The general problem proposed for investigation is the analysis of mammalian dosage compensation. In particular the project will be concerned with the molecular analysis of the X-inactivation mode of dosage compensation used in humans, mice and a number of other mammalian forms. Chromosomal dosage control is critical to normal growth and development, and X-inactivation brings about the essential equivalence of X-chromosome dosage in the somatic cells of females and males. No euploid fetus or individual with two active X-chromosomes has been observed, suggesting that X-chromosome dosage imbalance is lethal. There are three stages in the X-inactivation process: X-inactivation in the extra embryonic lineage at blastocyst formation, X-inactivation in the embryonic lineage during the very late blastocyst stage and reactivation of the inactive X-chromosome in the germ cell lineage near the time of oogenesis. At the molecular level three sequential steps must be considered: initiation of inactivation at a single site or control center, spreading of inactivation along the chromosome, and maintenance of the inactivated state throughout the cells' somatic history. Through the use of various X-linked probes, in situ nick translation and high voltage electron microscopy of sex chromatin, we will investigate these different levels of developmental control of X-inactivation. Initial studies will concentrate on differences between the active and inactive X-chromosomes regarding specific X-linked genes, primarily the phosphoglycerate kinase (PGK) and the replication enhancer plasmid genes. X-linked cDNA and genomic probes will be used to look for chromatin conformation and DNA modification differences between the active and inactive X-chromosomes. Materials to be studied will include cell cultures varying in X-chromosome composition and murine tissues including extra-embryonic and embryonic yolk sac tissues. Chromatin configuration will be investigated using nucleases and DNA modification will be studied with restriction enzyme and sequence analyses. In situ nick translation and high voltage electron microscopy will be used to study sex chromatin and a possible nuclear envelope attachment site of this structure.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Method to Extend Research in Time (MERIT) Award (R37)
Project #
5R37HD016659-11
Application #
3485109
Study Section
Mammalian Genetics Study Section (MGN)
Project Start
1982-09-01
Project End
1995-11-30
Budget Start
1992-12-01
Budget End
1993-11-30
Support Year
11
Fiscal Year
1993
Total Cost
Indirect Cost
Name
University of Washington
Department
Type
Schools of Arts and Sciences
DUNS #
135646524
City
Seattle
State
WA
Country
United States
Zip Code
98195
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Gartler, Stanley M; Varadarajan, Kartik R; Luo, Ping et al. (2006) Abnormal X: autosome ratio, but normal X chromosome inactivation in human triploid cultures. BMC Genet 7:41
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Miner, Brooks E; Stoger, Reinhard J; Burden, Alice F et al. (2004) Molecular barcodes detect redundancy and contamination in hairpin-bisulfite PCR. Nucleic Acids Res 32:e135
Laird, Charles D; Pleasant, Nicole D; Clark, Aaron D et al. (2004) Hairpin-bisulfite PCR: assessing epigenetic methylation patterns on complementary strands of individual DNA molecules. Proc Natl Acad Sci U S A 101:204-9
Gartler, Stanley M; Varadarajan, Kartik R; Luo, Ping et al. (2004) Normal histone modifications on the inactive X chromosome in ICF and Rett syndrome cells: implications for methyl-CpG binding proteins. BMC Biol 2:21
Hansen, R Scott (2003) X inactivation-specific methylation of LINE-1 elements by DNMT3B: implications for the Lyon repeat hypothesis. Hum Mol Genet 12:2559-67
Gartler, S M; Hansen, R S (2002) ICF syndrome cells as a model system for studying X chromosome inactivation. Cytogenet Genome Res 99:25-9
Matarazzo, Maria Rosaria; De Bonis, Maria Luigia; Gregory, Richard I et al. (2002) Allelic inactivation of the pseudoautosomal gene SYBL1 is controlled by epigenetic mechanisms common to the X and Y chromosomes. Hum Mol Genet 11:3191-8
Hassan, K M; Norwood, T; Gimelli, G et al. (2001) Satellite 2 methylation patterns in normal and ICF syndrome cells and association of hypomethylation with advanced replication. Hum Genet 109:452-62

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