Amander T. Clark - US grants

The goal of this project is to define the genetic and epigenetic signature that distinguishes human primordial germ cells (PGCs) from human embryonic stem cells (hESCs) with a specific focus on trimethylation (me3) of histone H3 lysine (K) 27, and function of the Polycomb Group (PcG) proteins on PGC formation. Human ESCs are the only genetically malleable human cell-based model for examining human germ cell formation. Germ cell derivation from the epiblast requires a delicate balance involving expression of pluripotent genes (such as NANOG and OCT4), suppression of somatic genes (such as HOXB1 and BRACHYURY) and functional unipotency to exclusively form gametes. Loss of germ line unipotency results in germ cell tumorigenesis. Therefore, disruption of the balance of factors that regulate germ cell derivation results in adverse clinical outcomes. In previous work Dr. Clark has shown that derivation of human germ cells from hESCs varies between independently derived lines. Her preliminary data now reveals that this can be correlated with differences in transcription of germ cell specific genes in undifferentiated ESCs as well as differential methylation of H3K27me3 at promoters of germ cell-expressed loci. In this Project, Dr Clark will continue her research on the role of the PcG proteins and H3K27me3 in modulating PGC formation in the following specific Aims: 1) Evaluating H3K27me3 and X reactivation during PGC development in two lines of female hESCs; 2) Deciphering the genome wide localization of H3K27me3 in four lines of hESCs, and PGCs derived from hESCs, and 3) Determining the function of the PcG repressive complex (PCR2) in PGC formation in four independently derived lines of hESCs. Sharing results on germ cell formation from hESCs with Projects 1 and 3 are essential to understanding hESC potential because the ability to form germ line is simultaneously reversed to form neural and hematopoietic lineages (loss of pluripotency and activation of somatic cell transcriptional programs). Thus identification of hESC lines that are capable of both faithful germ cell formation and robust somatic cell differentiation would constitute the best developmental models and therapeutic tools for future research.

DESCRIPTION (provided by applicant): The objective of this proposal is to generate germ cells from two federally approved lines of human embryonic stem cells (hESCs) called UC01 and UC06 (HSF-1 and HSF-6) in order to evaluate the role of transcriptional repression mediated by BLIMP1 and dimethylation of Histone H4 Arginine 3 (H4R3me2) on human germ cell formation. Understanding the molecular regulation of germ cell development is important to improving human health as abnormal germ cells can result in;infertility, which affects 10% of the reproductive age population in the United States, germ cell tumors, which are the most common cancer type to afflict males between the ages of 15 and 35, and birth defects, which occur in children born to parents with abnormal germ line development. In this proposal, we aim to evaluate whether defects in transcriptional repression during the initial stages of germ cell formation in the human fetus result in compromised germ cell development. The role of transcriptional repression will be evaluated in three Specific Aims. Specific Aim 1 involves determining the transcriptional signature of human germ cells from week 6-9 of human fetal gestation. This will be achieved by expression profiling germ cells isolated from human fetal gonads by FACS. Results from this Specific Aim will be used to further clarify the stage of germ cell formation acquired with hESC differentiation, to identify additional cell surface markers that can distinguish hESCs from germ cells, and as a foundation for evaluating the role of transcriptional repressors in germ cell development in vitro. In Specific Aim 2, we will evaluate the effect of a transcriptional repressor, BLIMP1 for a role in regulating human germ cell formation. It is known that BLIMP1 is essential for germ cell formation in murine models. In this Specific Aim, our goal is to knockdown and over express BLIMP1 in hESCs and assay human germ cell formation. This experiment will determine whether the yield of germ cells is reduced in genetically modified verses control ESCs. We will also FACS and isolate human germ cells from differentiating hESCs and evaluate transcription of germ cell specific genes, as well as genes associated with somatic cell differentiation. As a positive control and validation for the use of ESCs to study germ cell formation, we will compare our results to wild type and Blimp1 null mutant murine ESCs for their ability to form germ line in vitro. Finally in Specific Aim 3, the function of PRMT5 and Histone H4R3me2 in human germ cell derivation will be evaluated, and the binding sites of BLIMP1, PRMT5 and dimethylation of Histone H4R3 at promoters in ESCs and germ cells will be identified at a genome-wide level using Chromatin Immunoprecipitation (ChIP) followed by chip. PRMT5 is a protamine arginine methyltransferase that interacts with BLIMP1 to mediate dimethylation of histone H4R3 at loci that need to be repressed in order for germ cell formation to occur. Genes with promoters that share BLIMP1, PRMT5 and Histone H4R3me2 will be analyzed in future proposals. This proposal constitutes the first step from which additional downstream targets essential for normal human germ cell development can be evaluated. PUBLIC HEALTH RELEVANCE: Results from this proposal will be important for unraveling molecular mechanisms that lead specifically to human infertility.

Summary Germ cells are responsible for the passage of a parent's genome and epigenome from one generation to the next. Although the genome does not change after fertilization (except in instances of DNA damage) the epigenome in mammals is substantially altered through a process known as epigenetic remodeling. After embryo implantation, a second major wave of epigenetic remodeling occurs, this time in newly specified germ cells of the embryo called primordial germ cells (PGCs). The second wave of epigenetic remodeling, most notably erasure of DNA methylation from the epigenome is speculated to erase any acquired epialleles that could cause disease in future generations. In the last five years, my lab together with colleagues in the field discovered that DNA methylation remodeling in mouse and human PGCs is incomplete, involving stage- dependent combinations of DNA methylation erasure and DNA methylation protection. In our previous funding period, we showed that disrupting stage-dependent DNA methylation remodeling in PGCs results in germ cell loss and infertility. Given the importance of correctly staged DNA methylation remodeling to the biology of PGCs and the ability to reproduce, we are next interested in the underlying chromatin landscape responsible for dynamic DNA methylation protection and erasure. Results from this work will significantly enhance our knowledge of the epigenetic basis of reproduction. In this renewal, our overall hypothesis is that Polycomb repressor complex 2 upstream of Histone H3 Lysine 27 trimethylation (H3K27me3) (aim 1) and Tripartite motif 28 (Trim28) upstream of H3K9me3 (aim 2) play major roles in stage-dependent DNA methylation remodeling in PGCs. To address these hypotheses, we aim to use a combination of genomics, epigenomics and mouse modeling. In addition, we also aim to use single cell sequencing technologies (aim 3) to define the true epigenetic ground state of PGCs. In summary, identifying the epigenetic landscape of PGCs and the enzymes required to maintain it are critical to prioritizing future studies that disrupt the epigenome in PGCs during pregnancy, or the identification of epigenetic hot-spots in PGCs that could be tested for specific roles in infertility or transgenerational epigenetic inheritance in the future.

DESCRIPTION (provided by applicant): Approximately 1 in 10 couples of reproductive age in America are diagnosed with infertility. Infertility does not bias by race, gender or ethnicity. Underlying causes of human infertility are often unknown. However, abnormal formation, or irreversible damage to the lineage responsible for creating egg and sperm can cause infertility as an adult. We propose that one of the best models to understand mechanism of human egg and sperm differentiation (also known as germline differentiation) involve differentiating germline cells from pluripotent stem cells in vitro. Currently, the field of germline differentiation in vito is hindered by a lack of knowledge of human germline development in the embryo, low yield following in vitro differentiation, unknown variability in germline differentiation potential betwen lines, and failure to use the germline xenotransplantation model as a functional assay. To overcome these bottlenecks we propose to develop a comprehensive transcriptome and DNA methylome map of in vivo human germline cells during gestation using RNA- sequencing and whole genome bisulfite-sequencing. This will be used to transcriptionally and epigenetically stage germline cells acquired in vitro. Next, we propose to induce ectopic expression of PRDM14, NANOS3 and DAZL in a panel of nine well-characterized hESC lines by incorporating the genes into a safe harbor locus using genome-editing technology. We will measure germline identity using a next generation single-cell gene expression panel, and demethylation at loci that stably demethylate in early human germline development. Finally, we propose to use the germline xenotransplantation assay to transplant in vitro male germline cells into the testes of mice rendered infertile by chemotherapy. The endpoint of this assay will involve colony formation, proliferation and expression of mature germline markers. Xenotransplantation outcomes will be compared to control xenotransplantation of human spermatogonia and progenitor human germline cells from gestational stage testes. Results from this project will have a long lasting impact on the field of germline differentiation in vitro as it provides a measure of differentiation efficiency across multiple well characterized hESC lines, as well as incorporation of state-of-the art gene editing technology and functional assays to improve and confirm germline identity in vitro.

Summary Around 12% of women in the United States have problems getting pregnant and carrying a baby to term, with 7% of women and their partners being diagnosed as infertile. Some diagnoses of infertility can be overcome through Assisted Reproductive Technologies (ART), however these technologies require each partner to make functional gametes (eggs or sperm) for success. For those individuals incapable of making gametes, ART is not an option for overcoming a diagnosis of infertility. In this grant, we are developing a model for fertility restoration to the infertile patient using induced pluripotent stem cells (iPSCs). This basic research proposal is aimed at testing the hypothesis that prenatal and neonatal gonadal somatic cells are required to instruct male and female germline cell differentiation using non human primate (NHP) iPSCs. The proposal under consideration is based upon the scientific premise with mouse (m) models that male and female miPSCs differentiated into mouse primordial germ cell (PGC)-like cells (mPGCLCs) will undergo sex-specific differentiation when transplanted with prenatal gonadal somatic cells, or directly into neonatal niches. The success of this technology in the mouse was first built upon a fundamental understanding of mouse germline cell development, particularly during prenatal life. Here, we propose three aims in which we will use developmental biology, genomics and transplantation to understand the cell and molecular basis of sex- specific PGC differentiation in vivo using NHPs. This knowledge will be used to differentiate male and female NHP iPSCs towards PGCLCs that can undergo sex-specific differentiation using the signaling logic of the prenatal gonad, or alternatively following transplantation or culture with prenatal or neonatal gonadal somatic cells. At the conclusion of this proposal, we will have determined the extent to which NHP PGCLCs are capable of sex-specific differentiation within prenatal and neonatal gonadal niches. This work will be used to inform future studies aimed at recreating male and female gametogenesis from NHP's entirely in vitro.

Summary Human germline cells are essential for human reproduction as only these cells are capable of differentiating into gametes and transmitting DNA from parent to child. The pioneering cells of the human germline begin to form during prenatal life when a small number of embryonic cells are set aside around the time of embryo implantation and gastrulation in a process known as human primordial germ cell (hPGC) specification. This critical event in human germline cell development has a tremendous impact on an individual's future reproductive health as a failure in hPGC specification causes certain infertility. In this competitive renewal, the goal is to increase our fundamental knowledge on the cell and molecular basis of hPGC specification. Based on experimental results in the previous funding period, we aim to use human embryonic stem cells (hESCs) and human induced pluripotent stem cells (hiPSCs) and the differentiation of hPGC-like cells (hPGCLCs) to achieve this goal. The overall hypothesis is that non-rodent and human-specific molecular events have evolved to regulate hPGC specification. Given that the focus of this grant is largely on regions of the genome that are uniquely human, this project is perfectly suited to the use of human cell-based models. In aim 1, the hypothesis to be addressed is that TFAP2C-bound human-specific retrotransposons regulate hPGC specification. In aim 2, the hypothesis to be addressed is that the expression of TFAP2C bound retrotransposons are regulated by targeted changes to the epigenome during hPGCLC differentiation. In the third aim, we will evaluate the relationship between TFAP2C and SOX17 in hPGC specification, with the hypothesis that TFAP2C functions upstream of SOX17 in a lineage primed hPGC progenitor to regulate specification of hPGCs. In summary, this competitive renewal builds upon success from the first funding period to contribute essential knowledge on the identification of new loci in the human genome that have evolved to regulate the specification and identity of hPGCs.