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.

Agency
National Institute of Health (NIH)
Institute
National Institute of General Medical Sciences (NIGMS)
Type
Research Project (R01)
Project #
5R01GM050575-13
Application #
6476540
Study Section
Mammalian Genetics Study Section (MGN)
Program Officer
Carter, Anthony D
Project Start
1988-07-01
Project End
2003-11-30
Budget Start
2001-12-01
Budget End
2003-11-30
Support Year
13
Fiscal Year
2002
Total Cost
$451,653
Indirect Cost
Name
City of Hope/Beckman Research Institute
Department
Type
DUNS #
City
Duarte
State
CA
Country
United States
Zip Code
91010
Chen, Hsiu-Hua; Lebon, Jeanne; Riggs, Arthur D (2008) Analysis of RNA structure and RNA-protein interactions in mammalian cells by use of terminal transferase-dependent PCR. Methods Mol Biol 488:319-41
Chen, Zhao-Xia; Mann, Jeffrey R; Hsieh, Chih-Lin et al. (2005) Physical and functional interactions between the human DNMT3L protein and members of the de novo methyltransferase family. J Cell Biochem 95:902-17
Chen, Hsiu-Hua; Castanotto, Daniela; LeBon, Jeanne et al. (2004) In vivo detection of ribozyme cleavage products and RNA structure by use of terminal transferase-dependent PCR. Methods Mol Biol 252:109-24
Rodin, Sergei N; Riggs, Arthur D (2003) Epigenetic silencing may aid evolution by gene duplication. J Mol Evol 56:718-29
Shively, L; Chang, L; LeBon, J M et al. (2003) Real-time PCR assay for quantitative mismatch detection. Biotechniques 34:498-502, 504
Chong, Suyinn; Riggs, Arthur D; Bonifer, Constanze (2002) The chicken lysozyme chromatin domain contains a second, widely expressed gene. Nucleic Acids Res 30:463-7
Dai, S M; O'Connor, T R; Holmquist, G P et al. (2002) Ligation-mediated PCR: robotic liquid handling for DNA damage and repair. Biotechniques 33:1090-7
Chong, Suyinn; Kontaraki, Joanna; Bonifer, Constanze et al. (2002) A Functional chromatin domain does not resist X chromosome inactivation: silencing of cLys correlates with methylation of a dual promoter-replication origin. Mol Cell Biol 22:4667-76
Riggs, A D (2002) X chromosome inactivation, differentiation, and DNA methylation revisited, with a tribute to Susumu Ohno. Cytogenet Genome Res 99:17-24
Komura , J; Ikehata, H; Hosoi, Y et al. (2001) Mapping psoralen cross-links at the nucleotide level in mammalian cells: suppression of cross-linking at transcription factor- or nucleosome-binding sites. Biochemistry 40:4096-105

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