Michael P. Kladde

The ZEB1 pathway links glioblastoma initiation, invasion and chemoresistance

Glioblastoma remains one of the most lethal types of cancer, and is the most common brain tumour in adults. In particular, tumour recurrence after surgical resection and radiation invariably occurs regardless of aggressive chemotherapy. Here, we provide evidence that the transcription factor ZEB1 (zinc finger E‐box binding homeobox 1) exerts simultaneous influence over invasion, chemoresistance and tumourigenesis in glioblastoma. ZEB1 is preferentially expressed in invasive glioblastoma cells, where the ZEB1‐miR‐200 feedback loop interconnects these processes through the downstream effectors ROBO1, c‐MYB and MGMT. Moreover, ZEB1 expression in glioblastoma patients is predictive of shorter survival and poor Temozolomide response. Our findings indicate that this regulator of epithelial‐mesenchymal transition orchestrates key features of cancer stem cells in malignant glioma and identify ROBO1, OLIG2, CD133 and MGMT as novel targets of the ZEB1 pathway. Thus, ZEB1 is an important candidate molecule for glioblastoma recurrence, a marker of invasive tumour cells and a potential therapeutic target, along with its downstream effectors.

SWI/SNF Has Intrinsic Nucleosome Disassembly Activity that Is Dependent on Adjacent Nucleosomes

The ATP-dependent chromatin remodeling complex SWI/SNF regulates transcription and has been implicated in promoter nucleosome eviction. Efficient nucleosome disassembly by SWI/SNF alone in biochemical assays, however, has not been directly observed. Employing a model system of dinucleosomes rather than mononucleosomes, we demonstrate that remodeling leads to ordered and efficient disassembly of one of the two nucleosomes. An H2A/H2B dimer is first rapidly displaced, and then, in a slower reaction, an entire histone octamer is lost. Nucleosome disassembly by SWI/SNF did not require additional factors such as chaperones or acceptors of histones. Observations in single molecules as well as bulk measurement suggest that a key intermediate in this process is one in which a nucleosome is moved toward the adjacent nucleosome. SWI/SNF recruited by the transcriptional activator Gal4-VP16 preferentially mobilizes the proximal nucleosome and destabilizes the adjacent nucleosome.

Polyphosphate Loss Promotes SNF/SWI- and Gcn5-Dependent Mitotic Induction of PHO5

ABSTRACT Approximately 800 transcripts in Saccharomyces cerevisiae are cell cycle regulated. The oscillation of ∼40% of these genes, including a prominent subclass involved in nutrient acquisition, is not understood. To address this problem, we focus on the mitosis-specific activation of the phosphate-responsive promoter, PHO5. We show that the unexpected mitotic induction of the PHO5 acid phosphatase in rich medium requires the transcriptional activators Pho4 and Pho2, the cyclin-dependent kinase inhibitor Pho81, and the chromatin-associated enzymes Gcn5 and Snf2/Swi2. PHO5 mitotic activation is repressed by addition of orthophosphate, which significantly increases cellular polyphosphate. Polyphosphate levels also fluctuate inversely with PHO5 mRNA during the cell cycle, further substantiating an antagonistic link between this phosphate polymer and PHO5 mitotic regulation. Moreover, deletion of PHM3, required for polyphosphate accumulation, leads to premature onset of PHO5 expression, as well as an increased rate, magnitude, and duration of PHO5 activation. Orthophosphate addition, however, represses mitotic PHO5 expression in a phm3Δ strain. Thus, polyphosphate per se is not necessary to repress PHO transcription but, when present, replenishes cellular phosphate during nutrient depletion. These results demonstrate a dynamic mechanism of mitotic transcriptional regulation that operates mostly independently of factors that drive progression through the cell cycle.

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.

Chromatin structure mapping in vivo using methyltransferases.

Publisher Summary This chapter discusses the mapping of chromatin structure in vivo using methyltransferases. Expression of prokaryotic methyltransferases in Saccharomyces cerevisiae (S. cerevisiae ), the genome of which contains no endogenous detectable methylation is used as a means of probing chromatin structure in living cells. A DNA fragment from E. coli containing the dam methyltransferase gene was introduced into the LYS2 locus of S. cerevisiae by transfection. The gene was expressed from a fortuitous promoter in an uncontrolled fashion, leading to the modification of adenines in the GATC sequence recognized by this methyltransferase. These studies were seminal in use of such enzymes as probes for chromatin structure in living cells. The use of a positive chemical method for the detection of modified residues under conditions of limited overall modification allows quantitative analysis of accessibility over long stretches of DNA. Although currently only demonstrated in S. cerevisiae , the general approaches outlined in this chapter should be portable to some other species for analysis of chromatin structure. A limitation in use for other species is the occurrence of endogenous CG cytidine methylation. Clearly the sites modified in normal cells are detected in the chemical analysis. Similar, although less dramatic, problems occur for GATC methylation, where one cytidine in four will occur in the sequence GATCG. For this reason, as well as enhancement of the level of resolution of mapping, an active search is underway for cytidine methyltransferases that recognize di- or trinucleotides that occur with higher frequency than CG.

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.

TRIM29 Suppresses TWIST1 and Invasive Breast Cancer Behavior

TRIM29 (ATDC) exhibits a contextual function in cancer, but seems to exert a tumor-suppressor role in breast cancer. Here, we show that TRIM29 is often silenced in primary breast tumors and cultured tumor cells as a result of aberrant gene hypermethylation. RNAi-mediated silencing of TRIM29 in breast tumor cells increased their motility, invasiveness, and proliferation in a manner associated with increased expression of mesenchymal markers (N-cadherin and vimentin), decreased expression of epithelial markers (E-cadherin and EpCAM), and increased expression and activity of the oncogenic transcription factor TWIST1, an important driver of the epithelial-mesenchymal transition (EMT). Functional investigations revealed an inverse relationship in the expression of TRIM29 and TWIST1, suggesting the existence of a negative regulatory feedback loop. In support of this relationship, we found that TWIST1 inhibited TRIM29 promoter activity through direct binding to a region containing a cluster of consensus E-box elements, arguing that TWIST1 transcriptionally represses TRIM29 expression. Analysis of a public breast cancer gene-expression database indicated that reduced TRIM29 expression was associated with reduced relapse-free survival, increased tumor size, grade, and metastatic characteristics. Taken together, our results suggest that TRIM29 acts as a tumor suppressor in breast cancer through its ability to inhibit TWIST1 and suppress EMT.

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.

Targeted cytosine methylation for in vivo detection of protein–DNA interactions

We report a technique, named targeted gene methylation (TAGM), for identifying in vivo protein-binding sites in chromatin. M.CviPI, a cytosine-5 DNA methyltransferase recognizing GC sites, is fused to a DNA-binding factor enabling simultaneous detection of targeted methylation, factor footprints, and chromatin structural changes by bisulfite genomic sequencing. Using TAGM with the yeast transactivator Pho4, methylation enrichments of up to 34- fold occur proximal to native Pho4-binding sites. Additionally, significant selective targeting of methylation is observed several hundred nucleotides away, suggesting the detection of long-range interactions due to higher-order chromatin structure. In contrast, at an extragenic locus lacking Pho4-binding sites, methylation levels are at the detection limit at early times after Pho4 transactivation. Notably, substantial amounts of methylation are targeted by Pho4-M.CviPI under repressive conditions when most of the transactivator is excluded from the nucleus. Thus, TAGM enables rapid detection of DNA–protein interactions even at low occupancies and has potential for identifying factor targets at the genome-wide level. Extension of TAGM from yeast to vertebrates, which use methylation to initiate and propagate repressed chromatin, could also provide a valuable strategy for heritable inactivation of gene expression.

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.