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.T he packaging of eukaryotic DNA into chromatin influences many DNA-associated processes, including transcriptional regulation. Within the chromatin fiber, each basic nucleosome unit bears important chemical and structural information. The mechanisms by which nucleosomes are assembled on DNA during replication, transcription, DNA repair, etc., can thus impact not only the integrity of chromatin structure but also patterns of gene expression and epigenetic inheritance. The H3-H4 tetramer core of each nucleosome is the more stable component and contains most of the relatively persistent and functionally important histone methylation marks. Much attention has therefore been given to the questions of how the H3-H4 tetramers are formed and maintained on chromatin.Early studies attempted to distinguish between a conservative assembly model, by which old and new histones form separate tetramers on replicating DNA, and a semiconservative assembly mechanism, by which existing tetramers are split into H3-H4 dimers, followed by association of new H3-H4 dimers to complete each nucleosome core (1-3). In the semiconservative model, the resulting tetramers would bear a mixture of old and new histones, allowing transmission of epigenetic information within the basic nucleosome unit. Though mechanistically attractive, the mixed tetramer model receiv...

Asymmetric nucleosomes flank promoters in the budding yeast genome

“…Tracking ''old'' and ''new'' histones had also demonstrated splitting of nucleosomes in yeast (Katan-Khaykovich and Struhl 2011) and in human cells (Huang et al 2013). Our results suggest that the half-nucleosome footprints at active loci in yeast are due to RSC action at these nucleosome positions, suggesting a possible mechanism for nucleosome splitting.…”

Chromatin features and the epigenetic regulation of pluripotency states in ESCs

“…2). In yeast, where the single H3 isoform is homologous to H3.3, (H3-H4) 2 tetramer splitting can be observed on transcriptionally active loci (Katan-Khaykovich and Struhl, 2011). The same is observed in mammalian cells, although the frequency of (H3.3-H4) 2 splitting appears to correlate more with enhancer specificity and potentially during DNA replication (Huang et al, 2013;Xu et al, 2010).…”

Partially Assembled Nucleosome Structures at Atomic Detail

“…A genomic-scale study in yeast (23) detected widespread subnucleosomal structures in dynamic chromatin, including what appear to be half-nucleosomes (consisting of one copy of each histone), hexasomes, and tetrasomes. Moreover, it was shown that in actively transcribing genes and their enhancers (which are also the sites of very active histone exchange), preexisting (H3$H4) 2 tetramers might split into dimers, resulting in mixed nucleosomes composed of old and new histones (86,87). The enhanced presence of mixed tetramers at sites of active transcription suggests that nucleosome perturbation by Pol II involves a transfer of parental H3$H4 as dimers.…”