Identification of a developmentally stable domain of paternal-specific methylation upstream of the imprinted murine H19 gene / Kimberly D. Tremblay.

Format:

Book

Manuscript

Microformat

Thesis/Dissertation

Author/Creator:

Tremblay, Kimberly D.

Contributor:

Bartolomei, Marisa S., advisor.

University of Pennsylvania.

Language:

English

Subjects (All):

Penn dissertations--Biology.

Biology--Penn dissertations.

Biology.

Academic Dissertations as Topic.

Medical Subjects:

Biology.

Academic Dissertations as Topic.

Local Subjects:

Penn dissertations--Biology.

Biology--Penn dissertations.

Physical Description:

xi, 176 pages : illustrations ; 29 cm

Production:

1998.

Summary:

Genomic imprinting is defined as the differential expression of the maternally and paternally inherited alleles of a gene based upon their parental origin. This unusual expression pattern requires that the two alleles be distinguished or marked. A candidate for the imprinting mark is DNA methylation, which occurs at CpG dinucleotides in mammals. In support of this hypothesis, the maternally expressed H19 gene is hypermethylated in sperm and on the inactive paternal allele in somatic tissues, but is hypomethylated on the maternal allele in somatic tissues. However, to serve as the mark which designates the imprint, this differential methylation must also be present in the gametes and maintained during all stages of development, particularly during preimplantation development when a genome-wide demethylation event occurs. Using a sensitive PCR/SSCP assay, I demonstrate that several CpG dinucleotides, found within methylation-sensitive restriction sites, are unmethylated in oocytes and paternally methylated during preimplantation development. Furthermore, all of these CpGs are located within a 2.0 kb region 2.0 kb upstream from the H19 transcription start site. Using bisulfite mutagenesis coupled with genomic sequencing to investigate this region further in midgestation embryos, I show that 52 CpGs, comprising 1.9 kb, are strikingly differentially methylated during midgestation development. Moreover, at least 12 of the cytosine residues in this domain are exclusively methylated on the paternal allele in blastocysts. In contrast, CpG dinucleotides within a 350 bp promoter proximal region are less differentially methylated during midgestation and, like most of the genome, largely devoid of methylation on both alleles in blastocysts. I also demonstrate the exclusive expression of the maternal H19 allele in the embryos that exhibit paternal-specific methylation of the upstream 1.9 kb domain. Because this methylation pattern is set up in the gametes, maintained during preimplantation development, and preserved during midgestation as well as in adult tissues, I propose that the 1.9 kb differentially methylated domain is important for imprinted expression and serves as the mark which distinguishes the two alleles of H19.

Notes:

Supervisor: Marisa S. Bartolomei.

Thesis (Ph.D. in Biology) -- University of Pennsylvania, 1998.

Includes bibliographical references.

Local Notes:

University Microfilms order no.: 98-30001.

OCLC:

187471006