Chromatin Modification Requirements for 15-Lipoxygenase-1 Transcriptional Reactivation in Colon Cancer Cells*

15-Lipoxygenase-1 (15-LOX-1) contributes significantly to inflammation regulation and terminal cell differentiation. 15-LOX-1 is transcriptionally silenced in cancer cells, and its transcriptional reactivation (e.g. via histone deacetylase inhibitors (HDACIs)) is essential for restoring terminal cell differentiation to cancer cells. STAT-6 acetylation via the histone acetyltransferase KAT3B has been proposed to be necessary for 15-LOX-1 transcriptional activation. However, the exact mechanism underlying 15-LOX-1 transcriptional reactivation in cancer cells is still undefined, especially in regard to the contribution of 15-LOX-1 promoter histone modifications. We therefore examined the relative mechanistic contributions of 15-LOX-1 promoter histone modifications and STAT-6 to 15-LOX-1 transcriptional reactivation by HDACIs in colon cancer cells. We found that: 1) histone H3 and H4 acetylation in the 15-LOX-1 promoter through KAT3B was critical to 15-LOX-1 transcriptional activation; 2) 15-LOX-1 transcription was activated independently from STAT-6; and 3) dimethyl-histone H3 lysine 9 (H3K9me2) demethylation in the 15-LOX-1 promoter via the histone lysine demethylase KDM3A was an early and specific histone modification and was necessary for activation of transcription. These findings demonstrate that histone modification in the 15-LOX-1 promoter is important to 15-LOX-1 transcriptional silencing in colon cancer cells and that HDACIs can activate gene transcription via KDM3A demethylation of H3K9me2.


15-Lipoxygenase-1 (15-LOX-1) contributes significantly to inflammation regulation and terminal cell differentiation.
15-LOX-1 is transcriptionally silenced in cancer cells, and its transcriptional reactivation (e.g. via histone deacetylase inhibitors (HDACIs)) is essential for restoring terminal cell differentiation to cancer cells. STAT-6 acetylation via the histone acetyltransferase KAT3B has been proposed to be necessary for 15-LOX-1 transcriptional activation. However, the exact mechanism underlying 15-LOX-1 transcriptional reactivation in cancer cells is still undefined, especially in regard to the contribution of 15-LOX-1 promoter histone modifications. We therefore examined the relative mechanistic contributions of 15-LOX-1 promoter histone modifications and STAT-6 to 15-LOX-1 transcriptional reactivation by HDACIs in colon cancer cells. We found that: 1) histone H3 and H4 acetylation in the 15-LOX-1 promoter through KAT3B was critical to 15-LOX-1 transcriptional activation; 2) 15-LOX-1 transcription was activated independently from STAT-6; and 3) dimethyl-histone H3 lysine 9 (H3K9me2) demethylation in the 15-LOX-1 promoter via the histone lysine demethylase KDM3A was an early and specific histone modification and was necessary for activation of transcription. These findings demonstrate that histone modification in the 15-LOX-1 promoter is important to 15-LOX-1 transcriptional silencing in colon cancer cells and that HDACIs can activate gene transcription via KDM3A demethylation of H3K9me2.
To identify the crucial molecular mechanisms underlying 15-LOX-1 transcriptional reactivation in cancer cells, we examined the mechanisms by which HDACIs activate 15-LOX-1 transcription, given that HDACIs are the most potent class of agents known to induce this activation (23,28,32). In particular, we examined whether HDACIs activated 15-LOX-1 transcription via promoter histone modification or STAT-6, which has been proposed to be crucial to 15-LOX-1 transcriptional activation (33). In a series of experiments, we identified novel chromatin modification events in the and H4 in the 15-LOX-1 promoter. Caco-2 (A) and SW480 (B) cells were treated with depsipeptide (5 nM) or the vehicle solvent for depsipeptide (control). The cells were harvested 48 h after treatment and subjected to ChIP assays using specific antibodies against acetylated histone H3 (H3) or acetylated histone H4 (H4). ϩ and Ϫ indicate immunoprecipitation with and without H3 or H4 antibodies, respectively, and Input indicates total DNA before immunoprecipitation. Depsipeptide induced H3 and H4 acetylation in the 15-LOX-1 promoter. C, kinetics of effects of depsipeptide on H3 acetylation. Caco-2 cells were treated with depsipeptide (5 nM), harvested at the indicated times, and subjected to ChIP/real-time PCR assays using specific acetylated H3 antibodies. The
Cell Cultures and Treatments-HT29, SW480, HCT-116, RKO, and DLD-1 cells were grown in RPMI 1640 medium, and Caco-2 cells were grown in Eagle's minimal essential medium (Cambrex, Walkersville, MD) with l-glutamine in a humidified atmosphere containing 5% CO 2 at 37°C. Both types of medium contained 10% fetal bovine serum and were supplemented with 1% penicillinstreptomycin as described previously (21). Depsipeptide, a selective HDAC1 and HDAC2 inhibitor (37,38), and SAHA, a nonselective HDAC inhibitor (38), were dissolved in dimethyl sulfoxide and added to the culture media at the indicated concentrations.
RNA Extraction and Real-time PCR-Total RNA was extracted from cells using TRI reagent (Molecular Research Center Inc., Cincinnati, OH). RNA from each sample was reverse transcribed and then measured quantitatively by realtime PCR using a comparative C t method, as described previously (31).
Chromatin Immunoprecipitation (ChIP) and ChIP/Realtime PCR Assays-Cross-linking was performed by adding formaldehyde to the cell culture medium to a final concentration of 1% and incubating for 10 min at 37°C. ChIP assays were performed using a commercial kit according to the manufacturer's protocol (Upstate Cell Signaling Solutions). Chromatin was immunoprecipitated using the indicated antibodies. ChIP assays using an agarose gel electrophoresis method were performed similarly to what was described previously (31). For studying STAT-6 binding to the 15-LOX-1 promoter, we used primers that amplified a 182-bp fragment of the human 15-LOX-1 promoter that covers a specific STAT-6 binding site located 952 bp upstream of the 15-LOX-1 start codon (39): 5Ј-TAA TTC ACT CTG GTG GGG TGG-3Ј (sense) and 5Ј-CAG TTT CTT TTT GGG CTG GA-3Ј (antisense). The relative enrichment of transcription factors or histone modifications (e.g. KDM3A, H3K9me2) was measured using ChIP/real-time PCR with the following primers and FAM dyelabeling probe: forward, GTGTTTTCGGTCCAAATCCTTT-TCT; reverse, GAGAGCAGGGAGTGGAAACC; probe, CCTCCCGTCAAGATAGT to amplify the Ϫ280 bp to Ϫ212 bp region of the 15-LOX-1 promoter relative to the ATG site. Histone acetylation levels in the 15-LOX-1 promoter were measured by ChIP/real-time PCR with the following primers and FAM labeling probe: forward, GAGACCTGCCACCCA-CATT; reverse, GAGCTAAATAACCCAGCCTGAGA; probe, CCGACCCCAAGCGAC to amplify the Ϫ120 bp to Ϫ47 bp region of the 15-LOX-1 promoter relative to the ATG site. Realtime PCR was performed as described previously (32).
Statistical Analyses-We used the t test for two-group comparisons. For analyses involving single factors and more than two groups, we performed a one-way analysis of variance. If the overall analysis of variance test result was significant, we performed pairwise comparisons, adjusting for multiplicities using the Bonferroni correction. We analyzed data involving the simultaneous consideration of two factors using two-way analysis of variance. We first tested the interaction effect, and if it was statistically significant, we subsequently performed specific comparisons to investigate which differences were driving this effect, using the Bonferroni correction to adjust for the multiple testing problem. An individual comparison was considered significant only if the p value was less than 0.05/k, with k representing the number of comparisons performed. If the interaction effect was not significant, we tested the individual main effects. Then, if those were significant, we determined which pairwise comparisons were significant, again adjusting for multiplicities using the Bonferroni correction. We used the Pearson correlation method to correlate differences in the levels of the studied variables (e.g. 15-LOX-1 mRNA expression level, histone H3 acetylation in the 15-LOX-1 promoter) between depsipeptide-or SAHA-treated and control cells. All of the tests were two-sided and conducted at the p Յ 0.05 level. Quantitative data log transformation was used to account for the normal distributional assumptions underlying the methods. The data were analyzed using SAS software (SAS Institute, Cary, NC).
Recruitment of KDM3A to the 15-LOX-1 Promoter Is Required for 15-LOX-1 Transcriptional Activation-Depsipeptide significantly increased the recruitment of KDM3A, a demethylase of H3K9me2, to the 15-LOX-1 promoter in Caco-2 cells (Fig. 5A; p ϭ 0.004). KDM3A siRNA transfection into Caco-2 cells significantly reduced KDM3A recruitment to the 15-LOX-1 promoter with depsipeptide compared with the recruitment seen with nonspecific siRNA (Fig. 5B; p Ͻ 0.0001). The H3K9me2 level was significantly increased in KDM3A siRNA-transfected cells treated with depsipeptide, compared with the level in cells transfected with nonspecific siRNA and treated identically with depsipeptide ( Fig. 5C; p Ͻ 0.0001). KDM3A siRNA transfection reduced the 15-LOX-1 mRNA level in cells treated with depsipeptide compared with the level in cells transfected with nonspecific siRNA and treated with depsipeptide ( Fig. 5D; p Ͻ 0.0001).

DISCUSSION
Our new findings demonstrate that: 1)15-LOX-1 transcriptional activation in colon cancer cells requires specific histone modification events in the 15-LOX-1 promoter: H3K9me2 demethylation by KDM3A and histone H3 and H4 acetylation by KAT3B; and 2) HDACIs can activate transcription via KDM3A demethylation of H3K9me2.
Histone acetylation in the 15-LOX-1 promoter but not STAT-6 was necessary for 15-LOX-1 transcriptional activation. 15-LOX-1 transcriptional activation highly correlated with histone H3 acetylation as demonstrated by our testing in six colorectal cancer cell lines using both specific (depsipeptide (38)) and nonspecific (SAHA) HDACIs. By using KAT3B knockdown via siRNA to inhibit histone H3 and H4 acetylation, we confirmed that acetylation of histone H3 and H4 in the 15-LOX-1 promoter was necessary for 15-LOX-1 activation. The authors of a prior report proposed that KAT3B activates 15-LOX-1 transcription via STAT-6 acetylation (33). However, we found that 15-LOX-1 transcriptional activation required KAT3B acetylation of 15-LOX-1 promoter histones but not STAT-6. We found that STAT-6 down-regulation via siRNA, although successful in inhibiting the binding of STAT-6 to the 15-LOX-1 promoter, failed to influence depsipeptide activation of 15-LOX-1 transcription. These findings agree with those in a recent report showing that SAHA down-regulated STAT-6 expression (36), which suggested that STAT-6 does not contribute to transcriptional activation by HDACIs. STAT-6 has been reported to be necessary for IL-4 transcriptional activation of 12/15-LOX (the mouse homologue of 15-LOX-1) in mice (42) and for 15-LOX-1 transcription in human BEAS-2B transformed airway epithelial cells and A534 lung cancer cells (33,39). The fact that STAT-6 has been shown to be required for IL-4-induced but not HDACI-induced 15-LOX-1 transcriptional activation might reflect differences in the mechanisms of action between IL-4 and HDACIs. IL-4 binds to cell surface receptors and requires STAT-6 for signal transduction to influence nuclear events (43), whereas the HDACIs directly influence transcriptional events (38). In support of this notion, we found that depsipeptide activated 15-LOX-1 transcription much earlier than IL-4 did in A534 cells (supplemental Fig. S2).
Specific chromatin modification events in the 15-LOX-1 promoter activated 15-LOX-1 transcription. These events included very early removal of a transcriptional silencing histone modification, H3K9me2 (44,45), and an increase in H3K9ac, a transcriptional activation marker (46). The specificity of these histone changes is supported by the lack of changes in the other histone markers of transcriptional activation (e.g. H3K4me2 (44)) or transcriptional silencing (e.g. H3K9me1, H3K9me3, H3K27me2, H3K27me3, or H4K20me3 (44,47)) during 15-LOX-1 transcriptional activation. To our knowledge, the pattern we identified is the first specific chromatin modification pattern in the 15-LOX-1 promoter to have been identified, especially in regard to histone methylation. Previously published information on 15-LOX-1 promoter modifications is limited to nonspecific global H4 acetylation (28) and general histone H3 15-LOX-1 promoter acetylation (33).
We found that depsipeptide activated 15-LOX-1 transcription through demethylation of H3K9me2 by KDM3A, a recently identified specific H3K9me2 demethylase (48). Depsipeptide promoted KDM3A recruitment to the 15-LOX-1 promoter. Inhibition of this recruitment via KDM3A down-regulation by siRNA increased the H3K9me2 level in the 15-LOX-1 promoter and suppressed depsipeptide activation of 15-LOX-1 transcription. These findings indicate that depsipeptide recruitment of KDM3A to the 15-LOX-1 promoter and the subsequent H3K9me2 demethylation are necessary for activation of 15-LOX-1 transcription. Data are emerging to support the important role of histone demethylases in the transcriptional regulation of normal and pathologic molecular cellular events (49,50). The only prior report of a pharmaceutical intervention that enhanced the recruitment of KDM3A for transcriptional activation was a report on the hormonal androgen analogue R1881 (48). Our current results show that KDM3A demethylation of H3K9me2 appears not to be limited to transcriptional activation by hormonal agents but also to contribute to transcriptional activation by the HDACIs.
The strong correlation between the degree of 15-LOX-1 transcription activation in cancer cell lines and histone acetylation, although it supports the notion that histone deacetylation is necessary for maintaining 15-LOX-1 transcriptional silencing, still leaves open the possibility that other mechanisms might contribute to 15-LOX-1 transcriptional silencing in cancer cells. For example, we have recently found that the dissociation of DNA methyltransferase-1 from the 15-LOX-1 promoter can significantly influence 15-LOX-1 transcriptional activation in colon cancer cells (32). DNA methyltransferase-1 contributes to H3K9me2 formation during replication (51). Further studies are needed to evaluate the contribution of DNA methyltransferase-1 to H3K9me2 formation in the 15-LOX-1 promoter.
In summary, our findings demonstrate the important role of specific chromatin modification in the regulation of 15-LOX-1 transcription. These results provide a new model for the orchestrated chromatin modification events in the 15-LOX-1 promoter that are involved in 15-LOX-1 transcriptional activation: KDM3A recruitment to demethylate H3K9me2 and histone H3 and H4 acetylation via KAT3B. Further studies of these chromatin modification events in the 15-LOX-1 promoter will not only help to improve our understanding of the mechanism of 15-LOX-1 silencing in cancer cells but also help to identify novel molecular targets for inhibiting colonic tumorigenesis.