Vinclozolin Deregulates MicroRNAs in Germ Cells

19 p.-6 fig.In mammals, germ cell differentiation is initiated in the Primordial Germ Cells (PGCs) during fetal development. Prenatal exposure to environmental toxicants such as endocrine disruptors may alter PGC differentiation, development of the male germline and induce transgenerational epigenetic disorders. The anti-androgenic compound vinclozolin represents a paradigmatic example of molecule causing transgenerational effects on germ cells. We performed prenatal exposure to vinclozolin in mice and analyzed the phenotypic and molecular changes in three successive generations. A reduction in the number of embryonic PGCs and increased rate of apoptotic cells along with decrease of fertility rate in adult males were observed in F1 to F3 generations. Blimp1 is a crucial regulator of PGC differentiation. We show that prenatal exposure to vinclozolin deregulates specific microRNAs in PGCs, such as miR-23b and miR-21, inducing disequilibrium in the Lin28/let-7/Blimp1 pathway in three successive generations of males. As determined by global maps of cytosine methylation, we found no evidence for prominent changes in DNA methylation in PGCs or mature sperm. Our data suggest that embryonic exposure to environmental endocrine disruptors induces transgenerational epigenetic deregulation of expression of microRNAs affecting key regulatory pathways of germ cells differentiation.We thank M. Quesada, O. Barcón and M. Moreno for caring for the animals. We also acknowledged the support from "EpiGeneSys European Network of Excellence".Peer reviewe

Loss of Apc Rapidly Impairs DNA Methylation Programs and Cell Fate Decisions in Lgr5 + Intestinal Stem Cells

International audienceColorectal cancer initiation and progression result from the accumulation of genetic and epigenetic alterations. Although aberrant gene expression and DNA methylation profiles are considered hallmarks of colorectal cancer development, the precise timing at which these are produced during tumor establishment remains elusive. Here we investigated the early transcriptional and epigenetic changes induced by adenomatous polyposis coli (Apc) inactivation in intestinal crypts. Hyperactivation of the Wnt pathway via Apc inactivation in crypt base columnar intestinal stem cells (ISC) led to their rapid accumulation driven by an impaired molecular commitment to differentiation, which was associated with discrete alterations in DNA methylation. Importantly, inhibiting the enzymes responsible for de novo DNA methylation restored the responsiveness of Apc-deficient intestinal organoids to stimuli regulating the proliferation-to-differentiation transition in ISC. This work reveals that early DNA methylation changes play critical roles in the establishment of the impaired fate decision program consecutive to Apc loss of function. SIGNIFICANCE: This study demonstrates the functional impact of changes in DNA methylation to determine the colorectal cancer cell phenotype following loss of Apc function

Distinct oncogenes drive different genome and epigenome alterations in human mammary epithelial cells

Claire Fonti and Anne Saumet contributed equally to this work.International audienceMolecular subtypes of breast cancer are defined on the basis of gene expression and genomic/epigenetic pattern differences. Different subtypes are thought to originate from distinct cell lineages, but the early activation of an oncogene could also play a role. It is difficult to discriminate the respective inputs of oncogene activation or cell type of origin. In this work, we wished to determine whether activation of distinct oncogenic pathways in human mammary epithelial cells (HMEC) could lead to different patterns of genetic and epigenetic changes. To this aim, we transduced shp53 immortalized HMECs in parallel with the CCNE1, WNT1 and RASv12 oncogenes which activate distinct oncogenic pathways and characterized them at sequential stages of transformation for changes in their genetic and epigenetic profiles. We show that initial activation of CCNE1, WNT1 and RASv12, in shp53 HMECs results in different and reproducible changes in mRNA and micro-RNA expression, copy number alterations (CNA) and DNA methylation profiles. Noticeably, HMECs transformed by RAS bore very specific profiles of CNAs and DNA methylation, clearly distinct from those shown by CCNE1 and WNT1 transformed HMECs. Genes impacted by CNAs and CpG methylation in the RAS and the CCNE1/WNT1 clusters showed clear differences, illustrating the activation of distinct pathways. Our data show that early activation of distinct oncogenic pathways leads to active adaptive events resulting in specific sets of CNAs and DNA methylation changes. We, thus, propose that activation of different oncogenes could have a role in reshaping the genetic landscape of breast cancer subtypes

Exposure to Endocrine Disruptor Induces Transgenerational Epigenetic Deregulation of MicroRNAs in Primordial Germ Cells

<div><p>In mammals, germ cell differentiation is initiated in the Primordial Germ Cells (PGCs) during fetal development. Prenatal exposure to environmental toxicants such as endocrine disruptors may alter PGC differentiation, development of the male germline and induce transgenerational epigenetic disorders. The anti-androgenic compound vinclozolin represents a paradigmatic example of molecule causing transgenerational effects on germ cells. We performed prenatal exposure to vinclozolin in mice and analyzed the phenotypic and molecular changes in three successive generations. A reduction in the number of embryonic PGCs and increased rate of apoptotic cells along with decrease of fertility rate in adult males were observed in F1 to F3 generations. Blimp1 is a crucial regulator of PGC differentiation. We show that prenatal exposure to vinclozolin deregulates specific microRNAs in PGCs, such as <i>miR-23b</i> and <i>miR-21</i>, inducing disequilibrium in the <i>Lin28</i>/<i>let-7</i>/<i>Blimp1</i> pathway in three successive generations of males. As determined by global maps of cytosine methylation, we found no evidence for prominent changes in DNA methylation in PGCs or mature sperm. Our data suggest that embryonic exposure to environmental endocrine disruptors induces transgenerational epigenetic deregulation of expression of microRNAs affecting key regulatory pathways of germ cells differentiation.</p></div

Expression of <i>pri-let-7a</i>, <i>let-7a-1-3p</i> and <i>Lin28</i> in PGCs of mice exposed to VCZ.

<p>a-d) The graphs show the log₂ of the fold change of expression in PGCs of exposed embryos relative to the unexposed control embryos. e) LIN28 protein levels measured by Western blot in 13.5 dpc PGCs. The graph bars show the quantification of protein levels normalized to the unexposed control (value = 1). In the graphs the error bars represent the standard deviation (SD), (a) indicates a significant statistical difference of VD1 and VD2 compared to the control (p≤0.01), (b) indicates a significant statistical difference of VD1 compared to VD2 (p≤0.01).</p

DNA Methylation analysis in PGCs and spermatozoa of F1 mice exposed to VCZ.

<p>a) Distribution of the percentages of CpG methylation measured by RRBS in 13.5 dpc PGCs from control, VD1 and VD2 exposed embryos at the F1 generation. b) Distribution of the CG methylation measured in sequences hypomethylated in normal PGCs (defined as <20% methylation in control PGCs) or partially resistant to demethylation in PGCs (defined as >20% methylation in control PGCs). c) Evaluation of the methylation status of the promoters of <i>Lin28a</i>, <i>Lin28b</i> and <i>Blimp1</i> in 13.5dpc PGCs by single locus bisulfite sequencing (for <i>Lin28a</i> and <i>Lin28b</i>) and RRBS (for <i>Blimp1</i>). d) Pairwise comparison of CpG methylation measured in 400bp tiles in adult spermatozoa isolated from F1VD2 males compared to control males, which reveals global conservation of DNA methylation (Pearson correlation coefficient r = 1). The density of points increase from blue to dark red.</p

Levels of expression of <i>Blimp1</i> and regulatory miRNAs in PGCs of mice exposed to VCZ.

<p>a) Relative expression of <i>miR-23b</i> in PGCs of exposed embryos relative to the control embryos. b-c) Relative expression of the <i>Blimp1</i> mRNA (b) and BLIMP1 protein (c) in PGCs of exposed embryos relative to the control embryos. d) Relative expression of <i>miR-21</i>. The error bars represent the standard deviation (SD), (a) indicates a significant statistical difference of VD1 and VD2 compared to the control (p≤0.01), (b) indicates a significant statistical difference of VD1 compared to VD2 (p≤0.01).</p

Schematic model for the deregulation of PGC development by the <i>Lin28</i>/<i>let-7</i>/<i>Blimp1</i> pathway in the PGCs from mice exposed to VCZ.

<p>Red arrows indicate upregulation and green arrows indicate downregulation. miRNAs are illustrated in pink. A consequence of the downregulation of BLIMP1 is the reduction in the number of PGCs in embryonic testis.</p

Apoptosis in adult testis and PGCs of mice exposed to VCZ.

<p>a) Number of apoptotic cells counted per 10 tubules in the adult testis of control, VD1 and VD2 in F1 to F3 generations. b-d) Examples of apoptosis by TUNEL in testis sections of control (b), VD1 (c) and VD2 (d) animals. e) TUNEL positive signals after DNAse treatment of testis sections is shown as a positive control. f) Total number of PGCs isolated per testis by cell sorting with the surface marker SSEA-1 from control, VD1 and VD2 13.5dpc embryos. g) Histological evaluation of the number of PGCs in testis of 13.5dpc embryo by immunostaining with the marker SSEA-1. h) Evaluation of apoptosis in 13.5 dpc testis by TUNEL assay. i-k) Examples of co-detection of apoptosis by TUNEL and SSEA-1 positive PGCs cells by confocal microscopy analysis. i) Apoptosis detected in a PGC. j) Apoptosis detected in a somatic cell. k) None apoptotic cell detected. In the histograms, (a) indicates a significant statistical difference compared to the control value (p<0.01), and the error bars represent the standard deviation (SD).</p

Fertility and histopathological analysis in the testis of mice exposed to VCZ.

<p>a) Fertility rate after fetal exposure to the low dose (VD1) or the high dose (VD2) of VCZ, expressed as a percentage of fertile males along the three generations (F1, F2 and F3). b-e) Histological analysis of testis sections stained by hematoxilyn-eosin from 10 weeks old mice from the control group (b) or VCZ exposed group (c-e), show examples of impairment of seminiferous epithelium tubule (c), tubule disintegration with cells in the lumen (d), and hypertrophic cells with fragmented karyoplasm (e, red arrows).</p