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The roles of Dnmt1 cytosine methyltransferase proteins in genomic reprogramming duringmouse preimplantation development.

Ratnam, Sarayu (2004) The roles of Dnmt1 cytosine methyltransferase proteins in genomic reprogramming duringmouse preimplantation development. Doctoral Dissertation, University of Pittsburgh. (Unpublished)

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Abstract

Inheritance of DNA methylation on imprinted genes depends on the Dnmt1 (cytosine-5-)methyltransferase protein. Methylation patterns on imprinted genes are maintained by oocytespecificDnmt1o isoform at the 8-cell stage of preimplantation development. Methylationpatterns in postimplantation embryos are maintained by the Dnmt1s isoform. To determine ifDnmt1s can functionally replace Dnmt1o, we expressed Dnmt1s in oocytes and discovered thatDnmt1s can maintain genomic imprints in the absence of Dnmt1o. However, the ability ofDnmt1s to maintain imprinting is dependant on the level of oocyte Dnmt1s. Though Dnmt1s andDnmt1o have equivalent maintenance methyltransferase functions in oocytes, the unstable natureof oocyte Dnmt1s, in comparison to oocyte Dnmt1o, leads to levels lower than what are requiredto maintain methylation at the 8-cell stage. We also determined that in cloned embryos, Dnmt1oundergoes none of its expected trafficking to 8-cell stage nuclei. Instead, these embryos exhibita mosaic pattern of Dnmt1s expression. Defects in intracellular trafficking of Dnmt1o andmisexpression of Dnmt1s, along with the intrinsic instability of Dnmt1s, might contribute toivaberrant DNA methylation in cloned embryos, thus raising concerns about the use of currentcloning technologies for therapeutic cloning.Molecular mechanisms involved in the formation of ovarian teratomas were also analyzed.Unfertilized oocytes arrest at the MII stage of meiotic maturation. After fertilization, oocytescontinue into cell division. Premature activation of MII oocytes without fertilization, can lead toovarian teratoma formation. To better understand mechanisms governing the prevention ofspontaneous oocyte activation, we investigated the molecular defects leading to formation ofovarian teratomas in the Tgkd mouse model. Tgkd is a transgene insertional mutation that leadsto reduced levels of the Inpp4b protein in MII oocytes of hemizygous Tgkd females. WildtypeGV oocytes have less Inpp4b protein than MII oocytes, and a significant decrease in Inpp4b isalso seen after fertilization. Also, the dependence of ovarian teratoma formation on the mousestrain, emphasizes the role of a strain-specific modifier on chromosome 6, possibly IP31. Thus,it is possible that oocyte Inpp4b normally suppresses spontaneous MII oocyte activation,possibly by reducing levels of IP3, an intermediate in the oocyte activation mechanism, thatoccurs following fertilization.


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Details

Item Type: University of Pittsburgh ETD
Status: Unpublished
Creators/Authors:
CreatorsEmailPitt UsernameORCID
Ratnam, Sarayusarst58@pitt.eduSARST58
ETD Committee:
TitleMemberEmail AddressPitt UsernameORCID
Committee ChairChaillet, John Richardchaillet@pitt.eduCHAILLET
Committee CoChairFerrell, Robertrferrell@hgen.pitt.eduRFERRELL
Committee MemberSchmidt, Martinmcs2@mgb.pitt.edu
Committee MemberSurti, Urvashiusurti@mail.magee.edu
Date: 3 May 2004
Date Type: Completion
Defense Date: 1 April 2004
Approval Date: 3 May 2004
Submission Date: 22 April 2004
Access Restriction: No restriction; Release the ETD for access worldwide immediately.
Institution: University of Pittsburgh
Schools and Programs: Graduate School of Public Health > Human Genetics
Degree: PhD - Doctor of Philosophy
Thesis Type: Doctoral Dissertation
Refereed: Yes
Uncontrolled Keywords: cloning; Dnmt1; maintenance methylation; ovarian teratomas
Other ID: http://etd.library.pitt.edu/ETD/available/etd-04222004-135832/, etd-04222004-135832
Date Deposited: 10 Nov 2011 19:40
Last Modified: 15 Nov 2016 13:41
URI: http://d-scholarship.pitt.edu/id/eprint/7476

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