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

Abstract

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.

Fig. 1.

The TAGM strategy for identifying DNA–protein interactions in vivo. Hypothetical sites protected against methylation (arrowheads) or directly methylated (asterisks) are indicated.

Fig. 2.

Pho4 specifically targets M.CviPI to the PHO5 promoter. (A) Time course of targeted methylation. Cultures expressing Pho4-M.CviPI and Pho4 (lanes 8–17) or mut Zif-M.CviPI as a free MTase control (lanes 1–7) were grown under repressive conditions in high P_i medium (lanes 1, 8, and 9; only these +P_i samples from the full time course are shown, because all others for both MTase fusions were identical), then washed and transferred to P_i-free medium to activate PHO genes (lanes 2–7 and 11–17). Genomic DNA isolated from cells removed at the indicated times was analyzed for ^5meC levels at GC sites on the lower strand of the PHO5 promoter by a modification (5) of bisulfite genomic sequencing (19, 20). The locations of the two known Pho4-binding sites (filled bars), the UASp1 E box (CACGTT), and UASp2 E box (CACGTG), as well as positioned nucleosome –2 (27) (partial ellipse), are shown. The distance (base pairs) of each GC site from the respective proximal edge of UASp1 in the nuclease hypersensitive region (27) (GC sites from –405 to –331 relative to the PHO5 ATG) or UASp2 (GC sites from –290 to –241) are also indicated on the right. The same number of total counts was loaded in each lane. In strains expressing either MTase fusion, the ratios of ^5meC between several sites (•) in a given lane were similar, identifying sites to which methylation is nontargeted or targeted indirectly (see Fig. 1). Normalization of ^5meC levels to an accessible histone-free site remote from UASp1, site 43 (←), enables lane-to-lane comparisons (see B) and demonstrates protection against methylation (▸) as well as efficient targeting of M.CviPI to three GC sites (*) by bound Pho4. Selective targeting of ^5meC to these latter three sites is highly reproducible, as evidenced in lanes 9–17 and in five additional experiments analyzing one +P_i and a 4-h -P_i sample. Note that, after 2 h, high levels of methylation in the Pho4-M.CviPI samples lead to considerable departure from single-hit kinetics and underestimation of signal intensity from –344 to –241. (B) Quantitative scans of bisulfite genomic sequencing data. (Upper) Selected lanes (as indicated) in A are scanned (PHO5 UASp1). Methylation levels can be normalized to that at site 43. (Lower) Scans (PHO5 UASp2) were obtained by reextension of the same PCR products used in the analysis in A with primer PHO5b1–969 that anneals between sites 26b and 37. The same number of total counts was loaded in each lane. Symbols are as in A.(C) Initial rates of methylation are linear. Quantification of absolute ^5meC frequencies (percentage of total summed product intensities) of the indicated sites from the data in A, lanes 10–13. (D) M.CviPI is specifically targeted by Pho4 to PHO5 and not to CAR1 at early times after PHO activation. CAR1 sequences (+159 to +558) were amplified from a subset of the bisulfite-treated samples analyzed in A and analyzed for ^5meC levels. Only four GC sites at CAR1 are shown; the ratios among eight additional sites are also identical. (E) TAGM detects Pho4 binding more sensitively than ChIP analysis. Immunoselected (lanes 2–5) and nonimmunoselected (lane 1, input) samples from either wild-type PHO4 (lane 5, no tag) or 3Myc-PHO4 (lanes 2–5) strains that contain a wild-type PHO5 promoter and a mutated promoter (pho5 ΔUASs) were analyzed by competitive PCR. The folds of enrichment (PHO5:pho5 ΔUASs), normalized to the input ratio, are given.

Fig. 5.

M.CviPI is targeted by Pho4 to the PHO8 (A) and PHO84 (B) promoters. ^5meC levels were determined at PHO8 and PHO84 from cells expressing either mut Zif- or Pho4-M.CviPI grown in the presence (+) and absence (-) of P_i, as indicated. Shown are the quantitative scans of the phosphorimage obtained from the gel (same total counts per lane). GC sites to which M.CviPI directly targeted methylation (*), GC sites protected against methylation (▴), and Pho4-binding sites (filled bars), are labeled. ^5meC levels can be compared with the sites marked with arrows. The positions of nucleosomes (nuc -3 and nuc -4, partial ellipses), previously mapped at PHO8 (41), are shown. From the data in B, we infer the disruption of two nucleosomes in the analyzed PHO84 region (increased methylation on activation at seven GC sites, 36–221 bp from UAS E; compare scan 2 to scan 1 in B). To augment peak heights, quantification of the run-off products has been omitted. A region in scan 5 where the signal is underestimated due to departure from single-hit kinetics is bracketed.

Fig. 3.

Targeting of C5 methylation by Pho4-M.CviPI to the upper strand of the PHO5 promoter. The same bisulfite-treated samples used in the analysis in Fig. 2 A were used in the PCR amplification. Scans of the phosphorimage of the gel that was loaded with the same number of total counts per lane are shown. (Left) The brackets above scans 1 and 3 (PHO5 UASp1) indicate a nonspecific primer extension pause that occurred in samples 1–5 or only sample 3, respectively. See the legend to Fig. 2 A for symbol definitions.

Fig. 4.

Pho4 targets M.CviPI at a distance. (A) Determination of ^5meC levels upstream of PHO5 UASs. PHO5 sequences were amplified from a subset of the bisulfite-treated samples analyzed in Fig. 2 A to assay for ^5meC levels. The two asterisks at the top of the gel indicate sites 13 and 26a that are directly targeted by Pho4-M.CviPI near UASp1. Symbols are as in Fig. 2A, except that double (••) and triple GC sites (•••) that did not resolve during electrophoresis are also indicated. Site 319 used for normalization in B is marked as well (→). (B) Quantification of preferential targeting of M.CviPI by Pho4 to site 335, but not to site 278. The mean ± standard error of ^5meC levels for the indicated sites (normalized to site 319) for mut Zif-M.CviPI (n = 3) and Pho4-M.CviPI (n = 6) is shown.