DNA methylation patterns are reprogrammed in primordial germ cells and in preimplantation embryos by demethylation and subsequent de novo methylation. It has been suggested that epigenetic reprogramming may be necessary for the embryonic genome to return to a pluripotent state. We have carried out a genome-wide promoter analysis of DNA methylation in mouse embryonic stem (ES) cells, embryonic germ (EG) cells, sperm, trophoblast stem (TS) cells, and primary embryonic fibroblasts (pMEFs). Global clustering analysis shows that methylation patterns of ES cells, EG cells, and sperm are surprisingly similar, suggesting that while the sperm is a highly specialized cell type, its promoter epigenome is already largely reprogrammed and resembles a pluripotent state. Comparisons between pluripotent tissues and pMEFs reveal that a number of pluripotency related genes, including Nanog, Lefty1 and Tdgf1, as well as the nucleosome remodeller Smarcd1, are hypomethylated in stem cells and hypermethylated in differentiated cells. Differences in promoter methylation are associated with significant differences in transcription levels in more than 60% of genes analysed. Our comparative approach to promoter methylation thus identifies gene candidates for the regulation of pluripotency and epigenetic reprogramming. While the sperm genome is, overall, similarly methylated to that of ES and EG cells, there are some key exceptions, including Nanog and Lefty1, that are highly methylated in sperm. Nanog promoter methylation is erased by active and passive demethylation after fertilisation before expression commences in the morula. In ES cells the normally active Nanog promoter is silenced when targeted by de novo methylation. Our study suggests that reprogramming of promoter methylation is one of the key determinants of the epigenetic regulation of pluripotency genes. Epigenetic reprogramming in the germline prior to fertilisation and the reprogramming of key pluripotency genes in the early embryo is thus crucial for transmission of pluripotency.

Malformations of the septum, outflow tract and aortic arch are the most common congenital cardiovascular defects and occur in mice lacking Cited2, a transcriptional coactivator of TFAP2. Here we show that Cited2 -/-mice also develop laterality defects, including right isomerism, abnormal cardiac looping and hyposplenia, which are suppressed on a mixed genetic background. Cited2 -/-mice lack expression of the Nodal target genes Pitx2c, Nodal and Ebaf in the left lateral plate mesoderm, where they are required for establishing laterality and cardiovascular development. CITED2 and TFAP2 were detected at the Pitx2c promoter in embryonic hearts, and they activate Pitx2c transcription in transient transfection assays. We propose that an abnormal Nodal-Pitx2c pathway represents a unifying mechanism for the cardiovascular malformations observed in Cited2 -/-mice, and that such malformations may be the sole manifestation of a laterality defect.Genetic, developmental and molecular studies over the past decade have identified a number of DNA-binding transcription factors that have key roles in cardiac morphogenesis and in the pathogenesis of common congenital heart defects 1 . The role of transcriptional coactivators, molecules that connect DNA-binding transcription factors to the core transcriptional machinery, in cardiac development has only recently become apparent. These coactivators are exemplified by the paralogous genes EP300 and CREBBP 2 . Mutations in CREBBP cause Rubinstein-Taybi Syndrome 3 and are frequently associated with cardiac malformations 4 . EP300 and CREBBP interact with high affinity with a ubiquitously expressed cytokine and hypoxia-inducible transcriptional coactivator called CITED2 (also called p35srj and Mrg1) [5][6][7][8] . Binding of CITED2 to EP300 competitively inhibits the binding of the transcription factor HIF1A to EP300, blocking hypoxia-activated gene transcription 5,8 . Cited2 is essential for normal development of the heart, adrenals and nervous system 9-13 and for fibroblast proliferation 14 . Mice lacking Cited2 die prenatally with diverse cardiovascular malformations, including atrial and ventricular septal defects, double-outlet right ventricle, common arterial trunk and aberrant aortic arches.In addition to functioning as a transcriptional repressor of HIF1A, CITED2 also physically interacts with and coactivates TFAP2 (transcription factor AP2, also called Tcfap2) and LIM-domain containing transcription factors by linking them to EP300 and CREBBP 9,15,16 . Mutations in Tcfap2a and TFAP2B (Char syndrome) result in cardiac and aortic arch malformations 17,18 , suggesting that coactivation of TFAP2 by CITED2, EP300 and CREBBP is necessary for the normal development of these structures 9 . An alternative explanation for the development of cardiac malformations in mice lacking Cited2 is dysregulation of hypoxia-activated gene transcription 12 .The cardiovascular malformations resulting from deficiency of Cited2 encompass a diverse and variable spectrum that is not explained by effects on...

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