In human lymphoid cells lines, it has been predicted that the expression of only 2% of cellular genes is modulated in response to histone acetylation (62
). The expression of some genes, like c-myc
, is down-regulated by TSA or TPX, whereas GAPDH (glyceraldehyde-3-phosphate dehydrogenase) is not affected by any of these chemicals. In contrast, human immunodeficiency virus (HIV) expression is induced following histone hyperacetylation (55
). Induction of HIV type 1 expression is associated in part with the disruption of a specific nucleosome positioned at the transcriptional start in the viral promoter in response to TSA or TPX (62
). Transcriptional silencing of other viruses like Epstein-Barr virus, human cytomegalovirus, and Kaposi's sarcoma-associated herpesvirus also appears to involve deacetylation mechanisms (11
In the present report, we examined the role of deacetylase inhibitors (TSA and TPX) in BLV expression in vitro and in ex vivo-isolated sheep and cattle PBMCs. We show that inhibition of HDACs induced BLV expression as observed for a minority (2%) of cellular genes. In pools of D17 cell lines, treatment with TSA resulted in an increase in BLV promoter activity when the LTR reporter was stably integrated in the chromosomal DNA. Since these in vitro observations might be devoid of biological relevance, we next extended our assays in vivo using two different species susceptible to BLV infection and pathogenesis. We have demonstrated that, in cattle and sheep, inhibition of deacetylation caused a marked activation of BLV expression. This induction of expression was not due to a decrease in the levels of apoptosis of the B lymphocytes. Enhancement of viral expression induced by TSA was correlated with a slight increase in the percentage of apoptotic B lymphocytes, as is frequently reported for other cell types (14
). Incubation with PMA-ionomycin, however, almost completely impeded cell death in sheep PBMCs cultures. In this case, increase in viral expression could simply be the result of enhanced cell viability. It should be mentioned that TSA also activates viral expression directly in BLV-infected lymphocytes without apparent cooperation of T cells in the cultures (Fig. ).
Histone acetylation results from an equilibrium between opposing activities of HAT and HDAC. Previous reports have shown that increase in HIV expression in response to TSA correlates with enhancement in the level of histone acetylation (55
). Hence, the induction of BLV expression by TSA reported here could be due to an increase in histone acetylation. In mammals, several coactivators harbor an intrinsic HAT activity: CBP and p300 and associated cofactors (e.g., PCAF, p/CIP-ACTR, and SRC-1). Coactivators bearing HAT activity are recruited to cis
-regulatory regions by interaction with transcription factors, and they can enhance expression of the corresponding genes by acetylating nearby nucleosomes (24
). These coactivators thus act as molecular links between transcription factors and the basal transcriptional machinery. A number of studies have shown that CBP interacts with CREB and NF-κB (12
). CREB and NF-κB could thus target HATs to the BLV promoter since these transcriptional factors specifically bind to the LTR. Furthermore, the implication of CBP and PCAF in the process of transcriptional activation by human T-cell leukemia virus (HTLV) Tax has been previously described (21
). We therefore speculate that a similar mechanism could also mediate BLV Tax transactivation. In contrast to acetyltransferases, the role of deacetylases in BLV or HTLV transcription has, however, not been described.
Deacetylases interact with sequence-specific DNA-binding transcriptional repressors, like Mad/Max, and inhibit transcription by deacetylating chromatin (38
). Factors Sin3 and N-CoR/SMRT appear to establish a link between deacetylases and the Mad/Max complex, which recognize specifically the regulatory E-box sequence (CANNTG) (4
). Interestingly, the BLV promoter possesses three E-box motifs located within the three TxREs enhancers. We therefore hypothesized that these E-box motifs could be involved in TSA response by recruiting HDACs to the LTR since our results demonstrate that deacetylase inhibitors activate BLV expression. Supporting this model, we have previously demonstrated that, in transient transfection experiments, mutation of all three E-boxes located in the U3 region provokes a slight but reproducible increase in LTR basal activity, suggesting the repressor role of the E-box element (48
). However, our data show that a mutation (in boldface type) within the E-box sequences (CANNTG
) did not, at least under our experimental conditions, alleviate the activation of the BLV promoter by TSA either in D17 stable cell lines (Table ) or in sheep PBMCs (Table ). Nevertheless, in other cell lines, mutations in the E-box motifs have been shown to decrease the TSA induction of the BLV promoter (C. Calomme and C. Van Lint, unpublished data), suggesting that TSA might have different effects depending on the cell types. Furthermore, mutation in the other transcription factor binding sites (IRF, GRE, NF1, NF2, CRE3x, and Δ21-pb) did not seem to impede the induction of BLV expression by TSA. Most importantly, this could be clearly demonstrated for the Ebox3x, Δ21-pb, and NF1 mutants in the context of primary lymphocytes isolated from sheep (Fig. ).
Histones are not the sole natural substrates for acetylation, since HATs are able to efficiently acetylate a growing list of cellular proteins. Indeed, CBP and PCAF can acetylate p53 (25
) as well as general transcription factors such as TFIIF and TFIIEβ (29
). Additionally, CBP, PCAF, and human GCN5 are also able to directly acetylate the HIV type 1 transactivator protein, Tat, leading to an increase of its transcriptional activity (13
). It is thus possible that the increase in BLV expression occurring ex vivo in response to TSA was in part a consequence of Tax acetylation. However, it should be stressed that our in vitro data demonstrate an activating effect of TSA in cell lines containing an integrated LTR in the absence of BLV proteins (Fig. ). TSA could thus induce BLV expression through acetylation of histones and/or nonhistone proteins such as Tax and other cellular factors. In this regard, we have demonstrated in a detailed separate study that TSA synergistically enhanced Tax-mediated transcriptional activation of the BLV promoter (in several cell lines), suggesting that Tax could be functionally regulated by posttranslational acetylation (T. L.-A. Nguyen and C. Van Lint, unpublished results).
In summary, the results presented here demonstrate that inhibition of deacetylation plays an important role in the regulation of BLV transcription. Deacetylation mechanisms could be implicated in the silencing strategy adopted by BLV to escape from the host immune response. In this context, induction of viral expression by TSA or others deacetylase inhibitors (such as valproic acid) could open novel prospects for therapeutical approaches not only for BLV but, more importantly, for the related HTLV type 1 virus.