Functional Analysis of the Embryonic Epigenome in a Non-rodent Model

Summary

Principal Investigator: Mark E Westhusin
Abstract: DESCRIPTION (provided by applicant): 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.
Funding Period: 2010-04-15 - 2015-02-28
more information: NIH RePORT

Top Publications

  1. pmc Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development
    Michael C Golding
    Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, TX 77843 4466, USA
    Mol Reprod Dev 78:306-17. 2011
  2. pmc Production of transgenic calves expressing an shRNA targeting myostatin
    K Tessanne
    Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A and M University, College Station, Texas, USA
    Mol Reprod Dev 79:176-85. 2012
  3. doi 3 OXIDATIVE STRESS INDUCED CHANGES IN EPIGENETIC MODIFYING GENE mRNA IN PRE-IMPLANTATION IN VITRO BOVINE EMBRYOS
    C A Burroughs
    Texas A and M University, College Station, TX, USA
    Reprod Fertil Dev 25:149. 2012
  4. pmc 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
    Mindy N Carnahan
    College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, TX 77843, USA
    Alcohol 47:109-20. 2013
  5. pmc Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer
    Charles R Long
    Department of Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, Texas
    Mol Reprod Dev 81:183-93. 2014
  6. doi 122 suppression of epigenetic modifiers alters the bovine embryonic developmental program during in vitro culture
    M D Snyder
    Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, TX, USA
    Reprod Fertil Dev 26:175. 2013
  7. pmc Prenatal alcohol exposure and cellular differentiation: a role for Polycomb and Trithorax group proteins in FAS phenotypes?
    Kylee J Veazey
    Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, Texas, USA
    Alcohol Res 35:77-85. 2013

Research Grants

  1. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013

Detail Information

Publications7

  1. pmc Examination of DNA methyltransferase expression in cloned embryos reveals an essential role for Dnmt1 in bovine development
    Michael C Golding
    Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, TX 77843 4466, USA
    Mol Reprod Dev 78:306-17. 2011
    ..Our results indicate an essential role for Dnmt1 during bovine preimplantation development, and suggest proper transcriptional reprogramming of this gene family in SCNT embryos...
  2. pmc Production of transgenic calves expressing an shRNA targeting myostatin
    K Tessanne
    Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine, Texas A and M University, College Station, Texas, USA
    Mol Reprod Dev 79:176-85. 2012
    ..This is the first report of transgenic livestock produced by direct injection of a recombinant lentivirus, and expressing transgenes encoding shRNAs targeting an endogenous gene (myostatin) for silencing...
  3. doi 3 OXIDATIVE STRESS INDUCED CHANGES IN EPIGENETIC MODIFYING GENE mRNA IN PRE-IMPLANTATION IN VITRO BOVINE EMBRYOS
    C A Burroughs
    Texas A and M University, College Station, TX, USA
    Reprod Fertil Dev 25:149. 2012
    ..These data suggest that embryos surviving oxidative stress may exhibit epigenetic changes that could influence subsequent development...
  4. pmc 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
    Mindy N Carnahan
    College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, TX 77843, USA
    Alcohol 47:109-20. 2013
    ..Data presented here will aid in the design of future experiments using stem cells to study the transcriptional processes driving differentiation, and model the developmental impact of teratogens...
  5. pmc Reshaping the transcriptional frontier: epigenetics and somatic cell nuclear transfer
    Charles R Long
    Department of Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, Texas
    Mol Reprod Dev 81:183-93. 2014
    ..Interestingly, while resetting somatic transcription and associated epigenetic marks are absolutely required for development of SCNT embryos, life does not demand perfection...
  6. doi 122 suppression of epigenetic modifiers alters the bovine embryonic developmental program during in vitro culture
    M D Snyder
    Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, TX, USA
    Reprod Fertil Dev 26:175. 2013
    ..Ongoing experiments analysing DNA methylation and histone modifications through immunocytochemistry and global gene expression via RT-qPCR will further explore the establishment and maintenance of these genes in the embryonic epigenome. ..
  7. pmc Prenatal alcohol exposure and cellular differentiation: a role for Polycomb and Trithorax group proteins in FAS phenotypes?
    Kylee J Veazey
    Department of Veterinary Physiology and Pharmacology, College of Veterinary Medicine and Biomedical Sciences, Texas A and M University, College Station, Texas, USA
    Alcohol Res 35:77-85. 2013
    ..Characterizing the involvement of Polycomb and Trithorax group complexes in the etiology of fetal alcohol spectrum disorders will undoubtedly enhance understanding of the role that epigenetic programming plays in this complex disorder...

Research Grants30

  1. CENTER FOR GASTROINTESTINAL BIOLOGY AND DISEASE
    Robert S Sandler; Fiscal Year: 2013
    ..Through all of its activities, the Center improves communication, promotes collaboration, develops careers and generally enriches the intellectual climate for digestive disease research. ..