DNA in eukaryotic cells is packaged into nucleosomes, the structural unit of chromatin. Both DNA and bulk histones are extremely long-lived, because old DNA strands and histones are retained when chromatin duplicates. In contrast, the Drosophila HSP70 genes rapidly lose histone H3 and acquire variant H3.3 histones as these genes are induced. Histone replacement does not occur at artificial HSP70 promoter arrays, demonstrating that transcription is required for H3.3 deposition. The H3.3 histone is enriched in all active chromatin and throughout large transcription units, implying that deposition occurs during transcription elongation. Strikingly, the stability of chromatin-bound H3.3 differs between loci: H3.3 turns over at continually active rDNA genes, but becomes stable at induced HSP70 genes that have shut down. It is concluded that H3.3 deposition is coupled to transcription, and continues while a gene is active. Repeated histone replacement suggests a mechanism to both maintain the structure of chromatin and access to DNA at active genes (Schwartz, 2005).

Transcriptional regulation in eukaryotes requires the coordinated recruitment of RNA polymerase II along with a variety of activators, repressors, chromatin-remodeling factors, and other chromatin-associated proteins. Histone variants have also been implicated in transcriptional regulation. All eukaryotic genomes encode four conserved core histones that package bulk chromatin, but a fraction of chromatin is packaged by alternate histones. Three lines of evidence support the argument that an alternate H3 histone subtype in animals -- the variant H3.3 -- plays a role in transcriptionally active chromatin: (1) a pulse of epitope-tagged H3.3 protein localized to gene-rich chromatin in Drosophila cells (Ahmad 2002); (2) H3.3 became enriched at a transgene array after its induction (Janicki, 2004); (3) H3.3 is enriched for covalent histone modifications associated with active chromatin, while repressive modifications are enriched on the H3 histone (McKittrick, 2004). These observations suggest that H3.3 is a common component of active chromatin (Schwartz, 2005).

Studies of the H3.3 variant have also suggested that distinctive nucleosome assembly occurs in active chromatin. Recent biochemical and in vivo assays demonstrate that there are both DNA replication-coupled and replication-independent (RI) nucleosome assembly pathways in eukaryotic nuclei. DNA replication-coupled assembly serves to package newly synthesized DNA into nucleosomes, but the existence of RI assembly pathways implies that previously chromatinized templates are also loading new histones. One RI pathway exclusively deposits newly synthesized histone H4 and the H3.3 variant and operates specifically at active chromatin in interphase nuclei (Ahmad, 2002; Tagami, 2004). However, the relationship between transcriptional activity and H3.3 has not been defined. It is unknown if H3.3 deposits during or after transcription of a gene, or how frequently RI assembly occurs. Conceivably, occasional RI assembly would have little effect on the physical properties of chromatin and the stability of histone modifications. Alternatively, the structure of active chromatin might be disrupted if RI assembly occurs frequently (Schwartz, 2005).

This study demonstrates that H3.3 deposition is triggered by gene induction, and deposition occurs while genes are transcribed. H3.3 deposition is also associated with histone removal and degradation that depends on the activity of a locus. Repeated rounds of replacement may function to destabilize active chromatin. The progressive degradation of H3.3 histone subtypes is distinct from the conservative exchange of H2A/H2B subtypes, and explains constitutive synthesis of new H3.3 histone (Schwartz, 2005).

Cells use two pathways for the assembly of nucleosomes. The replication-coupled pathway suffices to package the bulk genome as DNA is replicated, using histones synthesized from the major canonical genes. The RI pathway acts on transcriptionally active loci and specifically uses the H3.3 histone variant (Ahmad, 2002; Janicki 2004; Tagami 2004). This study shows that RI assembly begins within minutes after gene induction and operates while genes are active. The binding of transcription factors to artificial promoter arrays is insufficient to trigger H3.3 deposition, implying that RI assembly is tied to a late step in gene induction. The findings that H3.3 becomes distributed throughout long transcribed genes suggest that RNA polymerase progression itself is involved in histone displacement and replacement. Finally, the stability of H3.3 in chromatin depends on transcriptional status: At continually active loci, H3.3 deposits and then leaves, but when genes become repressed, incorporated H3.3 becomes stable. This dependence is explained if transcription causes the displacement of chromatin-bound histones (Schwartz, 2005).

In vitro experiments show that RNA polymerases often pause when they run into a nucleosome. Transcription through a nucleosome requires the transient disruption of histone-DNA contacts so that the polymerase can progress. A key question is whether the loss of these contacts releases histones from DNA as the polymerase passes, or if the histones remain associated. The current view is that accessory factors assist polymerases by moving histone octamers along the template, or by returning any histones that are displaced. However, the rapid stimulation of H3.3 replacement by gene induction implies that, at least sometimes, displaced histones are not restored. The enrichment of H3.3 at all active chromatin in Drosophila indicates that the loss of displaced histones is a general feature of transcription from chromatin templates (Schwartz, 2005).

There are two processes that might link histone variant replacement to transcription. (1) The polymerase may displace histones, thus producing a naked template suitable for RI assembly machinery. Such a dependence on prior nucleosome disassembly would effectively restrict H3.3 replacement to active genes. (2) Factors that accompany elongating RNA polymerase may catalyze histone replacement. Support for this idea comes from the detection of transcriptional regulators in isolated H3.3 predeposition complexes (Tagami, 2004). Tagami suggested that these regulators might serve as protein interfaces to recruit H3.3 to sites of active transcription. Indeed, a protein interaction between the histone chaperone CAF large subunit and the DNA replication fork protein PCNA serves to enhance replication-coupled deposition of histone H3 (Shibahara, 1999). An analogous role might be performed by transcription factors that interact with the HIRA chaperone in H3.3 predeposition complexes. Additionally, the histone-binding proteins SPT5 and SPT6 are known to localize to transiently induced HSP70 genes, and are thought to manipulate nucleosomes for transcription (Andrulis, 2000). Such factors might drive histone replacement to clear DNA for polymerase passage. SPT5 and SPT6 are not known to localize to sites of RNA Pol I transcription, so there may also be analogous polymerase-specific factors that clear DNA. Since SPT5 and SPT6 leave HSP genes after transient induction, replacement would also cease and the genes would retain high levels of H3.3. Thus, H3.3 replacement leaves behind an extremely stable marker of past transcriptional activity (Schwartz, 2005).

The observation that chromatin-bound H3.3 has a short half-life (~24 h) is in stark contrast to the half-life of histones from bulk chromatin. Measurements of in vivo histone exchange have demonstrated recycling of H2A and H2B between sites within a nucleus, and at least one complex is capable of exchanging these histones between chromatin and histone chaperones. However, recycling of H3 and H4 was not detected, and in current experiments bulk H2B-GFP does not degrade. The fate of H3.3 when it is displaced from chromatin distinguishes H3.3 replacement from other kinds of chromatin exchange and remodeling, and suggests that replication-independent nucleosome assembly and disassembly is not an exchange reaction. This appears to be a general property of replacement H3 histones, because alfalfa replacement H3 also has a fast rate of turnover (Waterborg 1993). The rapid turnover of replacement H3 histones explains why all eukaryotes have constitutively expressed H3.3-type genes (Thatcher, 1994; Malik, 2003), because a continuous supply will be required to replenish active chromatin. Perhaps the purpose of specific histone degradation is to prevent exchange between chromatin sites. Chromatin-bound H3 and H4 are heavily modified, and it is suggested that H3/H4 subparticles are degraded to prevent moving modified histones to new sites. In contrast, the relative paucity of modifications on the H2A and H2B histones means that their exchange between sites has no detrimental effect (Schwartz, 2005).

Two other instances of histone degradation have been well-characterized. Chromatin-bound centromeric histones in budding yeast (CSE4) are subject to proteasome-mediated degradation that may limit this histone variant to centromeres (Collins 200). A separate system that degrades excess soluble histones has also been described (Gunjan, 2003). The current experiments do not distinguish whether H3.3 is degraded as it is displaced from chromatin or once it is soluble. Degradation of the histone may occur by a proteosome-catalyzed mechanism, or by a general response to damaged proteins. However, histone degradation in differentiated cells appears to discriminate between histone subtypes, because H2B-GFP is not similarly degraded. Since replication-independent histone deposition uses specialized chromatin assembly factors, the results raise the possibility that there are histone disassembly factors that specifically degrade H3.3. This could be tested with drugs that block known degradation pathways (Schwartz, 2005).

The H3 and H3.3 histones are extremely similar, differing by only four amino acid residues in Drosophila. Three of these specify whether the histone can be used for replication-coupled or RI nucleosome assembly, but these residues are not accessible in the complete nucleosome (Ahmad, 2002). This extreme similarity raises a paradox: What can be the function of replication-independent assembly, since new assembly results in a virtually identical nucleosome? In fact, the similarity between H3 and H3.3 distinguishes it from other histone variants like H2A.Z, which do introduce structural differences into the nucleosome. A possible resolution to this paradox comes from observations of the dynamics of RI assembly. Replication-coupled histone deposition only occurs once every cell cycle at a site, but RI deposition is a continual process at active genes. Thus, inactive chromatin retains the same histones for long periods of time, but active chromatin repeatedly shed its histones. Rapid turnover of histones at active genes has three implications for chromatin structure (Schwartz, 2005).

(1) The maintenance of any modification state in active chromatin will require ongoing activity of histone-modifying enzymes to modify newly arriving histones. Acetyl modifications do rapidly turn over in vivo, and can be removed enzymatically, but histone replacement will also contribute to turnover rates. Replacement may enhance the specificity of histone-modifying enzymes by continually removing modifications from active chromatin. Histone replacement also provides a mechanism to remove histone modifications that may not be metabolized. One example of this is the activation of heterochromatic gene arrays (Ahmad, 2002; Janicki 2004), where chromatin is marked by methylation at Lys 9 of the H3 tail (H3K9me). Activation of these gene arrays results in the coincident disappearance of H3K9me and deposition of H3.3, as if removal of the methylation mark occurs by histone replacement. In contrast, some histone modifications that are set during gene expression remain present after transcription and histone replacement ceases. Indeed, the retention of H3.3 at previously active chromatin may require that the variant maintain its extreme similarity with H3 so that they both can be repressed by the same modification systems (Schwartz, 2005).

(2) Continual histone replacement means that active regions have little time to 'mature' their chromatin between rounds of histone deposition. Nascent chromatin produced by replication-coupled assembly matures into a nuclease-resistant form within 1 h, and this delay is thought to reflect the rates for removing predeposition modifications, establishing new modifications, and condensing chromatin. If the rates for modification and condensation after RI assembly are similar to replicated chromatin, rapid histone turnover will continually reset chromatin to an 'immature' uncondensed state (Schwartz, 2005).

(3) High DNA accessibility is a characteristic feature of active chromatin, and this feature may simply result from ongoing histone replacement. This idea is supported by recent experiments in budding yeast demonstrating that the inducible accessibility of the PHO5 promoter is due to loss of nucleosomes. Earlier studies observed a slight decrease in the density of histone H4 at activated HSP70 genes, and concluded that transcription occurs without displacing histones from chromatin. However, a small decrease could result if displaced histones are rapidly replaced with new ones. Indeed, in budding yeast the density of histones appears to drop at very high transcription rates. Such transient structural instability of active chromatin would allow efficient polymerase elongation (Schwartz, 2005).

cDNA clone length - 772

Bases in 5' UTR - 97

Exons - 2

Bases in 3' UTR - 264

PROTEIN STRUCTURE

Amino Acids - 136

Structural Domains

The histone H3 family contains an evolutionary conserved variant, H3.3, that differs in sequence in only five amino acids from the canonical H3. There are four differences between Drosophila H3 and H3.3.

date revised: 6 August 2005

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