Recent evidence suggests that DNA is differentially methylated in uterine leiomyoma versus adjacent normal myometrial tissue; however, these findings are predominantly reported in small studies and analysis of individual candidate genes such as ESR1, which has been shown to be hypomethylated in leiomyomas 
. We particularly paid attention to the ESR1 gene, but we did not observe any differential DNA methylation patterns between leiomyoma and myometrium. Hypomethylation of ESR1 in leiomyoma was reported using a group of Japanese subjects; thus the difference between our findings and theirs could be attributed to racial differences. Similar racial differences have also been reported for the aromatase mRNA levels and promoter usage in uterine leiomyomas 
More recently published reports have attempted to demonstrate differential DNA methylation in leiomyomas; one study examined differences across the X chromosome in a single subject supporting the concept of epigenetic regulation in uterine leiomyoma. However, the other study was insufficient to identify differences in DNA methylation, which could be due to the small number of samples investigated 
. Here, we report the first genome-wide analysis of differential DNA-methylation and mRNA expression in uterine leiomyoma and adjacent normal myometrial tissues from 18 matched pairs, all from African American subjects to limit biological heterogeneity, and avoid epigenetic variations among ethnic groups. The following real-time RT-PCR validation of mRNA expression and bisulfite sequencing validation of DNA methylation of these genes were performed on both a subset of the original African American samples and additional new samples from Caucasian subjects. Additionally, in vitro
cultured experiments utilized primary cells from both ethnic groups. We have not observed any apparent differences with respect to the 3 studied genes between samples from African- and Caucasian-American subjects suggesting that the findings may be applicable to both ethnic groups. This conclusion, however, should be taken with some caution due to the low number of Caucasian subjects. Further studies are needed to make a more definitive conclusion.
Our study confirms the link between epigenetic DNA modifications and gene expression in uterine leiomyomas, by demonstrating the effects of promoter DNA methylation on gene silencing, particularly in three tumor suppressors known to be involved in reproductive tumorigenesis. Though our work is the first to examine genome-wide analysis of DNA methylation in uterine leiomyoma and there are no existing data against which we can compare our results, our mRNA expression profiles are consistent with previously published reports 
In our genome-wide analysis, we observed that 1,031 transcriptional regulatory regions were differentially methylated and only 525 mRNA species were transcriptionally altered in uterine leiomyoma compared with myometrial tissue. This degree of mismatch between DNA methylation and steady-state mRNA levels was expected since changes in DNA methylation may not always lead to changes in steady-state mRNA levels for the following potential reasons. (i) DNA methylation alone may not be sufficient to alter mRNA expression, and other events such as changes in the structure of chromatin formed on a methylated template are needed to render it transcriptionally altered. (ii) The availability and binding capacity of specific transcription factors are needed to regulate the rate of mRNA transcription from a gene promoter. (iii) Finally, other factors that regulate the half-life of a certain transcript will determine its steady-state levels. Consequently, it is expected that changes in steady-state mRNA levels are regulated only partially by DNA methylation.
Although, we attempted to account for differences in the menstrual cycle, the majority of the samples included in our analysis (14/18) were obtained during the proliferative phase. Moreover, it is challenging to date the endometrium for cycle phase since many women with uterine leiomyomas have irregular cycles with prolonged bleeding. The small number of secretory phase samples did not permit us to compare biological differences as a function of the cycle phase. Since the correlation between differences in DNA methylation and gene expression was evaluated in paired samples from the same patient, the effect of cycle phase on this analysis was further minimized.
In this study, we noted a key epigenetic mechanism whereby increased promoter methylation leads to transcriptional suppression in uterine leiomyoma compared with matched normal myometrial tissues. The second predominant mechanism was hypomethylation associated with overexpression of genes indicating an overall inverse relationship between DNA methylation and gene expression in uterine leiomyoma. However, we also observed some genes to be hypermethylated and upregulated, and other genes to be hypomethylated and downregulated. The absence of an inverse relationship between promoter DNA methylation and mRNA expression in this minor group of genes is consistent with previously published data. For example, methylation of one particular CpG island in the NR5A1 gene is associated with transcriptional suppression, whereas methylation of another CpG island located 4 kb downstream is associated with overexpression of NR5A1 mRNA 
. It is conceivable that the effects of a single methylated CpG island on gene expression may be either gene-specific or location-specific within the same gene.
We verified the effects of promoter DNA methylation on transcriptional inhibition of three tumor suppressor genes namely, KLF11, DLEC1, and KRT19. KLF11 is a transcription factor and a member of the transforming growth factor beta (TGFβ) family, which is involved in key cellular functions such as apoptosis, proliferation, and differentiation 
. KLF11 is expressed in a number of human tissues, and it is repressed in several human cancers. It inhibits neoplastic transformation and cell growth both in vivo and in vitro
. We previously demonstrated the downregulation of KLF11 expression in uterine leiomyoma tissues compared with normal matched myometrial tissue 
. Although the mechanism involved in KLF11-regulated cell proliferation is not fully understood, we demonstrated for the first time that KLF11 is epigenetically regulated by DNA methylation, with hypermethylation correlating with a repressed state in uterine leiomyoma. Recently, KLF11 was also shown to be aberrantly hypermethylated in myelodysplastic syndromes. It has been suggested that KLF11 inhibits gene expression through a Sin3a-HDAC interacting domain and recruitment of the corepressor mSin3a 
. We plan to investigate this mechanism further, and identify the DNMTs and DNA methyl binding proteins that are involved in silencing of KLF11.
DLEC1 is an epigenetically modified tumor suppressor gene 
. DLEC1 is localized in the cytoplasm ubiquitously expressed in all human tissues, and repressed in several human cancers. Hypermethylation of the DLEC1 promoter is associated with its transcriptional repression in a wide variety of malignant tumors originating from lung, esophagus, kidney, ovary, nasopharynx, and liver 
. The DLEC1 promoter region contains a CpG island in the first exon, and we demonstrated here that methylation of this CpG is responsible for the repression of DLEC1 expression in uterine leiomyoma. Our analysis revealed a strong association between silencing of DLEC1 expression and promoter hypermethylation in uterine leiomyoma; in addition, treatment of addition of cultured primary uterine leiomyoma smooth muscle cells with a DNMT inhibitor restored DLEC1 expression. The DLEC1 gene encodes a 166 kDa protein, whose biologic function remains unknown due to lack of homology to any known conserved proteins or domains 
. In the future, we plan to characterize the biological function of DLEC1 in uterine leiomyoma.
KRT19 is an intermediate filament protein responsible for the structural integrity of epithelial cells, this genes encodes a 40-kDa protein 
. In mammalian cells, keratin filaments are organized in a complex network spreading from the nucleus to the cytoplasmic membrane. KRT19 is also known as an epigenetically regulated tumor suppressor gene, which has frequently demonstrated promoter hypermethylation associated with transcriptional downregulation in several cancerous tumors such as neuroblastomas, squamous cell carcinoma of the head and neck region and renal cell carcinomas 
. Also, it is one of the most common used markers for real-time RT-PCR detection of tumor cells disseminated in lymph nodes, peripheral blood and bone marrow of breast cancer patients 
Using genome-wide analyses of DNA methylation in uterine leiomyoma we hope to define a specific epigenetic profile that could inform the development of diagnostic biomarkers for uterine leiomyoma as well as identify potential therapeutic targets. Because DNA methylation is reversible, epigenetic modifying drugs could be used in the medical management of uterine leiomyoma. Importantly, aberrant DNA methylation and other epigenetic abnormalities may represent a critical initial mechanism that triggers transformation of a single myometrial cell that will eventually give rise to a monoclonal leiomyoma tumor. Understanding the mechanism underlying the pathogenesis of uterine leiomyoma will be critical for developing new preventive and therapeutic approaches to the disease.