During pregnancy three generations of DNA co-exist. Mum's, baby's and the germ line of baby. During the first and second trimester the majority of methylated cytosines from the DNA of baby's progenitor germ line cells, called primordial germ cells (PGCs) are removed. This act is essential to remove errors in methylation acquired during gametogenesis in the parents, and/or during early development of baby after fertilization. If errors in cytosine methylation are not removed, the abnormally methylated alleles have a risk of being inherited as disease epialleles in the following generation. Given that the environment can stably influence the genome, including the genome of baby's PGCs in utero, there is a need to understand the mechanisms that regulate DNA demethylation in PGCs in order to develop strategies to guard against the transmission of disease epialleles in future generations. Recently my group discovered that DNA demethylation in human PGCs is regulated in two phases however the mechanisms underlying demethylation globally (phase 1) and locally (phase 2) are unclear. In this project we aim to uncover new details on the dynamics of DNA demethylation in human PGCs and use conditional deletions of mouse PGCs in vivo as well as differentiation of mouse PGCs from embryonic stem cells in vitro to address hypotheses regarding the specific mechanisms responsible for demethylation in the mammalian germ line.
In aim 1 we will identify the dynamic removal of cytosine methylation at base resolution for the very first time in human PGCs and address the hypothesis that Ubiquitin- like, containing PHD and RING finger domains, 1 (Uhrf1) repression by protein arginine methyltransferase 5 (Prmt5) is responsible for the phase 1 DNA demethylation in mammals.
In aim 2 using a conditional deletion in mouse PGCs we will address the hypothesis that Dnmt1 maintains cytosine methylation at discreet loci in PGCs in the absence of its major cofactor Uhrf1.
In aim 3, we turn to phase 2 demethylation to directly address the hypothesis that conversion of 5-methylcytosine to 5-hydroxymethylcytosine by Tet methylcytosine dioxygenases has a functional role in the demethylation of imprinting control centers in PGCs. Taken together, results from this grant will lead to new insights into the mechanisms that regulate germ line epigenetic inheritance, and in future work our goal will be to prevent epialleles from being acquired and transmitted.

Public Health Relevance

Mammalian primordial germ cells remove cytosine methylation to prevent disease epialleles from being transmitted to the next generation. This grant seeks to interrogate the sites and basic mechanisms by which cytosine methylation is removed from the mammalian primordial germ cell genome by mapping cytosine demethylation at base resolution in human PGCs, differentiating embryonic stem cells into primordial germ cells, as well as the use of transgenic mouse technology.

Agency
National Institute of Health (NIH)
Institute
Eunice Kennedy Shriver National Institute of Child Health & Human Development (NICHD)
Type
Research Project (R01)
Project #
2R01HD058047-06A1
Application #
8777631
Study Section
Special Emphasis Panel (ZRG1-EMNR-T (02))
Program Officer
Ravindranath, Neelakanta
Project Start
2008-04-01
Project End
2019-04-30
Budget Start
2014-08-01
Budget End
2015-04-30
Support Year
6
Fiscal Year
2014
Total Cost
$319,550
Indirect Cost
$112,050
Name
University of California Los Angeles
Department
Biochemistry
Type
Schools of Arts and Sciences
DUNS #
092530369
City
Los Angeles
State
CA
Country
United States
Zip Code
90095
Hargan-Calvopina, Joseph; Taylor, Sara; Cook, Helene et al. (2016) Stage-Specific Demethylation in Primordial Germ Cells Safeguards against Precocious Differentiation. Dev Cell 39:75-86
Gkountela, Sofia; Zhang, Kelvin X; Shafiq, Tiasha A et al. (2015) DNA Demethylation Dynamics in the Human Prenatal Germline. Cell 161:1425-36
Morselli, Marco; Pastor, William A; Montanini, Barbara et al. (2015) In vivo targeting of de novo DNA methylation by histone modifications in yeast and mouse. Elife 4:e06205
Clark, Amander T (2015) DNA methylation remodeling in vitro and in vivo. Curr Opin Genet Dev 34:82-7
Calvopina, Joseph Hargan; Cook, Helene; Vincent, John J et al. (2015) The Aorta-Gonad-Mesonephros Organ Culture Recapitulates 5hmC Reorganization and Replication-Dependent and Independent Loss of DNA Methylation in the Germline. Stem Cells Dev 24:1536-45
Li, Ziwei; Yu, Juehua; Hosohama, Linzi et al. (2015) The Sm protein methyltransferase PRMT5 is not required for primordial germ cell specification in mice. EMBO J 34:748-58
Oliveros-Etter, Marisabel; Li, Ziwei; Nee, Kevin et al. (2015) PGC Reversion to Pluripotency Involves Erasure of DNA Methylation from Imprinting Control Centers followed by Locus-Specific Re-methylation. Stem Cell Reports 5:337-49
Gkountela, Sofia; Li, Ziwei; Chin, Chee Jia et al. (2014) PRMT5 is required for human embryonic stem cell proliferation but not pluripotency. Stem Cell Rev 10:230-9
Pastor, William A; Stroud, Hume; Nee, Kevin et al. (2014) MORC1 represses transposable elements in the mouse male germline. Nat Commun 5:5795
Gkountela, Sofia; Clark, Amander T (2014) A big surprise in the little zygote: the curious business of losing methylated cytosines. Cell Stem Cell 15:393-4

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