Russell P. Darst

Bisulfite sequencing of DNA.

Exact positions of 5‐methylcytosine (m5C) 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. Curr. Protoc. Mol. Biol. 91:7.9.1‐7.9.17.

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.

Slx5 Promotes Transcriptional Silencing and Is Required for Robust Growth in the Absence of Sir2

ABSTRACT 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 column, 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 slx5Δ 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.

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.

Coupling Phosphate Homeostasis to Cell Cycle-Specific Transcription: Mitotic Activation of Saccharomyces cerevisiae PHO5 by Mcm1 and Forkhead Proteins

ABSTRACT 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 G2/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 G1. 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/G1 induction, and (iii) couple the mitotic cell cycle progression machinery to cellular phosphate homeostasis.

Maneuver at the transcription start site: Mot1p and NC2 navigate TFIID/TBP to specific core promoter elements

The process of transcription initiation in eukaryotic cells is complex and our view of how the transcription apparatus is assembled at the promoter has changed over the last decade. For most genes transcribed by RNA polymerase II (Pol II) general transcription factors and Pol II assemble at the core promoter and initiate transcription at specific sites. The number of distinct core promoter elements is increasing and so is the number of specific proteins that act through these elements. Core promoter elements include the TATA box, TFIIB recognition element (BRE), the initiator (INR), and the downstream promoter element (DPE). Transcription factors that act through these elements are TATA binding protein (TBP) and its associated factors (TAFs), Negative Cofactor 2 (NC2), Mot1p, TFIIB, and TFIIA. Recent observations suggest that TBP, Mot1p, and NC2 exert positive and negative effects on transcription complex assembly depending on the presence or absence of specific core promoter elements. It is proposed here that Mot1p and NC2 together function as a remodeling complex that positions TFIID and perhaps other protein complexes at specific core promoter elements.

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. Curr. Protoc. Mol. Biol. 95:21.22.1‐21.22.18.

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.