Transcription Factor TFAP2C Regulates Major Programs Required for Murine Fetal Germ Cell Maintenance and Haploinsufficiency Predisposes to Teratomas in Male Mice

<div><p>Maintenance and maturation of primordial germ cells is controlled by complex genetic and epigenetic cascades, and disturbances in this network lead to either infertility or malignant aberration. Transcription factor TFAP2C has been described to be essential for primordial germ cell maintenance and to be upregulated in several human germ cell cancers. Using global gene expression profiling, we identified genes deregulated upon loss of <i>Tfap2c</i> in embryonic stem cells and primordial germ cell-like cells. We show that loss of <i>Tfap2c</i> affects many aspects of the genetic network regulating germ cell biology, such as downregulation of maturation markers and induction of markers indicative for somatic differentiation, cell cycle, epigenetic remodeling and pluripotency. Chromatin-immunoprecipitation analyses demonstrated binding of TFAP2C to regulatory regions of deregulated genes (<i>Sfrp1, Dmrt1</i>, <i>Nanos3</i>, <i>c-Kit</i>, <i>Cdk6</i>, <i>Cdkn1a</i>, <i>Fgf4</i>, <i>Klf4</i>, <i>Dnmt3b</i> and <i>Dnmt3l</i>) suggesting that these genes are direct transcriptional targets of TFAP2C in primordial germ cells. Since <i>Tfap2c</i> deficient primordial germ cell-like cells display cancer related deregulations in epigenetic remodeling, cell cycle and pluripotency control, the <i>Tfap2c</i>-knockout allele was bred onto 129S2/Sv genetic background. There, mice heterozygous for <i>Tfap2c</i> develop with high incidence germ cell cancer resembling human pediatric germ cell tumors. Precursor lesions can be observed as early as E16.5 in developing testes displaying persisting expression of pluripotency markers. We further demonstrate that mice with a heterozygous deletion of the TFAP2C target gene <i>Nanos3</i> are also prone to develop teratomas. These data highlight TFAP2C as a critical and dose-sensitive regulator of germ cell fate.</p></div

Teratoma development in <i>Tfap2c</i> heterozygous mice in 129S2/Sv genetic background.

<p>(A) Quantitative RT-PCR with RNA isolated from E12.5 genital ridges of wt and <i>Tfap2c^+/−</i> embryos was performed. Expression levels of <i>Tfap2c</i> and <i>p21</i> were normalized to <i>Gapdh</i>. qRT-PCR was performed in biological duplicates. Error bars indicate standard deviation. (B) Percentage of total tumor incidence in <i>Tfap2c</i> heterozygous 129S2/Sv male mice. The seventh generation in 129S2/Sv shows 82% (n = 51) testicular tumors. Red bar: bilateral cases (35%), blue bar: unilateral tumors (47%). (C) Gross pathology of testicular teratoma in <i>Tfap2c^+/−</i> male mice. (D–G) HE-staining of testicular teratomas of 3–6 month old mice. Tumors show immature glia (D), mature cartilage, muscle (E), respiratory epithelium (F) and squamous epithelium (G). Scale bars: 200 µm.</p

Validation of cDNA microarray data.

<p>(A) Quantitative RT-PCR of a subset of markers with RNA isolated from ctrl and <i>Tfap2c^−/−</i>PGCLCs. Expression levels were normalized to <i>βActin</i> and expression level of <i>ctrl</i> PGCLCs were set to 1. qRT-PCR was performed in biological triplicates. Error bars indicate standard deviation. (B) Quantitative RT-PCR of a subset of markers with RNA isolated from TCam-2 cells after siRNA mediated knockdown of TFAP2C. Expression levels were normalized to GAPDH and expression levels of scrambled-siRNA-transfection were set to 1. qRT-PCR was performed in biological triplicate. Error bars indicate standard deviation. (C) ChIP/qPCR analysis for Tfap2c was performed with four biological replicates of PGCLCs. The qPCR results were calculated with the percentage input method and ChIP analyses with IgG antibody served as control and were set to 1. Error bars indicate standard deviation. ChIP analysis demonstrates increased binding of TFAP2C at indicated loci. (A) – (C) Marker genes were grouped in categories: somatic differentiation (category I), germ cell maintenance and maturation (category II), cell cycle regulation (category III), pluripotency (category IV) and epigenetic modification (category V).</p

Genes regulated in PGCLCs and ESCs by TFAP2C.

<p>Affymetrix microarray gene expression analysis performed with RNA extracted from #1-<i>ctrl</i>; #1-<i>Tfap2c^−/−</i> and #2-<i>ctrl</i>; #2-<i>Tfap2c^−/−</i> ESCs and PGCLCs. (A) Hierarchical clustering. The red bars cluster the ESCs, green bars the <i>Tfap2c^−/−</i>PGCLCs and blue bars ctrl PGCLCs, respectively. The shorter the horizontal bar that connects two branches the closer are the populations. (B) Heat map was performed with the probes whose range or variation across all samples was at least 3. Color bar on top codifies the gene expression in log2 scale. Red and blue indicate higher and lower relative expression. (C-D) Pairwise scatter plot of global gene expression in ctrl versus <i>Tfap2c^−/−</i>PGCLCs (C) and ESCs (D). Black lines indicate 1.5 fold-change in log2 scale of gene expression levels between paired PGCLCs and ESCs. Color bars on the side display the scattering density with light blue indicating lower and blue higher scatter density. Genes upregulated in <i>Tfap2c^−/−</i> samples are shown as red dots; genes downregulated are shown as green dots. R² = Fisher’s correlation coefficient. (E) Venn diagram; in PGCLCs 455 genes are deregulated; in ESCs 26 genes are deregulated. The intersection part show the commonly deregulated genes (n = 13) by TFAP2C in PGCLCs and ESCs (Fold-change >1.5 in log2 scale).</p

Teratoma development in <i>Nanos3</i> heterozygous mice in 129S2/Sv genetic background.

<p>(A) Percentage of total tumor incidence in <i>Nanos3</i> heterozygous 129S2/Sv male mice. 45% (n = 20) showed testicular tumors. Red bar: bilateral cases (5%); blue bar: unilateral cases (40%). Testes of control (wt) and <i>Nanos3</i> heterozygous (cre/+) mice (inset in A). (B–D) HE-staining of testicular teratomas of 4–8 month old mice. Tumors show tissues of all three germ layers: muscle (B), squamous epithelium (C) and respiratory epithelium (D). Scale bars: 50 µm.</p

Categorized deregulated genes in <i>Tfap2^−/−</i>PGCLCs based on gene ontology analysis.

<p>bold: genes with fold-change >1.5 in log2 scale; regular: genes with fold-change ≤1.5 in log2 scale.</p

Schematic of the genes and programs regulated by TFAP2C in primordial germ cells.

<p>Black arrows indicate pathways transactivated and induced by TFAP2C and black lines with terminal bars indicate pathways repressed by TFAP2C during development of primordial germ cells. Genes listed in respective pathways indicate direct regulation as demonstrated by ChIP analyses.</p

Phenotypic characterization of parental strains after six weeks of CCl₄ injections.

<p>Liver fibrosis was assessed by morphometric (A) and biochemical (B) measurement of hepatic collagen (Hyp) contents. Hepatic inflammation was measured by serum ALT activities (C). Sirius red staining of hepatic collagen showed circumferential fibrosis in C57BL/6J mice (D) and pronounced fibrosis in DBA/2J mice (E), corresponding to mean F-scores of 2.0±0.1 and 3.9±0.1, respectively.</p

Chromosomal regions of pQTLs with significant genome-wide LRS values determined by single QTL scans and CIM.

<p>Abbreviations and definitions: <b>pQTL (chr):</b> chromosomal position of quantitative trait locus; <b>LRS (max):</b> likelihood ratio statistic, maximum association between genotype and phenotype variation; <b>SNP (max):</b> single nucleotide polymorphism with maximum LRS in QTL region; <b>1.5 LOD support interval (Mb):</b> chromosomal region in Megabases spanning QTL position; <b>Additive allele effect</b>: estimate of a change in the average phenotype by substitution of one parental allele by another at a given marker position; <b>(−)</b> values indicate an increase of phenotype by C57BL/6J allele, <b>(+)</b> values an increase of phenotype by DBA/2J allele; <b>Dataset:</b> dataset in which the QTL was identified; <b>Hyp</b>: hydroxyproline; CIM: composite interval mapping.</p

Summary of fibrosis-associated pQTL and eQTL regions.

<p>Abbreviations and definitions: <b>pQTL (chr):</b> position of phenotypic (p) QTL; <b>pQTL Position (Mb)</b>: chromosomal position in Megabases; <b>LRS (max):</b> likelihood ratio statistic, maximum association detected in pQTL analysis; <b>Size of pQTL region (Mb):</b> size of 1.5 LOD support interval of the QTL; <b>Genes in pQTL region</b>: all genes localized in a pQTL region; <b>eQTLs in pQTL region</b>: regulatory genetic markers in pQTL region; <b><i>cis</i></b><b>QTLs in pQTL region</b>: genetic markers in the pQTL region, regulating genes within a 10 Mb distance; <b><i>cis</i></b><b>QTGs in pQTL region</b>: genes in the pQTL region (regulated by markers within a 10 Mb distance) with LRS≥12.0.</p