Methylscaper: an R/Shiny app for joint visualization of DNA methylation and nucleosome occupancy in single-molecule and single-cell data.

SUMMARY: Differential DNA methylation and chromatin accessibility are associated with disease development, particularly cancer. Methods that allow profiling of these epigenetic mechanisms in the same reaction and at the single-molecule or single-cell level continue to emerge. However, a challenge lies in jointly visualizing and analyzing the heterogeneous nature of the data and extracting regulatory insight. Here, we present methylscaper, a visualization framework for simultaneous analysis of DNA methylation and chromatin accessibility landscapes. Methylscaper implements a weighted principal component analysis that orders DNA molecules, each providing a record of the chromatin state of one epiallele, and reveals patterns of nucleosome positioning, transcription factor occupancy, and DNA methylation. We demonstrate methylscaper's utility on a long-read, single-molecule methyltransferase accessibility protocol for individual templates (MAPit-BGS) dataset and a single-cell nucleosome, methylation, and transcription sequencing (scNMT-seq) dataset. In comparison to other procedures, methylscaper is able to readily identify chromatin features that are biologically relevant to transcriptional status while scaling to larger datasets. AVAILABILITY AND IMPLEMENTATION: Methylscaper, is implemented in R (version > 4.1) and available on Bioconductor: https://bioconductor.org/packages/methylscaper/, GitHub: https://github.com/rhondabacher/methylscaper/, and Web: https://methylscaper.com. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Multiplex mapping of chromatin accessibility and DNA methylation within targeted single molecules identifies epigenetic heterogeneity in neural stem cells and glioblastoma.

Human tumors are comprised of heterogeneous cell populations that display diverse molecular and phenotypic features. To examine the extent to which epigenetic differences contribute to intratumoral cellular heterogeneity, we have developed a high-throughput method, termed MAPit-patch. The method uses multiplexed amplification of targeted sequences from submicrogram quantities of genomic DNA followed by next generation bisulfite sequencing. This provides highly scalable and simultaneous mapping of chromatin accessibility and DNA methylation on single molecules at high resolution. Long sequencing reads from targeted regions maintain the structural integrity of epigenetic information and provide substantial depth of coverage, detecting for the first time minority subpopulations of epigenetic configurations formerly obscured by existing genome-wide and population-ensemble methodologies. Analyzing a cohort of 71 promoters of genes with exons commonly mutated in cancer, MAPit-patch uncovered several differentially accessible and methylated promoters that are associated with altered gene expression between neural stem cell (NSC) and glioblastoma (GBM) cell populations. In addition, considering each promoter individually, substantial epigenetic heterogeneity was observed across the sequenced molecules, indicating the presence of epigenetically distinct cellular subpopulations. At the divergent MLH1/EPM2AIP1 promoter, a locus with three well-defined, nucleosome-depleted regions (NDRs), a fraction of promoter copies with inaccessible chromatin was detected and enriched upon selection of temozolomide-tolerant GBM cells. These results illustrate the biological relevance of epigenetically distinct subpopulations that in part underlie the phenotypic heterogeneity of tumor cell populations. Furthermore, these findings show that alterations in chromatin accessibility without accompanying changes in DNA methylation may constitute a novel class of epigenetic biomarker.

Epigenetic diversity of Kaposi's sarcoma-associated herpesvirus.

Spontaneous lytic reactivation of Kaposi's sarcoma-associated herpesvirus (KSHV) occurs at a low rate in latently infected cells in disease and culture. This suggests imperfect epigenetic maintenance of viral transcription programs, perhaps due to variability in chromatin structure at specific loci across the population of KSHV episomal genomes. To characterize this locus-specific chromatin structural diversity, we used MAPit single-molecule footprinting, which simultaneously maps endogenous CG methylation and accessibility to M.CviPI at GC sites. Diverse chromatin structures were detected at the LANA, RTA and vIL6 promoters. At each locus, chromatin ranged from fully closed to fully open across the population. This diversity has not previously been reported in a virus. Phorbol ester and RTA transgene induction were used to identify chromatin conformations associated with reactivation of lytic transcription, which only a fraction of episomes had. Moreover, certain chromatin conformations correlated with CG methylation patterns at the RTA and vIL6 promoters. This indicated that some of the diverse chromatin conformations at these loci were epigenetically distinct. Finally, by comparing chromatin structures from a cell line infected with constitutively latent virus, we identified products of lytic replication. Our findings show that epigenetic drift can restrict viral propagation by chromatin compaction at latent and lytic promoters.

MethylViewer: computational analysis and editing for bisulfite sequencing and methyltransferase accessibility protocol for individual templates (MAPit) projects.

Bisulfite sequencing is a widely-used technique for examining cytosine DNA methylation at nucleotide resolution along single DNA strands. Probing with cytosine DNA methyltransferases followed by bisulfite sequencing (MAPit) is an effective technique for mapping protein-DNA interactions. Here, MAPit methylation footprinting with M.CviPI, a GC methyltransferase we previously cloned and characterized, was used to probe hMLH1 chromatin in HCT116 and RKO colorectal cancer cells. Because M.CviPI-probed samples contain both CG and GC methylation, we developed a versatile, visually-intuitive program, called MethylViewer, for evaluating the bisulfite sequencing results. Uniquely, MethylViewer can simultaneously query cytosine methylation status in bisulfite-converted sequences at as many as four different user-defined motifs, e.g. CG, GC, etc., including motifs with degenerate bases. Data can also be exported for statistical analysis and as publication-quality images. Analysis of hMLH1 MAPit data with MethylViewer showed that endogenous CG methylation and accessible GC sites were both mapped on single molecules at high resolution. Disruption of positioned nucleosomes on single molecules of the PHO5 promoter was detected in budding yeast using M.CviPII, increasing the number of enzymes available for probing protein-DNA interactions. MethylViewer provides an integrated solution for primer design and rapid, accurate and detailed analysis of bisulfite sequencing or MAPit datasets from virtually any biological or biochemical system.

WIF1 is a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.

Aberrant activation of the Wnt/ß-catenin signaling axis is a prominent oncogenic mechanism in numerous cancers including cervical cancer. Wnt inhibitory factor-1 (WIF1) is a secreted protein that binds Wnt and antagonizes Wnt activity. While the WIF1 gene is characterized as a target for epigenetic silencing in some tumor types, WIF1 expression has not been examined in human cervical tissue and cervical cancer. Here, we show that WIF1 is unmethylated and its gene product is expressed in normal cervical epithelium and some cultured cervical tumor lines. In contrast, several cervical cancer lines contained dense CpG methylation within the WIF1 gene, and expression of both WIF1 transcript and protein was restored by culturing cells in the presence of the global DNA demethylating agent 5-aza-2'-deoxycytidine. Using single-molecule MAPit methylation footprinting, we observed differences in chromatin structure within the WIF1 promoter region between cell lines that express and those that do not express WIF1, consistent with transcriptional activity and repression, respectively. The WIF1 promoter was aberrantly methylated in ∼60% (10 of 17) high-grade highly undifferentiated squamous cell cervical tumors examined, whereas paired normal tissue showed significantly lower levels of CpG methylation. WIF1 protein was not detectable by immunohistochemistry in tumors with quantitatively high levels of WIF1 methylation. Of note, WIF1 protein was not detectable in two of the seven unmethylated cervical tumors examined, suggesting other mechanisms may contribute WIF1 repression. Our findings establish the WIF1 gene as a frequent target for epigenetic silencing in squamous cell carcinoma of the cervix.

High Fractional Occupancy of a Tandem Maf Recognition Element and Its Role in Long-Range ß-Globin Gene Regulation.

Enhancers and promoters assemble protein complexes that ultimately regulate the recruitment and activity of RNA polymerases. Previous work has shown that at least some enhancers form stable protein complexes, leading to the formation of enhanceosomes. We analyzed protein-DNA interactions in the murine ß-globin gene locus using the methyltransferase accessibility protocol for individual templates (MAPit). The data show that a tandem Maf recognition element (MARE) in locus control region (LCR) hypersensitive site 2 (HS2) reveals a remarkably high degree of occupancy during differentiation of mouse erythroleukemia cells. Most of the other transcription factor binding sites in LCR HS2 or in the adult ß-globin gene promoter regions exhibit low fractional occupancy, suggesting highly dynamic protein-DNA interactions. Targeting of an artificial zinc finger DNA-binding domain (ZF-DBD) to the HS2 tandem MARE caused a reduction in the association of MARE-binding proteins and transcription complexes at LCR HS2 and the adult ßmajor-globin gene promoter but did not affect expression of the ßminor-globin gene. The data demonstrate that a stable MARE-associated footprint in LCR HS2 is important for the recruitment of transcription complexes to the adult ßmajor-globin gene promoter during erythroid cell differentiation.

Integrated DNA methylation and chromatin structural analysis at single-molecule resolution.

Chromatin limits the accessibility of DNA to trans-acting factors in transcription, replication, and repair. Although transcriptional variation between cells in a population may contribute to survival and disease, most assays of chromatin structure recover only population averages. We have developed DNA methyltransferases (MTases) as probing agents of DNA accessibility in chromatin, either expressed in vivo in budding yeast or as recombinant enzymatic probes of nuclei isolated from mammalian cells. In this chapter, we focus on the use of recombinant MTase (M) M.CviPI to probe chromatin accessibility in nuclei isolated from mammalian cell lines and animal tissue. This technique, named methylation accessibility protocol for individual templates (MAPit), reports protein-DNA interactions at single-molecule resolution. The single-molecule readout allows identification of chromatin subpopulations and rare epigenetic variants within a cell population. Furthermore, the use of M.CviPI in mammalian systems gives a comprehensive view of both chromatin structure and endogenous DNA methylation in a single assay.

DNA methyltransferase accessibility protocol for individual templates by deep sequencing.

A single-molecule probe of chromatin structure can uncover dynamic chromatin states and rare epigenetic variants of biological importance that bulk measures of chromatin structure miss. In bisulfite genomic sequencing, each sequenced clone records the methylation status of multiple sites on an individual molecule of DNA. An exogenous DNA methyltransferase can thus be used to image nucleosomes and other protein-DNA complexes. In this chapter, we describe the adaptation of this technique, termed Methylation Accessibility Protocol for individual templates, to modern high-throughput sequencing, which both simplifies the workflow and extends its utility.

Simultaneous single-molecule detection of endogenous C-5 DNA methylation and chromatin accessibility using MAPit.

Bisulfite genomic sequencing provides a single-molecule view of cytosine methylation states. After deamination, each cloned molecule contains a record of methylation within its sequence. The full power of this technique is harnessed by treating nuclei with an exogenous DNMT prior to DNA extraction. This exogenous methylation marks regions of accessibility and footprints nucleosomes, as well as other DNA-binding proteins. Thus, each cloned molecule records not only the endogenous methylation present (at CG sites, in mammals), but also the exogenous (GC, when using the Chlorella virus protein M.CviPI). We term this technique MAPit, methylation accessibility protocol for individual templates.

Simultaneous single-molecule mapping of protein-DNA interactions and DNA methylation by MAPit.

Sites of protein binding to DNA are inferred from footprints or spans of protection against a probing reagent. In most protocols, sites of accessibility to a probe are detected by mapping breaks in DNA strands. As discussed in this unit, such methods obscure molecular heterogeneity by averaging cuts at a given site over all DNA strands in a sample population. The DNA methyltransferase accessibility protocol for individual templates (MAPit), an alternative method described in this unit, localizes protein-DNA interactions by probing with cytosine-modifying DNA methyltransferases followed by bisulfite sequencing. Sequencing individual DNA products after amplification of bisulfite-converted sequences permits assignment of the methylation status of every enzyme target site along a single DNA strand. Use of the GC-methylating enzyme M.CviPI allows simultaneous mapping of chromatin accessibility and endogenous CpG methylation. MAPit is therefore the only footprinting method that can detect subpopulations of molecules with distinct patterns of protein binding or chromatin architecture and correlate them directly with the occurrence of endogenous methylation. Additional advantages of MAPit methylation footprinting as well as considerations for experimental design and potential sources of error are discussed.

Bisulfite sequencing of DNA.

Exact positions of 5-methylcytosine (m(5)C) on a single strand of DNA can be determined by bisulfite genomic sequencing (BGS). Treatment with bisulfite ion preferentially deaminates unmethylated cytosines, which are then converted to uracil upon desulfonation. Amplifying regions of interest from deaminated DNA and sequencing products cloned from amplicons permits determination of methylation at single-nucleotide resolution along single DNA molecules, which is not possible with other methylation analysis techniques. This unit describes a BGS technique suitable for most DNA sources, including formaldehyde-fixed tissue. Considerations for experimental design and common sources of error are discussed.

Coupling phosphate homeostasis to cell cycle-specific transcription: mitotic activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead proteins.

Cells devote considerable resources to nutrient homeostasis, involving nutrient surveillance, acquisition, and storage at physiologically relevant concentrations. Many Saccharomyces cerevisiae transcripts coding for proteins with nutrient uptake functions exhibit peak periodic accumulation during M phase, indicating that an important aspect of nutrient homeostasis involves transcriptional regulation. Inorganic phosphate is a central macronutrient that we have previously shown oscillates inversely with mitotic activation of PHO5. The mechanism of this periodic cell cycle expression remains unknown. To date, only two sequence-specific activators, Pho4 and Pho2, were known to induce PHO5 transcription. We provide here evidence that Mcm1, a MADS-box protein, is essential for PHO5 mitotic activation. In addition, we found that cells simultaneously lacking the forkhead proteins, Fkh1 and Fkh2, exhibited a 2.5-fold decrease in PHO5 expression. The Mcm1-Fkh2 complex, first shown to transactivate genes within the CLB2 cluster that drive G(2)/M progression, also associated directly at the PHO5 promoter in a cell cycle-dependent manner in chromatin immunoprecipitation assays. Sds3, a component specific to the Rpd3L histone deacetylase complex, was also recruited to PHO5 in G(1). These findings provide (i) further mechanistic insight into PHO5 mitotic activation, (ii) demonstrate that Mcm1-Fkh2 can function combinatorially with other activators to yield late M/G(1) induction, and (iii) couple the mitotic cell cycle progression machinery to cellular phosphate homeostasis.

Slx5 promotes transcriptional silencing and is required for robust growth in the absence of Sir2.

The broadly conserved Sir2 NAD(+)-dependent deacetylase is required for chromatin silencing. Here we report the discovery of physical and functional links between Sir2 and Slx5 (Hex3), a RING domain protein and subunit of the Slx5/8 complex, [corrected] which is a ubiquitin E3 ligase that targets sumoylated proteins. Slx5 interacted with Sir2 by two-hybrid and glutathione S-transferase-binding assays and was found to promote silencing of genes at telomeric or ribosomal DNA (rDNA) loci. However, deletion of SLX5 had no detectable effect on the distribution of silent chromatin components and only slightly altered the deacetylation of histone H4 lysine 16 at the telomere. In vivo assays indicated that Sir2-dependent silencing was functionally intact in the absence of Slx5. Although no previous reports suggest that Sir2 contributes to the fitness of yeast populations, we found that Sir2 was required for maximal growth in slx5Delta mutant cells. A similar requirement was observed for mutants of the SUMO isopeptidase Ulp2/Smt4. The contribution of Sir2 to optimal growth was not due to known Sir2 roles in mating-type determination or rDNA maintenance but was connected to a role of sumoylation in transcriptional silencing. These results indicate that Sir2 and Slx5 jointly contribute to transcriptional silencing and robust cellular growth.

Mot1 activates and represses transcription by direct, ATPase-dependent mechanisms.

Mot1 is an essential yeast Snf2/Swi2-related ATPase that exerts both positive and negative effects on gene expression. In vitro, Mot1 can disrupt TATA-binding protein-DNA complexes in an ATP-dependent reaction. This activity can explain Mot1-mediated transcriptional repression, but how Mot1 activates transcription is unknown. We demonstrate that, remarkably, Mot1 is localized in vivo to promoters for both Mot1-repressed and Mot1-activated genes. Moreover, Mot1 ATPase activity is required for both activation and repression of gene activity. These findings suggest a novel function for the Mot1 ATPase at activated genes, perhaps involving ATP-driven reorganization of the preinitiation complex. Mot1 regulates the expression of approximately 3% of yeast genes in cells grown in rich medium. Most of these genes are repressed by Mot1, consistent with Mot1's ATP-dependent TATA-binding protein-DNA dissociating activity. Additionally, approximately 77% of the Mot1-repressed genes are involved in the diauxic shift, stress response, mating, or sporulation. The gene sets controlled by NC2 and Srb10 are strongly correlated with the Mot1-controlled set, suggesting that these factors cooperate in transcriptional control on a global scale.

Mot1 regulates the DNA binding activity of free TATA-binding protein in an ATP-dependent manner.

Mot1 is an essential Snf2/Swi2-related Saccharomyces cerevisiae protein that binds the TATA-binding protein (TBP) and removes TBP from DNA using ATP hydrolysis. Mot1 functions in vivo both as a repressor and as an activator of transcription. Mot1 catalysis of TBP.DNA disruption is consistent with its function as a repressor, but the Mot1 mechanism of activation is unknown. To better understand the physiologic role of Mot1 and its enzymatic mechanism, MOT1 mutants were generated and tested for activity in vitro and in vivo. The results demonstrate a close correlation between the TBP.DNA disruption activity of Mot1 and its essential in vivo function. Previous results demonstrated a large overlap in the gene sets controlled by Mot1 and NC2. Mot1 and NC2 can co-occupy TBP.DNA in vitro, and NC2 binding does not impair Mot1-catalyzed disruption of the complex. Residues on the DNA-binding surface of TBP are important for Mot1 binding and the Mot1.TBP binary complex binds very poorly to DNA and does not dissociate in the presence of ATP. However, the binary complex binds DNA well in the presence of the transition state analog ADP-AlF(4). A model for Mot1 action is proposed in which ATP hydrolysis causes the Mot1 N terminus to displace the TATA box, leading to ejection of Mot1 and TBP from DNA.