Effectors of lysine 4 methylation of histone H3 in Saccharomyces cerevisiae are negative regulators of PHO5 and GAL1-10.

Post-translational modifications of histone amino-terminal tails are a key determinant in gene expression. Histone methylation plays a dual role in gene regulation. Methylation of lysine 9 of histone H3 in higher eukaryotes is associated with transcriptionally inactive heterochromatin, whereas H3 lysine 4 methylation correlates with active chromatin. Methylation of lysine 4 of H3 via Set1, a component of the Saccharomyces cerevisiae COMPASS complex, is regulated by the transcriptional elongation Paf1-Rtf1 and histone ubiquitination Rad6-Bre1 complexes, which are required for the expression of a subset of genes. This suggests that lysine 4 methylation of histone H3 may play an activating role in transcription; however, the mechanism of Set1 function remains unclear. We show here that H3 lysine 4 methylation also negatively regulated gene expression, as strains without Set1 showed enhanced expression of PHO5, wherein chromatin structure plays an important transcriptional regulatory role. Di- and trimethylation of H3 lysine 4 was detected at the PHO5 promoter, and a strain expressing a mutant version of histone H3 with lysine 4 changed to arginine, (which cannot be methylated) exhibited PHO5 derepression. Moreover, PHO5 was derepressed in strains that lacked components of either the Paf1-Rtf1 elongation or Rad6-Bre1 histone ubiquitination complexes. Lastly, PHO84 and GAL1-10 transcription was also increased in set1Delta cells. These results suggest that H3 methylation at lysine 4, in conjunction with transcriptional elongation, may function in a negative feedback pathway for basal transcription of some genes, although being a positive effector at others.

Mapping chromatin structure in vivo using DNA methyltransferases.

Cytosine-5 DNA methyltransferases (C5 DMTases) are effective reagents for analyzing chromatin and footprinting DNA-bound factors in vivo. Cytosine methylation in accessible regions is assayed positively by the PCR-based technique of bisulfite sequencing. In this article, we outline two complementary uses for the DNA methyltransferase CviPI (M.CviPI, GC specificity) in probing chromatin organization. First, we describe the use of the naturally occurring, free enzyme as a diffusible probe to map changes in nucleosome structure and to footprint factor interactions at cis-regulatory sequences. In a second application, termed targeted gene methylation (TAGM), the DMTase is targeted via in-frame fusion to a DNA-binding factor. The rapid accumulation of DNA methylation enables highly sensitive detection of factor binding. Both strategies can be applied with any C5 DMTase, such as M.SssI, which also possesses a short-recognition specificity (CG). A description of methods for constructing C5 DMTase-expressing strains of Saccharomyces cerevisiae and analyzing chromatin regions is provided. We also include comprehensive protocols for the isolation and bisulfite treatment of genomic DNA as well as the subsequent bisulfite sequencing steps. Data demonstrating the efficacy of both DMTase probing techniques, theoretical considerations, and experimental analyses are presented at GAL1 and PHO5.

Site-selective in vivo targeting of cytosine-5 DNA methylation by zinc-finger proteins.

Cytosine-5 DNA methylation is a critical signal defining heritable epigenetic states of transcription. As aberrant methylation patterns often accompany disease states, the ability to target cytosine methylation to preselected regions could prove valuable in re-establishing proper gene regulation. We employ the strategy of targeted gene methylation in yeast, which has a naturally unmethylated genome, selectively directing de novo DNA methylation via the fusion of C5 DNA methyltransferases to heterologous DNA-binding proteins. The zinc-finger proteins Zif268 and Zip53 can target DNA methylation by M.CviPI or M.SssI 5-52 nt from single zinc-factor binding sites. Modification at specific GC (M.CviPI) or CG (M.SssI) sites is enhanced as much as 20-fold compared with strains expressing either the free enzyme or a fusion protein with the zinc-finger protein moiety unable to bind to DNA. Interestingly, methylation is also selectively targeted as far as 353 nt from the zinc-finger protein binding sites, possibly indicative of looping, nucleosomes or higher-order chromatin structure. These data demonstrate that methylation can be targeted in vivo to a potentially broad range of sequences using specifically engineered zinc-finger proteins. Further more, the selective targeting of methylation by zinc-finger proteins demonstrates that binding of distinct classes of factors can be monitored in living cells.

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.