Recovery of transcriptionally active chromatin restriction fragments by binding to organomercurial-agarose magnetic beads. A rapid and sensitive method for monitoring changes in higher order chromatin structure during gene activation and repression.

The unfolding of nucleosomes along transcriptionally active DNA sequences uncovers previously shielded cysteinyl-thiol groups of histone H3 molecules located at the center of the nucleosome core. This change in conformation and SH reactivity of nucleosomes along transcribed DNA sequences makes it possible to separate active from inactive nucleosomes by mercury affinity chromatography. The binding of thiol-reactive nucleosomes to an organomercurial-agarose column has been shown previously to reflect, with accuracy, both the timing and extent of transcription of the associated DNA sequences (Chen, T. A., and Allfrey, V. G. (1987) Proc. Natl. Acad. Sci. U. S. A. 84, 5252-5256). Here, we extend this experimental approach to the analysis of higher order chromatin structures. Large chromatin fragments released by treating isolated nuclei with restriction endonucleases are fractionated on mercurated agarose magnetic beads that capture nucleosomes with accessible histone H3 thiols, but do not react with the hidden H3 thiols of the compactly beaded nucleosomes of inactive genes. The SH-reactive domains of c-myc and other genes are rapidly separated from the non-SH-reactive restriction fragments by the magnetic bead technique. The new method also overcomes a major limitation of mercurated agarose column chromatography, which is not suitable for studies of higher order chromatin structure because large chromatin fragments occlude the mercury column; occlusion is not a problem in magnetic separations using suspended mercurated agarose beads. Here, we describe the synthesis of mercurated agarose magnetic beads with high capacity for SH groups and test their application to the recovery of chromatin restriction fragments of c-myc and the growth arrest gene gas1.

Nucleosome structural changes during derepression of silent mating-type loci in yeast.

Mutant a and alpha yeast cells were created with histone H3 containing cysteine in place of alanine 110. Because transcriptionally active nucleosomes "unfold" to reveal the histone H3-thiol groups at the center of the core, the active nucleosomes of the mutant strain can be isolated by mercury-affinity chromatography. We compared the unbound and mercury-bound nucleosomes of haploid H3-mutant strains expressing either the MAT alpha or the MATa mating-type locus. In a MAT alpha strain, the Hg-bound nucleosomes are enriched in MAT alpha DNA but lack the DNA of the transcriptionally silent HMRa mating-type locus. Conversely, in a MATa strain, the Hg-bound nucleosomes are enriched in MATa DNA sequences but deficient in HML alpha DNA. When the SIR3 gene, known to be required for silencing of the repressed mating-type loci, is mutated in the MAT alpha strain, transcription of the HMRa ensues, and its nucleosomes, as well as those of the MAT alpha locus, are retained by the organomercurial column. It follows that derepression of the silent mating-type locus, caused by the sir3 null mutation, is accompanied by an unfolding of its nucleosomes to reveal the histone H3 SH groups at their centers. Nucleosomes of the pheromone-encoding gene MFA2, a gene that is expressed in MATa cells but not in MAT alpha cells, are bound to the organomercurial column when isolated from MATa cells but not from MAT alpha cells. Thus, there is a good correlation between nucleosome unfolding and the renewed transcriptional activity at mating-type loci, and at MFA2, which had been silenced for prolonged periods. A close temporal correlation between nucleosome refolding and the cessation of transcription is not always observed in yeast, however, in contrast to observations in mammalian cells. For example, nucleosomes of the GAL1 gene are maintained in a "poised" or "primed" thiol-reactive state even when the gene is not being transcribed (Chen, T. A., Smith, M. M., Le, S., Sternglanz, R., and Allfrey, V. G. (1991) J. Biol. Chem. 266, 6489-6498). It follows that the unfolding of the nucleosome cores of the yeast H3 mutant is regulated by factors that are not temporally linked to the recruitment or traverse of the RNA polymerase complex, but which may determine the rate at which different domains of chromatin adapt to the need for transcription of the associated DNA sequences.