In this study, we examined the changes in histone variant distribution that accompany gene induction and attempted to determine the relationships between the exchange of H3 histones, changes in chromatin accessibility, and alterations in PTM patterns with transcriptional activation. Two genes with distinct expression patterns were examined, the CD69 gene, a poised gene that is rapidly induced following T-cell activation, and the heparanase gene, a gene that exhibits basal expression and more modest inducibility. The major finding of this study is that the transcriptional activation of these genes is accompanied by chromatin accessibility that occurs without nucleosome loss on inducible gene promoters. Instead, histone variant exchange takes place in the promoter regions with a loss of histone H3 and a gain of H3.3. In addition, a concomitant decline of H2A.Z levels accompanied gene induction. These events were most dramatic in the 5′-transcribed region of the CD69 gene. Our data lend support to a model whereby histone H2A.Z preferentially coexists with histone H3 on inducible gene promoters in the basal state, whereas it is more likely to be associated with histone variant H3.3 during the establishment of the transcriptionally active phase. Furthermore, H3.3 deposition appeared to be associated with enrichment of the histone PTM K9Ac and concomitant depletion of K4me2 and K9me2.
The CD69 gene represents a classic poised gene with docked Pol II on its promoter in the basal state, and this gene displays rapid induction following activation (16
). As with other inducible immune genes, such as the IL-2 gene, the CD69 gene exhibits low levels of chromatin accessibility prior to activation (41
). In contrast, the heparanase gene is expressed in resting T cells and can be further induced upon T-cell activation. Consistent with the basal expression of heparanase, the heparanase gene promoter exhibits considerable nuclease accessibility and Pol II occupancy in NS T cells. However, both Pol II occupancy and chromatin accessibility do increase further upon T-cell activation. The transcript induction kinetics and accessibility patterns of heparanase are similar to those of the granulocyte-macrophage colony-stimulating factor gene in T cells (20
). Thus, the CD69 and heparanase genes represent different classes of genes in terms of transcript induction kinetics and chromatin accessibility patterns.
Nevertheless, many of the chromatin changes that accompany the transcriptional induction of both of these genes are similar. While chromatin remodeling accompanies gene induction in both cases, these remodeling events are not accompanied by nucleosome loss. This is somewhat surprising, as other studies have demonstrated that nucleosome loss accompanies gene induction in T cells (7
). This difference may be because these studies demonstrating nucleosome loss were performed with murine cells and hence may reflect differences between the mouse and human system. Alternatively, it may be due to gene-specific differences in chromatin remodeling mechanisms. Nevertheless, this finding demonstrates that nucleosome loss is not required for chromatin remodeling to occur. Indeed, a recent genome-wide study with human CD4+
T cells demonstrated that transcriptional activation of other genes in T cells occurs without nucleosome loss, although this study did not investigate any changes in chromatin accessibility (45
The increase in the chromatin accessibility of both genes in activated T cells was accompanied not by nucleosome loss but rather by exchange of H3 histones. With a new H3.3-specific antibody, which we generated, it was found that H3.3 levels are absent or low on both genes in the basal state. However, the induction of both genes was accompanied by H3.3 deposition. Moreover, H3.3 was not restricted to the proximal promoter region, as extensive H3.3 deposition was observed both upstream of the promoter and in the transcribed region of the CD69 gene just downstream of the transcription initiation site. Similar distributions in H3.3 have been observed in genome-wide studies utilizing epitope-tagged H3.3 (32
). Our data thus further support the ideas that H3.3 deposition occurs upon transcription and that H3.3 is associated with transcriptionally active regions of the genome.
Chromatin remodeling and induction of both the heparanase and CD69 genes were also associated with the decline of H2A.Z, particularly in the 5′-transcribed region of the CD69 gene. In the resting state, H2A.Z enrichment near the TSS and its subsequent removal upon induction have been observed in other studies (3
). Our finding that H2A.Z-containing chromatin is present just downstream of the transcriptional start site on the poised inducible CD69 gene is analogous to the results of a recent genome-wide analysis of the location of H2A.Z-containing nucleosomes in Drosophila
). This latter study raised the possibility that this H2A.Z-containing nucleosome is involved in blocking the movement of the poised RNA Pol II complex. Our results support this notion because in response to an activation signal, the biochemical makeup of this downstream nucleosome within the CD69 gene is altered with the loss of H2A.Z. Hence, one could speculate that H2A.Z-containing nucleosomes on poised genes may block elongation by preventing the movement of RNA Pol II in resting T cells (13
The histone variant composition of a nucleosome was recently demonstrated to influence nucleosome stability. Specifically, H3.3-containing nucleosomes were shown to be less stable than their H3 counterparts, with H3.3/H2A.Z-containing nucleosomes being particularly sensitive to disruption (21
). Given that histone exchange accompanies chromatin accessibility across inducible genes in this study, it is therefore tempting to speculate that changes in nucleosome composition may impact upon chromatin accessibility. Thus, the extensive H3.3 deposition we observed upon gene induction will diminish nucleosome stability across the promoter and transcribed regions. Importantly, the H2A.Z present at the time of remodeling (4 h postactivation) on both gene promoters appeared to be predominantly associated with H3.3 rather than H3. Thus, the highly unstable H3.3/H2A.Z nucleosome also increases in frequency upon gene induction. However, while an increase in H3.3/H2A- and H3.3/H2A.Z-containing nucleosomes may facilitate nucleosome remodeling, this does not necessarily mean that H3.3 deposition is sufficient to trigger an increase in chromatin accessibility. While H3.3-containing nucleosomes may be more susceptible to remodeling, the recruitment of the appropriate ATP-dependent remodeling activities to the gene promoter is still likely to be required. Furthermore, it could be reasoned that H3.3 deposition in different gene regions plays different roles. While deposition of H3.3 onto promoters may facilitate an increase in chromatin accessibility, one could speculate that the enrichment of H3.3 in transcribed regions facilitates Pol II elongation.
Another way in which H3.3 deposition may promote chromatin accessibility is through carrying the K9Ac PTM onto its target regions. In fact, we demonstrated that the appearance of K9Ac over the CD69 gene strongly correlated with regions of H3.3 enrichment. This finding is in line with studies showing that K9Ac is strongly associated with H3.3 (18
) and represents the first in vivo association between H3.3 and K9Ac at the gene-specific level. As ATP-dependent chromatin remodeling enzymes, such as Brg1, contain an acetylated lysine binding bromodomain (34
), an increase in K9Ac may promote remodeler recruitment and subsequent accessibility increases. The observation that unincorporated H3.3 is enriched in K9Ac (30
) raises the possibility that H3.3 is “premodified” and carries K9Ac onto the regions of the CD69 gene upon deposition. Nevertheless, we cannot rule out the possibility that H3.3 is preferentially acetylated subsequent to incorporation into the CD69 region. Surprisingly, induction of the heparanase gene is actually accompanied by promoter deacetylation. While it could be argued that the more modest H3 loss and H3.3 deposition on the heparanase gene promoter make it more susceptible to deacetylase activity, our sequential ChIP assays indicate that the H3.3 deposited onto the heparanase gene promoter is also deacetylated. Therefore, opposing enzymatic activities can counterbalance the PTMs deposited on a promoter in association with H3.3. This also means that, despite a strong association (18
), H3.3 is not invariably marked with K9Ac. Deacetylation on the heparanase gene promoter clearly dampens transcriptional induction. As the transcript of the heparanase gene is highly stable, a large increase in transcriptional activity is not required for transcript accumulation to occur. Thus, heparanase gene promoter deacetylation upon T-cell activation may represent a biological mechanism for restraining gene induction.
We also found that H3.3 appeared depleted of the PTMs K9me2 and K4me2. The failure to detect any K9me2-modified H3.3 on either gene promoter at any time point was unexpected given previous data showing the presence of this PTM on H3.3 in human cell lines (30
). The observation that K9me2 is present on unincorporated H3.3 (30
) suggests that active demethylation may be occurring upon deposition onto these gene promoters. However, unincorporated H3.3 is depleted of K4me2 (30
), meaning that the loss of K4me2 that parallels initial H3.3 deposition onto the promoters of the CD69 and heparanase genes is not unexpected. The patterns of K4me2 at later time points do not conform to any clear pattern, indicating that whether or not H3.3 K4 dimethylation occurs subsequent to deposition is highly dependent on the gene region examined. Overall, we can conclude that PTM modulation by H3.3 deposition is only one of many factors governing the final pattern of histone PTMs adopted by a gene region. This is consistent with the observation that the PTM pattern associated with H3.3 changes markedly upon H3.3 deposition into chromatin (30
) and agrees with a recently proposed model in which chromatin environment controls histone variant PTM patterns (29
Importantly, histone variant exchange at gene promoters may have an unanticipated relevance to cancer biology. Heparanase is a degradative enzyme thought to play a key role in cancer cell invasion and metastasis (35
). The observation that a shift in histone variant usage across the heparanase gene promoter accompanies the acquisition of a metastatic phenotype in mammary carcinoma cells suggests that dysregulation of histone variant distribution may play a role in oncogenesis. This observation, coupled with our other data, further underscores the fundamental role that histone variants are likely to play in gene induction.