Splitting of H3-H4 tetramers at transcriptionally active genes undergoing dynamic histone exchange

Abstract

Nucleosome deposition occurs on newly synthesized DNA during DNA replication and on transcriptionally active genes via nucleosome-remodeling complexes recruited by activator proteins and elongating RNA polymerase II. It has been long believed that histone deposition involves stable H3-H4 tetramers, such that newly deposited nucleosomes do not contain H3 and H4 molecules with their associated histone modifications from preexisting nucleosomes. However, biochemical analyses and recent experiments in mammalian cells have raised the idea that preexisting H3-H4 tetramers might split into dimers, resulting in mixed nucleosomes composed of "old" and "new" histones. It is unknown to what extent different genomic loci might utilize such a mechanism and under which circumstances. Here, we address whether tetramer splitting occurs in a locus-specific manner by using sequential chromatin immunoprecipitation of mononucleosomes from yeast cells containing two differentially tagged versions of H3 that are expressed "old" and "new" histones. At many genomic loci, we observe little or no nucleosomal cooccupancy of old and new H3, indicating that tetramer splitting is generally infrequent. However, cooccupancy is detected at highly active genes, which have a high rate of histone exchange. Thus, DNA replication largely results in nucleosomes bearing exclusively old or new H3-H4, thereby precluding the acquisition of new histone modifications based on preexisting modifications within the same nucleosome. In contrast, tetramer splitting, dimer exchange, and nucleosomes with mixed H3-H4 tetramers occur at highly active genes, presumably linked to rapid histone exchange associated with robust transcription.

Fig. 1.

Experimental design. (A) The experimental strain YKY69 lacks the endogenous H3 genes and contains two centromeric plasmids expressing H3–VSVG from the methionine-repressible MET3 promoter and H3–HA from the galactose-inducible GAL1 promoter. (B) The control strain YKY64 is similar to YKY69, except that the two tagged versions of H3 are under the control of the native HHT2 promoter, resulting in their coexpression. (C) YKY69 cells are initially grown in medium lacking methionine and containing raffinose, leading to expression of only H3-VSVG (“old” histone). Methionine is added for 4.5 h to repress the MET3 promoter and terminate H3-VSVG expression (sample A). Cells are then treated with galactose to induce H3–HA expression (“new” histone) for 2.5 h, 4.5 h, and 6.5 h (samples B, C, and D, respectively). Sample E consists of YKY69 cells grown for 20 h in medium containing both methionine and galactose, resulting in expression of H3–HA. (D) Procedure for performing sequential ChIP of mononucleosomes.

Fig. 2.

Association of old and new H3 at different loci. YKY69 cells were treated to induce “old” H3-VSVG expression, followed by its repression, and then by induction of “new” H3–HA expression, as described in Fig. 1C. In samples A and E, only a single H3 form was induced and substantially incorporated (VSVG- and HA-tagged, respectively), while the chromatin of samples B–D contained both H3 forms, incorporated at different times. Sample F represents the control strain YKY64, coexpressing the two tagged H3 forms from their native promoter. The graphs show the average results of ChIP analysis using VSVG (A) or HA (B) antibodies. H3 occupancy was analyzed at a telomeric locus (TELVI-R) and six different genes, with the positions relative to the translation start site indicated.

Fig. 3.

Nucleosome cooccupancy of old and new H3. Samples from the experiments described in Figs. 1C and 2 were subjected to two rounds of immunoprecipitation using VSVG and HA antibodies. The graphs show the average results of sequential ChIP analysis, relative to the values of the single-induction sample E, regarded as experimental background. H3 cooccupancy was analyzed at a telomeric locus (TELVI-R) and six different genes, with the positions relative to the translation start site indicated.

Fig. 4.

Nucleosomal occupancy and cooccupancy of inducible H3 at active and inactive loci. Single and sequential ChIP samples from the experiments of Figs. 1, 2, and 3 were analyzed for association at additional loci. The graphs show the average results of (A) ChIP analysis using HA antibodies, as described in Fig. 2B, or (B) sequential ChIP using VSVG and HA antibodies, as described in Fig. 3. H3 occupancy was analyzed at different genes, with the positions relative to the translation start site or around the stop codon (STOP) indicated.