The long-term goal of the proposed research project is to characterize the factors and processes responsible for establishment of the mammalian epigenome, then decipher the mechanisms by which aberrations in epigenetic reprogramming which occur during preimplantation embryo development result in failed development or disease. Two key factors are associated with the epigenetic control of gene expression 1) DNA methylation and 2) histone modifications. Shortly after fertilization mammalian embryos undergo genome-wide epigenetic reprogramming by demethylation followed later by de novo remethylation. Aberrant epigenetic reprogramming has been clearly linked to failed embryo development and associated with serious human diseases. From the standpoint of human reproductive health and human disease, understanding the factors and mechanisms which control epigenetic reprogramming during early mammalian development is of critical importance. The objective of the proposed research is to generate scientific information that can be used to address these concerns. We propose to use a bovine model to pursue the following aims.
Specific Aim 1 is to characterize the expression and activity of DNA methyltransferases (DNMTs), histone methyltransferases (HMTs) and their associated genes during mammalian oocyte maturation and pre-implantation development. Expression of genes coding for these enzymes in addition to protein quantity, localization and trafficking will be analyzed in bovine ova and embryos.
Specific Aim 2 is to silence the expression of DNMTs and HMTs during bovine preimplantation development, then determine the effect on epigenetic reprogramming and embryo/fetal development. For study 1, RNA interference (RNAi) will be used transiently "knock down" expression of DNMTs and HMTs at the 1-cell stage of development. The percentage of embryos developing in vitro and embryo quality will be assessed. Global methylation in addition to methylation of specific target genes will also be analyzed. Microarray analysis and quantitative real-time PCR will be utilized to identify alterations in gene expression resulting from RNAi induced gene silencing. Immunocytochemistry will be employed to assess changes in protein. In a second study, siRNAs designed to silence the expression of DNMTs, HMTs and/or associated genes will be injected into one-cell bovine embryos which wil then be cultured to the blastocyst stage. These will be transferred into recipient females. Pregnancy rates will be compared between treatments and the normalcy of fetal development monitored by ultrasound. Conceptuses will be removed at 60 days of gestation to perform an extensive morphological and molecular analysis of placental and fetal tissues using methods similar to those described for study 1. Information generated in the proposed studies will be useful for generating guidelines and strategies to improve methods for assisted reproduction in humans, prevention of reproductive failure in addition to the prevention of human disease.

Public Health Relevance

The long-term goal of the proposed research project is to characterize the factors and processes responsible for establishment of the mammalian epigenome, then decipher the mechanisms by which aberrations in epigenetic reprogramming which occur during embryo development result in failed development.
The specific aims of this project involve the identification of key genes involved with epigenetic reprogramming during early mammalian development then employing RNAi based techniques to silence the expression of these genes and analyze the effects on embryo/fetal development.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
5R01HD058969-04
Application #
8435308
Study Section
Cellular, Molecular and Integrative Reproduction Study Section (CMIR)
Program Officer
Ravindranath, Neelakanta
Project Start
2010-04-15
Project End
2015-02-28
Budget Start
2013-03-01
Budget End
2014-02-28
Support Year
4
Fiscal Year
2013
Total Cost
$266,935
Indirect Cost
$84,727
Name
Texas Agrilife Research
Department
Veterinary Sciences
Type
Schools of Veterinary Medicine
DUNS #
847205713
City
College Station
State
TX
Country
United States
Zip Code
77843
Long, Charles R; Westhusin, Mark E; Golding, Michael C (2014) Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer. Mol Reprod Dev 81:183-93
Carnahan, Mindy N; Veazey, Kylee J; Muller, Daria et al. (2013) Identification of cell-specific patterns of reference gene stability in quantitative reverse-transcriptase polymerase chain reaction studies of embryonic, placental and neural stem models of prenatal ethanol exposure. Alcohol 47:109-20
Veazey, Kylee J; Muller, Daria; Golding, Michael C (2013) Prenatal alcohol exposure and cellular differentiation: a role for Polycomb and Trithorax group proteins in FAS phenotypes? Alcohol Res 35:77-85
Tessanne, K; Golding, M C; Long, C R et al. (2012) Production of transgenic calves expressing an shRNA targeting myostatin. Mol Reprod Dev 79:176-85
Golding, Michael C; Williamson, Gayle L; Stroud, Todd K et al. (2011) Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development. Mol Reprod Dev 78:306-17