Interaction between DMRT1 function and genetic background modulates signaling and pluripotency to control tumor susceptibility in the fetal germ line

Abstract

Dmrt1(doublesex and mab-3 related transcription factor 1) is a regulator of testis development in vertebrates that has been implicated in testicular germ cell tumors of mouse and human. In the fetal mouse testis Dmrt1 regulates germ cell pluripotency in a strain-dependent manner. Loss of Dmrt1 in 129Sv strain mice results in a >90% incidence of testicular teratomas, tumors consisting cells of multiple germ layers; by contrast, these tumors have never been observed in Dmrt1 mutants of C57BL/6J (B6) or mixed genetic backgrounds. To further investigate the interaction between Dmrt1 and genetic background we compared mRNA expression in wild type and Dmrt1 mutant fetal testes of 129Sv and B6 mice at embryonic day 15.5 (E15.5), prior to overt tumorigenesis. Loss of Dmrt1 caused misexpression of overlapping but distinct sets of mRNAs in the two strains. The mRNAs that were selectively affected included some that changed expression only in one strain or the other and some that changed in both strains but to a greater degree in one versus the other. In particular, loss of Dmrt1 in 129Sv testes caused a more severe failure to silence regulators of pluripotency than in B6 testes. A number of genes misregulated in 129Sv mutant testes also are misregulated in human testicular germ cell tumors (TGCTs), suggesting similar etiology between germ cell tumors in mouse and man. Expression profiling showed that DMRT1 also regulates pluripotency genes in the fetal ovary, although Dmrt1 mutant females do not develop teratomas. Pathway analysis indicated disruption of several signaling pathways in Dmrt1 mutant fetal testes, including Nodal, Notch, and GDNF. We used a Nanos3-cre knock-in allele to perform conditional gene targeting, testing the GDNF coreceptors Gfra1 and Ret for effects on teratoma susceptibility. Conditional deletion of Gfra1 but not Ret in fetal germ cells of animals outcrossed to 129Sv caused a modest but significant elevation in tumor incidence. Despite some variability in genetic background in these crosses, this result is consistent with previous genetic mapping of teratoma susceptibility loci to the region containing Gfra1. Using Nanos3-cre we also uncovered a strong genetic interaction between Dmrt1 and Nanos3, suggesting parallel functions for these two genes in fetal germ cells. Finally, we used chromatin immunoprecipitation (ChIP-seq) analysis to identify a number of potentially direct DMRT1 targets. This analysis suggested that DMRT1 controls pluripotency via transcriptional repression of Esrrb, Nr5a2/Lrh1, and Sox2. Given the strong evidence for involvement of DMRT1 in human TGCT, the downstream genes and pathways identified in this study provide potentially useful candidates for roles in the human disease.

Genome-wide identification of regulatory elements in Sertoli cells

“…We used previously published DMRT1 ChIP-seq data (Krentz et al, 2013) and assessed overlap with Sertoli DHSs using BedTools. For further details see the supplementary Materials and Methods.…”

The mammalian Doublesex homolog DMRT6 coordinates the transition between mitotic and meiotic developmental programs during spermatogenesis

“…Chromatin from testes of three adult wild-type B6 and 129Sv mixed genetic background mice was cross-linked with formaldehyde, sheared and immunoprecipitated with anti-DMRT6 antibody as described previously (Krentz et al, 2013).…”

“…This filtered gene list (supplementary material Table S2) was further annotated with the number of PubMed publications for each gene and the keyword 'testis' and whether that gene was associated with a peak in the DMRT1 and DMRT6 ChIP-Seq data. ChIP-Seq data were mapped to mm9 and peaks were identified using MACS (Feng et al, 2012) using a P-value cutoff of 10 −5 as described previously (Krentz et al, 2013). ChIP-Seq peaks were annotated with overlapping and nearest start features (supplementary material Table S3).…”

Reprogramming of germ cells into pluripotency

“…Conditional knockout mice demonstrate that the loss of Dmrt1 in PGCs, but not in Sertoli cells, leads to teratoma formation [80] . Pluripotencyrelated genes and Nodal pathway genes are upregulated, whereas the gliacell derived neurotrophic factor (GDNF) receptor genes including Ret and Gfra1 are downregulated in mutant fetal testes [81] . As deletion of Gfra1 in 129/Sv mice modestly increases the incidence of testicular teratomas [81] , the effects of Dmrt1 deletion are at least partly mediated by downregulation of GDNF signal.…”

“…Pluripotencyrelated genes and Nodal pathway genes are upregulated, whereas the gliacell derived neurotrophic factor (GDNF) receptor genes including Ret and Gfra1 are downregulated in mutant fetal testes [81] . As deletion of Gfra1 in 129/Sv mice modestly increases the incidence of testicular teratomas [81] , the effects of Dmrt1 deletion are at least partly mediated by downregulation of GDNF signal. Alternatively, enhanced RA signaling in germ cells lacking Dmrt1 may drive dedifferentiation, as RA treatment induces PGC reprogramming in vitro [25,77,79] .…”