Examining the Developmental Trajectory of an in Vitro Model of Mouse Primordial Germ Cells following Exposure to Environmentally Relevant Bisphenol A Levels.

BACKGROUND: Animal-based studies indicate that bisphenol A (BPA) exposure is detrimental to reproductive health, but its impact on the earliest stages of germ cell development remains poorly defined. OBJECTIVES: Using a murine in vitro model of early germ cell specification and differentiation, we sought to assess whether exposure to low levels of BPA prior to formation of primordial germ cells (PGCs) alters their differentiation trajectory and unique molecular program. METHODS: We used an established method of in vitro differentiation of mouse embryonic stem cells (ESCs) into epiblast-like cells (EpiLCs) followed by PGC-like cells (PGCLCs), which together recapitulate defined stages of early germ cell development. Cellular consequences were determined using hemocytometer-based cell counting, fixation, and intracellular staining, followed by flow cytometry/fluorescence-activated cell sorting (FACS) of cells exposed to increasing concentrations (range: 1 nM-10 µM) of BPA. To interrogate and characterize gene expression differences resulting from BPA exposure, we also generated RNA-seq libraries from RNA extracted from FACS-purified PGCLCs and performed transcriptome analysis using bioinformatics-based approaches. RESULTS: Exposure of EpiLCs to BPA resulted in higher numbers of cells that were associated with a higher proportion of cells in S-phase as well as a lower proportion undergoing apoptosis; this difference occurred in a concentration-dependent manner. Exposure also resulted in a greater fraction of EpiLCs showing signs of DNA damage. Remarkably, EpiLC exposure did not negatively affect PGC specification and resulted in a concentration-dependent effect on PGCLC proliferation in XX but not XY cells. PGCLC transcriptome analysis revealed an aberrant program with significant deregulation of X-linked genes and retrotransposon expression. Differential gene expression analysis also revealed the deregulation of genes associated with lipid metabolism as well as deregulated expression of genes associated with later stages of gametogenesis. CONCLUSIONS: To the best of our knowledge our findings represent the first characterization of the consequences of early BPA exposure on a model of mammalian PGC development, highlighting altered cell behavior, altered underlying pathways, and altered molecular processes. https://doi.org/10.1289/EHP8196.

Generation of a cancer testis antigen mCherry reporter HCT116 colorectal carcinoma cell line.

In the context of cancer immunotherapy, agents that target the immune system to cancer cells need to fulfil two criteria: 1) that they are only expressed on the desired target cell and 2) that they can elicit a potent immunological response. Cancer Testis Antigens are a large disparate family of factors ordinarily expressed in the germ-line but aberrantly expressed across multiple types of cancer. The ability to enforce their expression on tumour cells is an attractive strategy that could render such cells potent targets of the immune system, but very little is known about their regulation. We describe the generation of an mCherry reporter cell line using HCT116 colorectal carcinoma cells that we anticipate will be useful for screen-based approaches to identify novel regulators of CTA expression. Discoveries arising from their use could in future be exploited to enhance tumour cell immunogenicity and improve cancer immuno-therapy.

A novel requirement for DROSHA in maintenance of mammalian CG methylation.

In mammals, faithful inheritance of genomic methylation patterns ensures proper gene regulation and cell behaviour, impacting normal development and fertility. Following establishment, genomic methylation patterns are transmitted through S-phase by the maintenance methyltransferase Dnmt1. Using a protein interaction screen, we identify Microprocessor component DROSHA as a novel DNMT1-interactor. Drosha-deficient embryonic stem (ES) cells display genomic hypomethylation that is not accounted for by changes in the levels of DNMT proteins. DNMT1-mediated methyltransferase activity is also reduced in these cells. We identify two transcripts that are specifically upregulated in Drosha- but not Dicer-deficient ES cells. Regions within these transcripts predicted to form stem-loop structures are processed by Microprocessor and can inhibit DNMT1-mediated methylation in vitro. Our results highlight DROSHA as a novel regulator of mammalian DNA methylation and we propose that DROSHA-mediated processing of RNA is necessary to ensure full DNMT1 activity. This adds to the DROSHA repertoire of non-miRNA dependent functions as well as implicating RNA in regulating DNMT1 activity and correct levels of genomic methylation.

The Dnmt3L ADD Domain Controls Cytosine Methylation Establishment during Spermatogenesis.

A critical aspect of mammalian gametogenesis is the reprogramming of genomic DNA methylation. The catalytically inactive adaptor Dnmt3L is essential to ensuring this occurs correctly, but the mechanism by which it functions is unclear. Using gene targeting to engineer a single-amino-acid mutation, we show that the Dnmt3L histone H3 binding domain (ADD) is necessary for spermatogenesis. Genome-wide single-base-resolution DNA methylome analysis of mutant germ cells revealed overall reductions in CG methylation at repetitive sequences and non-promoter CpG islands. Strikingly, we also observe an even more severe loss of non-CG methylation, suggesting an unexpected role for the ADD in this process. These epigenetic deficiencies were coupled with defects in spermatogonia, with mutant cells displaying marked changes in gene expression and reactivation of retrotransposons. Our results demonstrate that the Dnmt3L ADD is necessary for Dnmt3L function and full reproductive fitness.

DNA methylation is dispensable for suppression of the agouti viable yellow controlling element in murine embryonic stem cells.

The agouti viable (Avy) locus is considered a model to understand how retroelements function as controlling elements in mammals. Epigenetic factors, principally CpG methylation, are widely held to play a dominant regulatory role in controlling the locus' activity. The purpose of this study was to examine its behavior in ES cells and determine if this locus could be exploited for use in screen-based investigations. We have derived multiple Avy ES cell lines from the C57BL/6 strain and generated a cell line carrying a GFP-reporter gene (Avy/AGFP). Use of the DNA demethylating drug 5-azacitidine on various ES cell lines does not induce either agouti or GFP expression. Methylation analysis reveals that although most lines display normal methylation at IAP elements in general, the Avy IAP element is essentially unmethylated. In addition, we find that different repeat compartments are epigenetically unstable in a number of derived cell lines.

Ectopic DNMT3L triggers assembly of a repressive complex for retroviral silencing in somatic cells.

UNLABELLED: Mammalian genomes are replete with retrotransposable elements, including endogenous retroviruses. DNA methyltransferase 3-like (DNMT3L) is an epigenetic regulator expressed in prospermatogonia, growing oocytes, and embryonic stem (ES) cells. Here, we demonstrate that DNMT3L enhances the interaction of repressive epigenetic modifiers, including histone deacetylase 1 (HDAC1), SET domain, bifurcated 1 (SETDB1), DNA methyltransferase 3A (DNMT3A), and tripartite motif-containing protein 28 (TRIM28; also known as TIF1ß and KAP1) in ES cells and orchestrates retroviral silencing activity with TRIM28 through mechanisms including, but not limited to, de novo DNA methylation. Ectopic expression of DNMT3L in somatic cells causes methylation-independent retroviral silencing activity by recruitment of the TRIM28/HDAC1/SETDB1/DNMT3A/DNMT3L complex to newly integrated Moloney murine leukemia virus (Mo-MuLV) proviral DNA. Concurrent with this recruitment, we also observed the accumulation of histone H3 lysine 9 trimethylation (H3K9me3) and heterochromatin protein 1 gamma (HP1γ), as well as reduced H3K9 and H3K27 acetylation at Mo-MuLV proviral sequences. Ectopic expression of DNMT3L in late-passage mouse embryonic fibroblasts (MEFs) recruited cytoplasmically localized HDAC1 to the nucleus. The formation of this epigenetic modifying complex requires interaction of DNMT3L with DNMT3A as well as with histone H3. In fetal testes at embryonic day 17.5, endogenous DNMT3L also enhanced the binding among TRIM28, DNMT3A, SETDB1, and HDAC1. We propose that DNMT3L may be involved in initiating a cascade of repressive epigenetic modifications by assisting in the preparation of a chromatin context that further attracts DNMT3A-DNMT3L binding and installs longer-term DNA methylation marks at newly integrated retroviruses. IMPORTANCE: Almost half of the mammalian genome is composed of endogenous retroviruses and other retrotransposable elements that threaten genomic integrity. These elements are usually subject to epigenetic silencing. We discovered that two epigenetic regulators that lack enzymatic activity, DNA methyltransferase 3-like (DNMT3L) and tripartite motif-containing protein 28 (TRIM28), collaborate with each other to impose retroviral silencing. In addition to modulating de novo DNA methylation, we found that by interacting with TRIM28, DNMT3L can attract various enzymes to form a DNMT3L-induced repressive complex to remove active marks and add repressive marks to histone proteins. Collectively, these results reveal a novel and pivotal function of DNMT3L in shaping the chromatin modifications necessary for retroviral and retrotransposon silencing.

The colorful history of active DNA demethylation.

Patterns of DNA cytosine methylation are subject to mitotic inheritance in both plants and vertebrates. Plants use 5-methylcytosine glycosylases and the base excision repair pathway to remove excess cytosine methylation. In mammals, active demethylation has been proposed to operate via several very different mechanisms. Two recent reports in Nature now claim that the demethylation process is initiated by the same enzymes that establish the methylation mark, the DNA methyltransferases DNMT3A and DNMT3B (Kangaspeska et al., 2008; Métivier et al., 2008).

Cytosine methylation: remaining faithful.

DNA methyltransferase-1 (DNMT1) has a higher specific activity on hemimethylated DNA than on unmethylated DNA, but this preference is too small to explain the faithful mitotic inheritance of genomic methylation patterns. New genetic studies in plants and mammals have identified a novel factor that increases the fidelity of maintenance methylation.

DNMT3L connects unmethylated lysine 4 of histone H3 to de novo methylation of DNA.

Mammals use DNA methylation for the heritable silencing of retrotransposons and imprinted genes and for the inactivation of the X chromosome in females. The establishment of patterns of DNA methylation during gametogenesis depends in part on DNMT3L, an enzymatically inactive regulatory factor that is related in sequence to the DNA methyltransferases DNMT3A and DNMT3B. The main proteins that interact in vivo with the product of an epitope-tagged allele of the endogenous Dnmt3L gene were identified by mass spectrometry as DNMT3A2, DNMT3B and the four core histones. Peptide interaction assays showed that DNMT3L specifically interacts with the extreme amino terminus of histone H3; this interaction was strongly inhibited by methylation at lysine 4 of histone H3 but was insensitive to modifications at other positions. Crystallographic studies of human DNMT3L showed that the protein has a carboxy-terminal methyltransferase-like domain and an N-terminal cysteine-rich domain. Cocrystallization of DNMT3L with the tail of histone H3 revealed that the tail bound to the cysteine-rich domain of DNMT3L, and substitution of key residues in the binding site eliminated the H3 tail-DNMT3L interaction. These data indicate that DNMT3L recognizes histone H3 tails that are unmethylated at lysine 4 and induces de novo DNA methylation by recruitment or activation of DNMT3A2.

Epigenetic decisions in mammalian germ cells.

Specific sequences are designated for de novo DNA methylation at CpG dinucleotides in mammalian germ cells. The result is the long-term transcriptional silencing of the methylated sequences, most of which are retrotransposons and CpG-rich sequences associated with imprinted genes. There is profound sexual dimorphism in both the nature of the sequences that undergo de novo methylation in germ cells and in the mechanism by which de novo methylation is regulated. The restriction of future gene expression by the imposition of heritable methylation patterns in germ cell genomes is characteristic of mammals but is rare in other taxa.

Critical roles for Rac1 and Rac2 GTPases in B cell development and signaling.

The Rac1 guanosine triphosphatase (GTPase) has been implicated in multiple cellular functions, including actin dynamics, proliferation, apoptosis, adhesion, and migration resulting from signaling by multiple receptors, including the B cell antigen receptor (BCR). We used conditional gene targeting to generate mice with specific Rac1 deficiency in the B cell lineage. In the absence of both Rac1 and the highly related Rac2, B cell development was almost completely blocked. Both GTPases were required to transduce BCR signals leading to proliferation, survival and up-regulation of BAFF-R, a receptor for BAFF, a key survival molecule required for B cell development and maintenance.