A significant volume of work has focused on identifying estrogen receptor binding sites using computational genomics (37
), promoter microarrays (26
), CpG island libraries (40
), ChIP cloning (25
) and ChIP-on-chip methods (24
). Although progesterone has important and complex effects on the female reproductive tract, comparatively little is known about genome-wide PR-binding sites. Using ChIP-cloning technology and progesterone-responsive primary leiomyoma cells, we identified 18 novel PR-binding sites, which corresponded to 20 genes proximal to these sites. Specifically, we showed that RU486 enhanced recruitment of PR, its coactivators SRC-1 and SRC-2, as well as key general transcription factors RNA PolII and Sp1, to both distal PR-binding region and basal promoter of the KLF11 gene and induced its expression. We also found that KLF11 is a robust suppressor of leiomyoma growth.
We did not identify any PR-binding sites within a basal promoter. Five percent of PR-binding sequences were located in far distal 5′-regions, 5% were in 3′-regions, and 80% of binding sites lay within an intron of a gene. Considering that introns comprise approximately 40% of the genome, this suggests a higher affinity of PR to coding regions of its target genes. This is consistent with our previous findings indicating that 46% of ERα binding sites were located within an intron of a gene in breast cancer cells (25
None of the reported PR target genes was pulled out in this study (14
). Some unavoidable limitations of the current ChIP cloning strategy such as, the affinity of the antibody used to pull down chromatin and the on-and-off interaction pattern of transcription factors with DNA may cause the incomplete cloning of all target genes (25
). The low endogenous PR levels in cultured LSM cells may also account for the low number of PR-binding sites cloned in this study (43
Regulation of half of the genes located proximately to these distant PR-binding sites by progesterone or RU486 suggested that these sequences are functionally active. PR-binding fragments located distal to genes or within introns might function through long-range interactions that involve looping of chromatin to bring the regulatory elements within the proximity of basal promoters.
Both proximal and distal regulatory regions of the KLF11 gene recruited not only PR and Sp1, but also RNA PolII upon RU486 treatment. Various models have been proposed to explain how a distal enhancer communicates with a proximal promoter: the looping, the tracking, and the linking models (44
). With regard to the distal-proximal communication of the KLF11 gene, our data support a looping model. We plan to use a chromosome conformation capture assay to investigate whether the KLF11 distal region and proximal promoter physically interact in intact cells.
RU486 stimulated the basal promoter activity but not the enhancer function of the distal region. RU486 also enhanced PR binding to the basal promoter but not to the distal region. The distal region, however, exhibits significant basal enhancer function, basal PR binding activity and RU486-induced SRC-2 recruitment, which is PR-dependent (). Taken together, these findings are suggestive that the distal PR binding site’s major function is the stabilization of the RU486/PR-dependent enhancer complex at the KLF11 promoter. It appears that an interaction between the basal promoter and the distal PR binding region would be necessary for RU486-dependent regulation of KLF11 expression.
We found that, at least for the target genes studied here, RU486 was a much more robust regulator compared with progesterone. Knockdown of PR or treatment with RU486 significantly increased KLF11 expression, whereas the native progesterone hormone minimally suppressed its expression. The synthetic progesterone agonist R5020, however, markedly suppressed KLF11 level ( and ). The difference between the effects of progesterone and R5020 may be due to possible rapid metabolization of progesterone in these cells.
Consistent with the notion that coactivators play major roles in determining PR activity (46
), we found that, through interacting with PR, RU486 enhanced the binding of SRC-1 and SRC-2 to the basal promoter and the distal PR-binding region, which gave rise to gene induction. RU486 function as an inducer of gene transcription is not without precedent. Shatnawi et al demonstrated that RU486 can stimulate expression of genes encoding folate receptor type α, p21 and p27 in a specific promoter context that has G/C-rich Sp1 binding elements but no classical PRE sites (22
). In line with this observation, we found that both distal PR-binding region and the basal promoter of the KLF11 gene, with multiple G/C-rich Sp1 binding sites in both regions, could recruit PR. Additionally, our data suggest that the biological activity of PR liganded by mixed antagonist/agonist compounds is complex and may depend on the promoter context and the availability of coregulators.
KLF11 is a transcription factor that serves as a downstream effector of TGFβ-dependent signaling. It is down-regulated in human cancers, inhibits cell growth in vitro
and in vivo
, and inhibits neoplastic transformation (34
). Consistent with these reports, we found that KLF11 expression was significantly down-regulated in leiomyoma tissues compared with adjacent matched myometrial tissues. Moreover, KLF11 inhibited LSM cell growth. While the mechanism involved in KLF11-regulated cell proliferation needs to be further clarified, our studies provide an initial insight into the mechanism by which progesterone or RU486 regulates leiomyoma growth and development. Given that progesterone exerts a number of essential functions in inhibition of estrogen-dependent endometrial carcinogenesis and promotion of breast cancer development, it is tempting to explore the regulation and function of KLF11 in these malignancies (47
In summary, using an unbiased approach, we uncovered KLF11 as a novel PR target responsive to progesterone antagonist RU486 in LSM cells. KLF11 mediated the anti-proliferative effects of RU486 in leiomyoma cells. These studies reveal a potential mechanism underlying the modulation of progesterone- and antiprogestin-regulated differentiation and proliferation in LSM cells. The PR-dependent down-regulation of KLF11 expression in LSM cells may represent a missing link in steroid signaling pathways that explains the pathogenesis of uterine leiomyoma. Furthermore, the identification of direct targets of KLF11 will be important to elucidate its role in uterine leiomyoma biology.