differentiation of cardiomyocytes relies on the efficient transmission and perception of differentiation cues from several signaling pathways that influence cardiomyocyte-specific gene expression programs. Signaling pathways also mediate intercellular communications to promote proper cardiomyocyte differentiation. We have reviewed the major signaling pathways involved in cardiomyocyte differentiation, including the BMP, Notch, sonic hedgehog, Hippo, and Wnt signaling pathways. Additionally, we highlight the differences between different cardiomyocyte cell lines and the use of these signaling pathways in the differentiation of cardiomyocytes from stem cells. Finally, we conclude by discussing open questions and current gaps in knowledge about the in vitro differentiation of cardiomyocytes and propose new avenues of research to fill those gaps.

Cardiomyocyte Differentiation from Mouse Embryonic Stem Cells by WNT Switch Method.

The differentiation of ESCs into cardiomyocytes in vitro is an excellent and reliable model system for studying normal cardiomyocyte development in mammals, modeling cardiac diseases, and for use in drug screening. Mouse ESC differentiation still provides relevant biological information about cardiac development. However, the current methods for efficiently differentiating ESCs into cardiomyocytes are limiting. Here, we describe the "WNT Switch" method to efficiently commit mouse ESCs into cardiomyocytes using the small molecule WNT signaling modulators CHIR99021 and XAV939 in vitro. This method significantly improves the yield of beating cardiomyocytes, reduces number of treatments, and is less laborious.

Chromatin domains, insulators, and the regulation of gene expression.

The DNA sequence elements called insulators have two basic kinds of properties. Barrier elements block the propagation of heterochromatic structures into adjacent euchromatin. Enhancer blocking elements interfere with interaction between an enhancer and promoter when placed between them. We have dissected a compound insulator element found at the 5' end of the chicken ß-globin locus, which possesses both activities. Barrier insulation is mediated by two kinds of DNA binding proteins: USF1/USF2, a heterodimer which recruits multiple enzyme complexes capable of marking histone on adjacent nucleosomes with 'activating' marks, and Vezf1, which protects against DNA methylation. We have found that the heterochromatic region upstream of the insulator element is maintained in its silent state by a dicer-dependent mechanism, suggesting a mechanism for Vezf1 function in the insulator. Enhancer blocking function in the ß-globin insulator element is conferred by a binding site for CTCF. Consistent with this property, CTCF binding was found some years ago to be essential for imprinted expression at the Igf2/H19 locus. Work in many laboratories has since demonstrated that CTCF helps stabilize long-range interactions in the nucleus. We have recently shown that in the case of the human insulin locus such an interaction, over a distance of ~300kb, can result in stimulation of a target gene which itself is important for insulin secretion. This article is part of a Special Issue entitled: Chromatin in time and space.

VEZF1 elements mediate protection from DNA methylation.

There is growing consensus that genome organization and long-range gene regulation involves partitioning of the genome into domains of distinct epigenetic chromatin states. Chromatin insulator or barrier elements are key components of these processes as they can establish boundaries between chromatin states. The ability of elements such as the paradigm beta-globin HS4 insulator to block the range of enhancers or the spread of repressive histone modifications is well established. Here we have addressed the hypothesis that a barrier element in vertebrates should be capable of defending a gene from silencing by DNA methylation. Using an established stable reporter gene system, we find that HS4 acts specifically to protect a gene promoter from de novo DNA methylation. Notably, protection from methylation can occur in the absence of histone acetylation or transcription. There is a division of labor at HS4; the sequences that mediate protection from methylation are separable from those that mediate CTCF-dependent enhancer blocking and USF-dependent histone modification recruitment. The zinc finger protein VEZF1 was purified as the factor that specifically interacts with the methylation protection elements. VEZF1 is a candidate CpG island protection factor as the G-rich sequences bound by VEZF1 are frequently found at CpG island promoters. Indeed, we show that VEZF1 elements are sufficient to mediate demethylation and protection of the APRT CpG island promoter from DNA methylation. We propose that many barrier elements in vertebrates will prevent DNA methylation in addition to blocking the propagation of repressive histone modifications, as either process is sufficient to direct the establishment of an epigenetically stable silent chromatin state.

Dnmt3bas coordinates transcriptional induction and alternative exon inclusion to promote catalytically active Dnmt3b expression.

Embryonic expression of DNMT3B is critical for establishing de novo DNA methylation. This study uncovers the mechanism through which the promoter-associated long non-coding RNA (lncRNA) Dnmt3bas controls the induction and alternative splicing of Dnmt3b during embryonic stem cell (ESC) differentiation. Dnmt3bas recruits the PRC2 (polycomb repressive complex 2) at cis-regulatory elements of the Dnmt3b gene expressed at a basal level. Correspondingly, Dnmt3bas knockdown enhances Dnmt3b transcriptional induction, whereas overexpression of Dnmt3bas dampens it. Dnmt3b induction coincides with exon inclusion, switching the predominant isoform from the inactive Dnmt3b6 to the active Dnmt3b1. Intriguingly, overexpressing Dnmt3bas further enhances the Dnmt3b1:Dnmt3b6 ratio, attributed to its interaction with hnRNPL (heterogeneous nuclear ribonucleoprotein L), a splicing factor that promotes exon inclusion. Our data suggest that Dnmt3bas coordinates alternative splicing and transcriptional induction of Dnmt3b by facilitating the hnRNPL and RNA polymerase II (RNA Pol II) interaction at the Dnmt3b promoter. This dual mechanism precisely regulates the expression of catalytically active DNMT3B, ensuring fidelity and specificity of de novo DNA methylation.

RyR2/IRBIT regulates insulin gene transcript, insulin content, and secretion in the insulinoma cell line INS-1.

The role of ER Ca2+ release via ryanodine receptors (RyR) in pancreatic ß-cell function is not well defined. Deletion of RyR2 from the rat insulinoma INS-1 (RyR2KO) enhanced IP3 receptor activity stimulated by 7.5 mM glucose, coincident with reduced levels of the protein IP3 Receptor Binding protein released with Inositol 1,4,5 Trisphosphate (IRBIT). Insulin content, basal (2.5 mM glucose) and 7.5 mM glucose-stimulated insulin secretion were reduced in RyR2KO and IRBITKO cells compared to controls. INS2 mRNA levels were reduced in both RyR2KO and IRBITKO cells, but INS1 mRNA levels were specifically decreased in RyR2KO cells. Nuclear localization of S-adenosylhomocysteinase (AHCY) was increased in RyR2KO and IRBITKO cells. DNA methylation of the INS1 and INS2 gene promotor regions was very low, and not different among RyR2KO, IRBITKO, and controls, but exon 2 of the INS1 and INS2 genes was more extensively methylated in RyR2KO and IRBITKO cells. Exploratory proteomic analysis revealed that deletion of RyR2 or IRBIT resulted in differential regulation of 314 and 137 proteins, respectively, with 41 in common. These results suggest that RyR2 regulates IRBIT levels and activity in INS-1 cells, and together maintain insulin content and secretion, and regulate the proteome, perhaps via DNA methylation.

Misregulation of the expression and activity of DNA methyltransferases in cancer.

In mammals, DNA methyltransferases DNMT1 and DNMT3's (A, B and L) deposit and maintain DNA methylation in dividing and nondividing cells. Although these enzymes have an unremarkable DNA sequence specificity (CpG), their regional specificity is regulated by interactions with various protein factors, chromatin modifiers, and post-translational modifications of histones. Changes in the DNMT expression or interacting partners affect DNA methylation patterns. Consequently, the acquired gene expression may increase the proliferative potential of cells, often concomitant with loss of cell identity as found in cancer. Aberrant DNA methylation, including hypermethylation and hypomethylation at various genomic regions, therefore, is a hallmark of most cancers. Additionally, somatic mutations in DNMTs that affect catalytic activity were mapped in Acute Myeloid Leukemia cancer cells. Despite being very effective in some cancers, the clinically approved DNMT inhibitors lack specificity, which could result in a wide range of deleterious effects. Elucidating distinct molecular mechanisms of DNMTs will facilitate the discovery of alternative cancer therapeutic targets. This review is focused on: (i) the structure and characteristics of DNMTs, (ii) the prevalence of mutations and abnormal expression of DNMTs in cancer, (iii) factors that mediate their abnormal expression and (iv) the effect of anomalous DNMT-complexes in cancer.

Simplified MethylRAD Sequencing to Detect Changes in DNA Methylation at Enhancer Elements in Differentiating Embryonic Stem Cells.

Differential DNA methylation is characteristic of gene regulatory regions, such as enhancers, which mostly constitute low or intermediate CpG content in their DNA sequence. Consequently, quantification of changes in DNA methylation at these sites is challenging. Given that DNA methylation across most of the mammalian genome is maintained, the use of genome-wide bisulfite sequencing to measure fractional changes in DNA methylation at specific sites is an overexertion which is both expensive and cumbersome. Here, we developed a MethylRAD technique with an improved experimental plan and bioinformatic analysis tool to examine regional DNA methylation changes in embryonic stem cells (ESCs) during differentiation. The transcriptional silencing of pluripotency genes (PpGs) during ESC differentiation is accompanied by PpG enhancer (PpGe) silencing mediated by the demethylation of H3K4me1 by LSD1. Our MethylRAD data show that in the presence of LSD1 inhibitor, a significant fraction of LSD1-bound PpGe fails to gain DNA methylation. We further show that this effect is mostly observed in PpGes with low/intermediate CpG content. Underscoring the sensitivity and accuracy of MethylRAD sequencing, our study demonstrates that this method can detect small changes in DNA methylation in regulatory regions, including those with low/intermediate CpG content, thus asserting its use as a method of choice for diagnostic purposes.