DNA methylation in mammals is involved in several essential processes including X chromosome inactivation, genomic imprinting, and host defense against mobile genetic elements. How methylation is targeted to specific sequences in the germ line and how specific methylation patterns are maintained during development is not fully understood. Genomic methylation is established in the gamete, decreases dramatically during preimplantation development, and is re-established after implantation of the blastocyst. However, the methylation present at imprinted loci is specifically maintained during preimplantation development. Imprinted genes are located in clusters, and within each gene cluster parent-of-origin specific expression is governed by an imprinting center (IC). The ICs of the maternally imprinted murine Snrpn, Kcnq1, and Igf2r gene clusters coincide with their differentially methylated domains (DMDs). We have shown that specific DMD sequences are required to establish differential methylation at an imprinted locus. Hybrid transgenes were generated using a non-imprinted derivative of the imprinted RSVIgmyc mouse transgene, Ig/myc, and sequences from endogenous imprinted gene DMDs. Addition of specific DMD sequences to the Ig/myc transgene restored its imprinting. Only the tandem repeats found within the Snrpn, Kcnq1, and Igf2r DMDs were capable of establishing maternal-specific transgene methylation. These experiments have also shown that the methylation on imprinted gene DMD sequences is specifically maintained during the early stages of preimplantation development. These results clearly demonstrate the importance of tandem repeats in the process of genomic imprinting. DNA methylation is also critical for silencing intracisternal A particle (IAP) transposition within the genome. It is thought that maintenance of IAP element methylation during preimplantation is critical to repress IAP element transcription and transposition. The methylation of IAP element long terminal repeat (LTR) sequences was analyzed at the blastocyst stage of preimplantation development using the bisulfite genomic sequencing technique. These experiments have shown that methylation is maintained on the majority of IAP elements at the blastocyst stage of preimplantation development. However, the methylation on specific IAP elements is completely lost at this time, including the methylation of single IAP element LTRs.
| Identifer | oai:union.ndltd.org:PITT/oai:PITTETD:etd-08212003-145414 |
| Date | 17 November 2003 |
| Creators | Reinhart, Bonnie Lynn |
| Contributors | Deborah Chapman, Karen Arndt, Gregg Homanics, Jeffrey Hildebrand, J. Richard Chaillet |
| Publisher | University of Pittsburgh |
| Source Sets | University of Pittsburgh |
| Language | English |
| Detected Language | English |
| Type | text |
| Format | application/pdf |
| Source | http://etd.library.pitt.edu/ETD/available/etd-08212003-145414/ |
| Rights | unrestricted, I hereby certify that, if appropriate, I have obtained and attached hereto a written permission statement from the owner(s) of each third party copyrighted matter to be included in my thesis, dissertation, or project report, allowing distribution as specified below. I certify that the version I submitted is the same as that approved by my advisory committee. I hereby grant to University of Pittsburgh or its agents the non-exclusive license to archive and make accessible, under the conditions specified below, my thesis, dissertation, or project report in whole or in part in all forms of media, now or hereafter known. I retain all other ownership rights to the copyright of the thesis, dissertation or project report. I also retain the right to use in future works (such as articles or books) all or part of this thesis, dissertation, or project report. |
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