Abstract
The human placenta is a highly specialized organ that is responsible for the survival of pregnancy. During its development, placental trophoblast cells invade into the uterine wall to establish a blood supply for the growing fetus. Previous studies have suggested similarities between the invasive phenotypes of trophoblasts and tumour cells; however, a key difference is that trophoblast invasion is under strict control. Given that epigenetic mechanisms have been linked with the silencing of key regulatory genes in cancer, we hypothesized that the epigenetic regulation of first-trimester placental trophoblasts may provide a mechanistic relationship between placental and cancer growth. Further, although the hypomethylated environment within the pseudo-malignant placenta is unique, its role in facilitating placental function is poorly understood. We sought to document placental-specific epigenetic modifications, taking into account that the origin of the placenta is determined during the earliest stages of embryonic development, when the inner-cell mass is first distinguished from the trophectoderm, and when the inner-cell mass further differentiates into the primitive endoderm and the epiblast.
A genome-wide methylation analysis was performed using methylated DNA immunoprecipitation (MeDIP) combined with hybridisation to promoter microarrays to identify differentially methylated gene promoters between first-trimester human placenta and peripheral blood DNA. The promoter methylation of 29 candidate genes was then quantified using Sequenom MassARRAY®. Differential methylation patterns were detected in placental tissues compared to both fetal and adult somatic tissues. The relationship between promoter methylation and gene expression was then assessed using real-time PCR and immunohistochemistry.
The promoter methylation of one gene, KCNH5, was found to be lineage-specific: low in all tissues derived from the extra-embryonic lineages (trophectoderm and primitive endoderm) and very high in tissues derived from the embryonic (epiblast) lineage. The dichotomous promoter methylation of KCNH5 was found to regulate the lineage-specific expression of alternative gene transcripts. Interestingly, the KCNH5 promoter that is used in tissues derived from the extra-embryonic lineages, and which shows dichotomous methylation, has recently evolved from a SINE retrotransposon that is present in only humans, old world monkeys and apes. To our knowledge, this the first example of a human transcript derived from the insertion of a SINE element. Finally, the lineage origin of the extra-embryonic mesenchyme has been a topic of longstanding debate. The combined epigenetic and expression profiles of KCNH5 in placental villous stroma provide compelling evidence that the extra-embryonic mesenchyme is derived from the primitive endoderm.
Retrotransposons are normally silenced by methylation to prevent genome dysfunction. However, the placenta is becoming increasingly known as a tissue in which retrotransposons are actively transcribed. We observed that the absence of retrotransposon-silencing by methylation permitted the emergence of a placental-specific transcript by allowing the retrotransposon to serve as an alternative promoter for KCNH5. Examination of additional retrotransposon-derived genes in the placenta (INSL4 and ERVWE1) confirmed that dichotomous methylation between embryonic and extra-embryonic lineages is a feature of early development. The finding that the retro-elements in these genes have escaped the normal silencing mechanism suggests that they may have functional roles that are unique to the invasive placentas of humans and recent primates.