DNA methyltransferase-based single-molecule (MAPit) assay for mapping protein-DNA interactions in vitro
互联网
Introduction
Analysis of protein-DNA interactions in the context of chromatin is pivotal for understanding the complex structure and functions of eukaryotic genomes. Packaging DNA with histones into nucleosomes impedes the binding of most trans -acting factors requiring access to their specific target sites. Alteration of nucleosome structure and organization is therefore a central feature of gene regulation. Factors gain access to their target sites when the intrinsically dynamic nucleosomes preferentially expose DNA sequences at the nucleosome termini by a process termed site exposure (Polach and Widom, 1995, 1996). In addition, nucleosome-mediated repression is relieved by multi-protein chromatin remodeling complexes that disrupt nucleosomal structure in an ATP-dependent manner. Nucleosomes are reorganized either by remodeler-directed repositioning, disassembly and/or exchange of H2A-H2B dimers for histone variants (Längst and Becker, 2004; Saha et al., 2006).
Since the emergence of the basic nucleosome model of chromosome structure, various methods have been developed for mapping and characterization of histone and non-histone protein-DNA interactions. Most often, enzymatic (e.g., micrococcal nuclease, DNase I, DNase II, restriction endonucleases, exonuclease III) or chemical (e.g., dimethyl sulfate, methidiumpropyl-EDTA, psoralens, ultraviolet light) footprinting techniques, which rely on DNA cleavage, are used to localize nucleosome positions and/or factor binding sites (Simpson, 1998). In general, these footprinting techniques are based on the premise that protein-DNA complexes are more resistant to nuclease digestion than free DNA. Large nucleosomal or small regulatory factor footprints are inferred by comparison of the patterns of DNA cleavage in protein-containing and -free samples.
Although significant progress has been made in elucidating basic chromatin structure and dynamics, several problems associated with classical footprinting techniques have severely restricted the scope of questions that can be addressed. First, these methods fail to detect heterogeneity between individual chromatin molecules in a sample as they report the average behavior of all molecules in the population. Second, techniques based on DNA cleavage cannot detect multiple footprints arising from coordinated or sequential binding of factors along an individual DNA molecule within a population of molecules. This is because, considering a single molecule, only the first cleavage site proximal to the probe can be mapped. Related to this, quantitative assessment of factor occupancy is subject to the constraints of Poisson single-hit kinetic conditions; random cleavage of ≤ 1 per molecule in at least 90% of the molecules in the population. However, biological processes are complex and often involve non-random situations leading to a multiple-hit regime where the probe modifies the same DNA molecule more than once. Lastly, nucleases exhibit strong cleavage preferences (Flick et al., 1986) that frequently limit accurate assignment of nucleosome or factor positions.
Efficient single-molecule techniques that do not damage DNA are therefore necessary to provide a detailed view of chromatin structure. DNA methyltransferases (DMTases) offer an attractive alternative for investigating chromatin architecture and deciphering dynamic chromatin-mediated processes. We recently developed a single-molecule assay termed MAPit (M ethyltransferase A ccessibility P rotocol for i ndividual t emplates) for analyzing a wide range of protein-DNA interactions by DNA methylation protection rather than nuclease-based strand scission (Jessen et al., 2006). Independently, a similar approach was developed, referred to as methyltransferase-based single-promoter analysis (MSPA) (Fatemi et al., 2005). We previously employed MAPit in living yeast cells to provide the first evidence for a stochastic and heterogeneous nature of chromatin transitions occurring during transactivation of the phosphate-responsive PHO5 promoter (Jessen et al., 2006). Here, we describe the application of this method to map nucleosome positions in biochemically reconstituted chromatin preparations either directly or after remodeling with the prototypical yeast ISW2 complex purified from budding yeast. This method should provide a powerful new approach for exploring mechanistic aspects of chromatin remodeling.
Back to top
Procedure
To map nucleosome positions using MAPit, nucleosomes are first reconstituted and probed directly or subjected to remodeling in the presence of a purified ATP-dependent chromatin remodeling complex. After terminating remodeling reactions, DMTases are added to methylate accessible DNA sites. Subsequently, samples are analyzed by bisulfite genome sequencing (BGS), which reveals methylation patterns of individually cloned molecules at single-nucleotide resolution (Clark et al., 1994; Frommer et al., 1992) (Fig. 1).
Preparation of Nucleosome Substrate
- Recombinant core histones (Xenopus laevis) are expressed, purified and refolded into the octamer using standard methods (Dyer et al., 2004).
- Double-stranded DNA fragments (note 1 ) are prepared with one Cy5-labeled and one unlabeled primer in a preparative PCR reaction.
- The large-scale PCR reactions are then concentrated (we use Millipore ultracel YM-50 filters) and purified by phenol-chloroform extraction.
- After ethanol precipitation, the final concentration of DNA is adjusted to 1 mg/ml for reconstitutions.
- Mononucleosomes are reconstituted on a micro-scale using rapid salt dilution method. Typically, we titrate the DNA-to-octamer in molar ratios of 0.3:1.0, 0.5:1, 0.75:1.0 and 1.0:1.0 in a 10 µl reaction containing 2 M NaCl and 10 µg of octamer and use the sample with the least amount of free DNA for further analysis. Higher molar ratios can also be employed to achieve maximal reconstitution of free DNA.
- After incubating for 25 min at 37°C, the salt concentration is serially diluted to 1.5 M, 1 M, 0.7 M and 0.3 M by the step-wise addition of 25 mM Tris-HCl, pH 8.0 at 10 min intervals.
- The efficiency of each nucleosome reconstitution is verified by polyacrylamidegel electrophoresis (PAGE) at 4°C on a 4% native gel buffered with 0.2X TBE.
- An image of the gel is documented using an appropriate imaging apparatus.
Nucleosome Remodeling
- Yeast ISW2 complex (or other desired activities) is purified as previously described (Tsukiyama et al., 1999).
-
Remodeling reactions can be performed as described in Zofall et al. (2004). A typical remodeling reaction of 75 µl contains (note 2 ):
Reconstituted nucleosome ~ 6 µl (1-18 pmol) ISW2 binding buffer 1X final ISW2 complex 3-84 nM final 10 mM ATP 0.6-0.8 mM final
Reactions are incubated at 30°C for 30 min and stopped by adding ATP-γ-S (5 mM final). - Reactions(4-6 µl) are analyzed by electrophoresis on a 5% non-denaturing PAGE buffered with 0.2X TBE at 4°C.
- Document the gel using an instrument capable of fluorescent imaging (note 3 ).
Probing Mononucleosomes with DMTases
Cytosine-5-specific DMTases that recognize specific dinucleotide sites (e.g., M.SssI with CG specificity (Renbaum et al., 1990) or M.CviPI with GC specificity (Xu et al., 1998) are particularly useful because of their high mapping resolution. C-5 methylation (m5 C) is required to provide a single-molecule view of DNA methylation via BGS.
[1] [2] [3] 下一页