Previous studies from our laboratory (15
) and others (24
) have shown that Thy-1 silencing is correlated with CpG hypermethylation in its promoter region. In this study, we examined the hypothesis that Thy-1 gene silencing is mediated by histone modifications as well. Chromatin histone modifications and DNA methylation are important components in the epigenome that regulate gene expression and biological processes in somatic cells (22
). Increasing data suggest that DNA methylation and histone deacetylation are coordinately regulated to modify gene expression (26
). The hyperacetylation effect of HDACi would be predicted to result in a general increase in gene expression as the treatment opens up the chromatin structure. However, studies have shown that treatment with HDAC inhibitors, like TSA, only significantly altered 2 to 5% of the genes (27
). TSA can affect the methylation status of some genes, but not the others (20
In this report, we showed that the histone deacetylation inhibitor TSA can induce histone acetylation, restores the expression of the fibrosis suppressor gene Thy-1 associated with both changes in chromatin marks and demethylation of the Thy-1 promoter region, and may increase genomic stability by increasing global DNA methylation. These data suggest that histone modifications and DNA methylation are coordinately regulated to change the biological behavior of fibrotic lung fibroblasts. In epigenetic regulation, histone modifications and DNA methylation can work together to silence or activate a gene. Histone deacetylase inhibitors, as well as DNA methyltransferase inhibitors, can be used together to achieve maximal effects in restoring expression of some genes. In our model, Thy-1 reexpression in Thy-1(−) cells after treatment with TSA never reached the same levels as in Thy-1(+) cells even with the addition of the demethylation agent 5-aza-2′deoxycytidine (AZA) (see
Figure E1 in the online supplement). We previously showed (15
) that AZA can partially restore Thy-1 expression in fibrotic lung fibroblasts. The addition of TSA in the culture together with AZA appears to be additive but does not restore Thy-1 expression to levels seen in Thy-1(+) cells (Figure E1). There are likely to be other mechanisms, transcriptional or posttranscriptional, that suppress Thy-1 expression in Thy-1(−) fibroblasts. For example, our earlier studies (14
) indicated that shedding can be an important mechanism for posttranscriptional regulation of Thy-1 expression.
There is abundant evidence showing that histone acetylation and DNA methylation interact (28
). Some studies have shown that TSA had no effect on DNA methylation (30
). Other studies have shown that TSA induces global and gene-specific DNA demethylation in human cancer cells (32
). In the latter study, the authors noted that the demethylation effect of TSA is gene selective. In another report, TSA and the DNMT inhibitor 5-azaCdR restored the expression of lysozyme, a secretory protein mainly expressed in glandular epithelial cells, but TSA did not change its DNA methylation status (33
). The authors conclude that DNA methylation and histone acetylation are simultaneously and independently operative in their model. TSA has been reported to induce promoter region demethylation in Neurspora crassa
) and in mammalian cells (19
). Although the exact mechanisms are unclear, in addition to TSA, other HDAC inhibitors have been reported to have demethylation activity (34
). In human endometrial cells, TSA and butyrate can suppress DNMT3B expression by decreasing the stability of DNMT3B mRNA (35
). Another HDACi that has much stronger activity than that of TSA, depsipeptide, has been found to decrease the binding of DNMT1 to the promoter of genes that were silent but not to affect the expression of DNMT1 (34
). Although we did not measure the activity and expression of each of the specific DNA methyltransferases, our data indicated that the overall activity of total DNMTs in treated Thy-1(−) cells was changed.
In human cancer genomes, a hallmark alteration is global hypomethylation, resulting in genomic instability, and tumor suppressor gene hypermethylation, resulting in gene silencing (36
). IPF has been proposed as a neoproliferative disorder of the lung, based on the fundamental pathological characteristics of IPF and cancer (37
). Thy-1(−) fibroblasts are more profibrotic (17
) and thus may have similar alterations to some cancer cells. We suspect that the higher overall DNA methylation levels observed in Thy-1(+) cells correspond with a more physiologically stable phenotype and are consistent with the observed higher overall DNMT activity (). TSA appears to partially reverse the Thy-1(−) cells' total DNA methylation levels as well as the total DNMT activity (). The exact mechanism remains unclear; DNA methylation is a complex process and involves multiple molecular interactions. Taken together, this study and our previous study (15
) showed that the expression of Thy-1 can be restored through DNA demethylation of the promoter region by the DNMT inhibitor 5-aza-2′deoxycytidine or by the histone deacetylation inhibitor TSA.
Because TSA is a histone deacetylase inhibitor and not a demethylation agent, the demethylation effect shown in our model may be the indirect effect of chromatin remodeling, which may alter the susceptibility of DNA to methylation. We further investigated which chromatin modifications are associated with Thy-1 before and after treatment with TSA. clearly shows that acetylated H3 and H4 are significantly higher in the TSA-treated cells than in the untreated control cells. By ChIP assay, we observed that acetylated histone H4 (H4Ac) and trimethylated histone H3 Lys-4 (H3K4Me3) directly correlated and that trimethylated histone H3 Lys-27 (H3K27Me3) inversely correlated with Thy-1 expression and changed after treatment with TSA. We do not know the exact mechanisms of the changes associated with histone modifications after treatment with TSA. The mechanism of decreased Thy-1 association with H3K27Me3 and enriched association with H3K4 me3 in TSA-treated cells is not clear because the TSA can induce histone acetylation but not methylation. Reports have shown that modification at one site of the histone tail can influence modification of a second site (38
); thus, it is possible that acetylated H3 and H4 may affect the other forms of modification of histone H3 and H4. It is also possible that TSA can directly affect histone-related enzymes. A study in human lung cancer cells using depsipeptide indicates that depsipeptide can suppress the expression of methyltransferases of H3K9 (34
). We do not know if the dissociation H3K27 from Thy-1 in TSA-treated Thy-1(−) cells is a direct or indirect effect of TSA on H3K27 methyltransferases; this deserves further exploration. In another report (3
) focused on the DNA repair protein MGMT, its methylation-mediated silencing seems to be associated with decreased acetylation of H3 and H4. Their study indicates a multiprotein complex containing methylation binding proteins and histone methyltransferases interacting with the gene promoter region resulting in a closed chromatin structure, thus inactivating the gene. This may be similar to what we have observed regarding Thy-1 gene expression in lung fibroblasts. As with increased acetylation of H3 and H4 by TSA treatment (), Thy-1 expression was restored and was associated with a relaxed chromatin structure ().
Although it is known that histone deacetylation and cytosine methylation communicate with each other and cause transcriptional silencing, it is not certain which is the initiating event. DNA methylation may be the main marker for gene silencing that triggers the events resulting in a repressive chromatin state, or the loss of histone acetylation may be the initial event followed by changes in DNA methyltransferase/demethylase activities to induce local gene hypermethylation and cause the silencing of the gene (25
). A study using the histone deacetylase inhibitor valproic acid (VPA) (41
) examined DNA methylation changes in some specific genes and noted that, after treatment with VPA, certain CpG sites of some genes showed increased methylation, whereas other CpG sites were demethylated. Together with our own data demonstrating methylation changes in a specific gene and global DNA methylation, this may suggest that HDACi are able to reprogram the gene epigenetic makeup through histone acetylation. It would be very useful to identify the specific HDAC involved in Thy-1 expression. However, TSA is a broad HDACi that inhibits class I and II HDACs. The possible specific HDAC(s) that are involved in the regulation of Thy-1 expression can be explored in the future by methods such as ChIP when the specific antibodies become available.
Finally, this study demonstrates that epigenetic modification using TSA results in reprogramming of at least one element of the profibrotic phenotype, the expression of α-SMA (). This result may be mediated via direct effects on the α-SMA promoter, as previous studies showed that TSA can affect α-SMA expression (44
), or this could be the result of effects on transcription of multiple profibrotic genes, though we have previously demonstrated that restoration of Thy-1 expression alone is sufficient to reverse the myofibroblastic phenotype of lung fibroblasts (17
Thy-1 has been proposed as a “fibrosis suppressor” gene (14
). This report showing that a HDACi can restore Thy-1 expression in fibrotic fibroblasts and change the phenotype of the cells suggests the possibility that drugs in this category could be used in fibrotic lung diseases such as IPF. Because other drugs in this family are in use for treatment of other diseases, this approach could offer a very exciting alternative for the treatment of this deadly disease.