X CHROMOSOME INACTIVATION AND DNA METHYLATION

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Epigenetic phenomena, which are heritable changes that do not irreversibly alter DNA base sequence, are increasingly becoming recognized as important for normal mammalian development, disease, and aging. Recent studies have firmly established that both in mouse and man some genes function differently depending on whether they come from the mother or father; by definition this is due to parental imprinting. The best characterized epigenetic mechanism in mammals is DNA modification by the formation of 5-methylcytosine. X chromosome inactivation (XCI) has long been known to display parental imprinting, stable somatic inheritance of chromatin activity states, and DNA methylation changes. Using XCI and imprinted genes as our experimental system, we will try to better understand the molecular mechanisms underlying epigenetic mechanisms. More specifically we propose to: (i) Improve and study application of Terminal transferase-Dependent PCR (TDPCR), a new procedure, to the analysis of DNA and RNA; (ii) continue investigation of a dynamic, stochastic model for DNA methylation and quantitatively determine methylation parameters such as the in vivo rate of de novo methylation and demethylation at several selected CpG sites. An assay based on TDPCR is expected to facilitate accomplishment of these studies; (iii) determine nuclease accessibility differences between active and inactive X-linked and imprinted genes using a novel LMPCR/SNuPE approach; and (iv) use mass spectrometry for the identification and characterization of DNA binding proteins and apply this methodology to the study of proteins involved in epigenetic mechanisms. X chromosome inactivation and parental imprinting play a role in several genetic diseases, including the major form of heritable mental retardation (fragile X syndrome) and some familial tumors. DNA methylation is becoming increasingly recognized as a significant factor for many cancers.