Methylation of the adenomatous polyposis coli (APC) gene in human placenta and hypermethylation in choriocarcinoma cells.

Methylation of the human APC gene promoter is associated with several different types of cancers and has also been documented in some pre-cancerous tissues. We have examined the methylation of APC gene promoters in human placenta and choriocarcinoma cells. This revealed a general hypomethylation of the APC-1b promoter and a pattern with monoallelic methylation of the APC-1a promoter in full term placental tissue. However, there was no evidence of a parent-of-origin effect, suggesting random post zygotic origin of methylation. Increased methylation of this promoter was observed in all choriocarcinoma-derived trophoblast cell lines, suggesting a trophoblastic origin of placental APC methylation and implicating APC hypermethylation in the development of this group of gestational tumours. Our demonstration of placental methylation of the APC-1a promoter represents the first observation of monoallelic methylation of this gene in early development, and provides further support for a role of canonical Wnt signalling in placental trophoblast invasiveness. This also implicates tumour suppressor gene silencing as an integral part of normal human placental development.

Centromere protein b-null mice display decreasing reproductive performance through successive generations of breeding due to diminishing endometrial glands.

Centromere protein B is a highly conserved constitutive protein found at centromeres. Gene knockout studies in mice have unexpectedly identified Cenpb as a candidate gene involved in uterine function. The present study further explores the role of Cenpb in mice by intermating Cenpb-null mice over several generations. Breeding studies and analysis of uterine tissue indicate that Cenpb-null mice lose reproductive fitness over a number of generations due to a significant reduction in endometrial glands. These results suggest that Cenpb may play an important function in the short- and long-term maintenance of uterine integrity.

A novel chromatin immunoprecipitation and array (CIA) analysis identifies a 460-kb CENP-A-binding neocentromere DNA.

Centromere protein A (CENP-A) is an essential histone H3-related protein that constitutes the specialized chromatin of an active centromere. It has been suggested that this protein plays a key role in the epigenetic marking and transformation of noncentromeric genomic DNA into functional neocentromeres. Neocentromeres have been identified on more than two-thirds of the human chromosomes, presumably involving different noncentromeric DNA sequences, but it is unclear whether some generalized sequence properties account for these neocentromeric sites. Using a novel method combining chromatin immunoprecipitation and genomic array hybridization, we have identified a 460-kb CENP-A-binding DNA domain of a neocentromere derived from the 20p12 region of an invdup (20p) human marker chromosome. Detailed sequence analysis indicates that this domain contains no centromeric alpha-satellite, classical satellites, or other known pericentric repetitive sequence motifs. Putative gene loci are detected, suggesting that their presence does not preclude neocentromere formation. The sequence is not significantly different from surrounding non-CENP-A-binding DNA in terms of the prevalence of various interspersed repeats and binding sites for DNA-interacting proteins (Topoisomerase II and High-Mobility-Group protein I). Notable variations include a higher AT content similar to that seen in human alpha-satellite DNA and a reduced prevalence of long terminal repeats (LTRs), short interspersed repeats (SINEs), and Alus. The significance of these features in neocentromerization is discussed.

Uterine dysfunction and genetic modifiers in centromere protein B-deficient mice.

Centromere protein B (CENP-B) binds constitutively to mammalian centromere repeat DNA and is highly conserved between humans and mouse. Cenpb null mice appear normal but have lower body and testis weights. We demonstrate here that testis-weight reduction is seen in male null mice generated on three different genetic backgrounds (denoted R1, W9.5, and C57), whereas body-weight reduction is dependent on the genetic background as well as the gender of the animals. In addition, Cenpb null females show 31%, 33%, and 44% reduced uterine weights on the R1, W9.5, and C57 backgrounds, respectively. Production of "revertant" mice lacking the targeted frameshift mutation but not the other components of the targeting construct corrected these differences, indicating that the observed phenotype is attributable to Cenpb gene disruption rather than a neighbouring gene effect induced by the targeting construct. The R1 and W9.5 Cenpb null females are reproductively competent but show age-dependent reproductive deterioration leading to a complete breakdown at or before 9 months of age. Reproductive dysfunction is much more severe in the C57 background as Cenpb null females are totally incompetent or are capable of producing no more than one litter. These results implicate a further genetic modifier effect on female reproductive performance. Histology of the uterus reveals normal myometrium and endometrium but grossly disrupted luminal and glandular epithelium. Tissue in situ hybridization demonstrates high Cenpb expression in the uterine epithelium of wild-type animals. This study details the first significant phenotype of Cenpb gene disruption and suggests an important role of Cenpb in uterine morphogenesis and function that may have direct implications for human reproductive pathology.

Expression and localization of thioredoxin during early implantation in the marmoset monkey.

Thioredoxin is a powerful redox protein expressed in invasive cytotrophoblasts and essential for blastocyst implantation in mice. Isolated marmoset thioredoxin cDNA showed that the deduced amino acid sequence differed from the human sequence by four amino acids. The close homology of thioredoxin in the two species enabled us to use monoclonal antibodies against human thioredoxin to detect marmoset thioredoxin in implantation sites, blastocysts and culture medium. Immunocytochemistry on marmoset implantation sites, on pregnancy days 12 and 15, showed that thioredoxin is highly expressed in uterine luminal epithelium, glands and in some endometrial stromal cells. In attached blastocysts, thioredoxin staining was detected in mural and polar trophoblast cells and both visceral and parietal endoderm, whereas no staining was present in the inner cell mass. A similar pattern of thioredoxin expression was detected in hatched blastocysts attached to Matrigel in tissue culture. Trophoblastic vesicles derived from blastocysts expressed thioredoxin in inner endoderm-like cells and outer trophoblast-like cells and secreted thioredoxin into the culture medium. These experiments have demonstrated thioredoxin expression during early stages of embryo-maternal interaction. We propose that thioredoxin protects the early placenta from oxidative damage and that the marmoset is a valuable model for studying thioredoxin regulation and function during implantation and blastocyst differentiation.