31 research outputs found
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 proteināDNA 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 proteināDNA 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
Rapid detection of functional expression of C-5-DNA methyltransferases in yeast.
We have previously employed the cytosine-5-DNA methyltransferase (MTase), M. Sss I, as a probe for chromatin architecture in intact cells. Although M. Sss I offers the highest resolution of any currently available MTase, the difficulty in establishing stable, methylation-positive strains poses a barrier to its general utility as a chromatin probe. We describe a simple screen for M. Sss I-expressing strains that eliminates the purification of PCR products amplified from bisulfite-treated DNA, use of radioisotopes, polyacrylamide sequencing gel electrophoresis, and autoradiography. The high throughput of the method now makes it feasible to introduce M. Sss I into a variety of wild-type and mutant genetic backgrounds
Tup1p represses Mcm1p transcriptional activation and chromatin remodeling of an a-cell-specific gene
In yeast, a number of regulatory proteins expressed only in specific cell types interact with general transcription factors in a combinatorial manner to control expression of cell-type-specific genes. We report a detailed analysis of activation and repression events that occur at the promoter of the a-cell-specific STE6 gene fused to a Ī²-galactosidase gene in a yeast minichromosome, as well as factors that control the chromatin structure of this promoter both in the minichromosome and in the genomic STE6 locus. Mcm1p results in chromatin remodeling and is responsible for all transcriptional activity from the STE6 promoter in both wild-type a and Ī± cells. MatĪ±2p cooperates with Tup1p to block both chromatin remodeling and Mcm1p-associated activation. While MatĪ±2p represses only Mcm1p, the Tup1p-mediated repression involves both Mcm1p-dependent and -independent mechanisms. Swi/Snf and Gcn5p, required for full induction of the STE6 gene, do not contribute to chromatin remodeling. We suggest that Tup1p can contribute to repression by blocking transcriptional activators, in addition to interacting with transcription machinery and stabilizing chromatin
Effects of Sin- versions of histone H4 on yeast chromatin structure and function.
Previous studies have identified single amino acid changes within either histone H3 or H4 (Sin- versions) that allow transcription in the absence of the yeast SWI-SNF complex. The histone H4 mutants are competent for nucleosome assembly in vivo, and the residues that are altered appear to define a discrete domain on the surface of the histone octamer. We have analyzed the effects of the Sin- versions of histone H4 on transcription and chromatin structure in vivo. These histone H4 mutants cause an increased accessibility of nucleosomal DNA to Dam methyltransferase and to micrococcal nuclease. Sin- derivatives of histone H4 also grossly impair the ability of nucleosomes to constrain supercoils in vivo. Nucleosome-mediated repression of the PHO5 gene is severely impaired by these histone H4 mutants; PHO5 expression is derepressed to 31% of the wild-type induced level. In contrast to the induction caused by nucleosome depletion, full PHO5 derepression by Sin- versions of histone H4 requires upstream regulatory elements. In addition, Sin- derivatives of histone H4 do not activate expression from CYC1 or GAL1 promoters that lack UAS elements. We propose that these Sin- mutations alter histone-DNA contact residues that play key roles in restricting the accessibility of nucleosomal DNA to transcription factors
Targeted cytosine methylation for in vivo detection of proteināDNA 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 DNAāprotein 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