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Tissue specific differentially methylated regions (TDMRs) were identified and localized in the mouse genome using second generation virtual RLGS (vRLGS). Sequenom MassARRAY quantitative methylation analysis was used to confirm and determine the fine structure of tissue specific differences in DNA methylation. TDMRs have a broad distribution of locations to intragenic and intergenic regions including both CpG islands, and non-CpG islands regions. Somewhat surprising, there is a strong bias for TDMR location in non-promoter intragenic regions. Although some TDMRs are within or close to repeat sequences, overall they are less frequently associated with repetitive elements than expected from a random distribution. Many TDMRs are methylated at early developmental stages, but unmethylated later, suggesting active or passive demethylation, or expansions of populations of cells with unmethylated TDMRs. This is notable during postnatal testis differentiation where many testis-specific TDMRs become progressively “demethylated”. These results suggest that methylation changes during development are dynamic, involve demethylation and methylation, and may occur at late stages of embryonic development or even postnatally.

BackgroundChanges in DNA methylation in the mammalian genome during development are frequent events and play major roles regulating gene expression and other developmental processes. It is necessary to identify these events so that we may understand how these changes affect normal development and how aberrant changes may impact disease.ResultsIn this study Methylated DNA ImmunoPrecipitation (MeDIP) was used in conjunction with a NimbleGen promoter plus CpG island (CpGi) array to identify Tissue and Developmental Stage specific Differentially Methylated DNA Regions (T-DMRs and DS-DMRs) on a genome-wide basis. Four tissues (brain, heart, liver, and testis) from C57BL/6J mice were analyzed at three developmental stages (15 day embryo, E15; new born, NB; 12 week adult, AD). Almost 5,000 adult T-DMRs and 10,000 DS-DMRs were identified. Surprisingly, almost all DS-DMRs were tissue specific (i.e. methylated in at least one tissue and unmethylated in one or more tissues). In addition our results indicate that many DS-DMRs are methylated at early development stages (E15 and NB) but are unmethylated in adult. There is a very strong bias for testis specific methylation in non-CpGi promoter regions (94%). Although the majority of T-DMRs and DS-DMRs tended to be in non-CpGi promoter regions, a relatively large number were also located in CpGi in promoter, intragenic and intergenic regions (>15% of the 15,979 CpGi on the array).ConclusionsOur data suggests the vast majority of unique sequence DNA methylation has tissue specificity, that demethylation has a prominent role in tissue differentiation, and that DNA methylation has regulatory roles in alternative promoter selection and in non-promoter regions. Overall, our studies indicate changes in DNA methylation during development are a dynamic, widespread, and tissue-specific process involving both DNA methylation and demethylation.

All living cells rely on intermediary metabolism to maintain an adequate state of energetics. Considerable progress has been made over the last century in defining many of the pathways and regulatory mechanisms of intermediary metabolism. Following from this mechanistic understanding, much insight has been gained into how organisms modulate the metabolism of their various cell types to achieve energy homeostasis during different physiologic states and developmental stages. In mammals, the prenatal period is less well characterized in terms of energy metabolism, principally due to technical difficulties associated with the small size of and limited accessibility to samples. The initial stages of prenatal development, when there is rapid growth and striking morphologic changes, stand out as a particularly important period for investigating energy metabolism. Furthermore, the increasing utilization of in vitro fertilization techniques for human and domestic animal reproduction mandates a better understanding of early embryonic metabolism to improve culture conditions. This review will provide an overview of the current understanding of intermediary metabolism associated with energy production in the early murine embryo, the most fully characterized of mammalian embryos. Several approaches that have been used to investigate embryonic metabolism will be considered, with a special emphasis being given to recently introduced mutations affecting pathways implicated in energy homeostasis of the early embryo. Substrate Utilization during Early EmbryogenesisSince the first reports of successful culture of rabbit embryos almost a century ago, much effort has been devoted to defining the substrates required for mammalian embryo viability and then characterizing how these substrates are utilized. Before considering studies investigating the metabolism of substrates in vitro, it is important to appreciate what is known about the substrates that are available to the embryo in vivo. The embryo begins life with an endowment of maternally provided substrates that are carried over from the oocyte. Unlike a number of vertebrate and invertebrate species, oocytes from many mammalian species, including the mouse, contain relatively limited amounts of glycogen or lipids (1). Mammalian oocytes do, however, have substantial stores of amino acids and protein. It has been estimated that the endogenous protein store within the one-cell mouse embryo could meet all of its energetic needs for the first several days of development (2). The observation that the murine embryo loses ϳ25% of its total protein content during the first 2-3 days of development indicates that endogenous protein is catabolized during early embryogenesis (3). Beyond the first several days of development, little is known about the relative amounts of endogenous substrates present in the embryo, and no studies have determined how these endogenous substrates are utilized.Studies of the female reproductive tract fluid in the mouse have identified a number of substrates that c...