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Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor.

Gely-Pernot A, Raverdeau M, Teletin M, Vernet N, Féret B, Klopfenstein M, Dennefeld C, Davidson I, Benoit G, Mark M, Ghyselinck NB - PLoS Genet. (2015)

Bottom Line: We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia.Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia.They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Génétique Fonctionnelle et Cancer, Illkirch, France; Centre National de la Recherche Scientifique (CNRS), UMR7104, Illkirch, France; Institut National de la Santé et de la Recherche Médicale (INSERM), U964, Illkirch, France; Université de Strasbourg (UNISTRA), Illkirch Cedex, France.

ABSTRACT
All-trans retinoic acid (ATRA) is instrumental to male germ cell differentiation, but its mechanism of action remains elusive. To address this question, we have analyzed the phenotypes of mice lacking, in spermatogonia, all rexinoid receptors (RXRA, RXRB and RXRG) or all ATRA receptors (RARA, RARB and RARG). We demonstrate that the combined ablation of RXRA and RXRB in spermatogonia recapitulates the set of defects observed both upon ablation of RAR in spermatogonia. We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia. Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia. They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation. Importantly, they indicate also that meiosis eventually occurs in the absence of a RAR/RXR pathway within germ cells and suggest that instructing this process is either ATRA-independent or requires an ATRA signal originating from Sertoli cells.

No MeSH data available.


Related in: MedlinePlus

RARG/RXRA heterodimers bind to Sall4 in testis chromatin, on an IR1 motif located in the first intron.(A) UCSC Genome Browser snapshot of the Sall4 locus in NCBI37/mm9 assembly, including tracks for anti-RAR ChIP-seq [34], RefSeq genes and mammalian conservation (from top to bottom). RARE points to the RAR-binding region identified according to Moutier et al. [34]. (B) Alignments of the DNA sequences from the indicated species and corresponding to the region containing the RAR-binding region in mouse Sall4. The dotted arrows indicate orientations of the core motifs. Stars and grey boxes highlight the DNA residues and the RAR binding sequences that are conserved in all 6 species, respectively. (C) Left panel: relative expression of Sall4a mRNA quantified by RT-qPCR in whole testes from control (white bars) and Rarg−/− (grey bars) mice at PN5. Error bars represent s.e.m. (n = 9); * p < 0.05. Right panel: schematic representation of Sall4 locus and analysis of DNA recovered from testis chromatin immunoprecipitated using antibodies directed against RNApol2, all RAR or all RXR isotypes (RAR or RXR, respectively). The untranslated exon and the transcription start site (TSS) are depicted by an open box and a broken arrow, respectively. The locations of primers used are indicated at −11 kb and in the RAR-binding region (RARE). Mean fold enrichment of three experiments at RARE binding site (grey bars) is relative to the amount of DNA recovered at −11 kb (set at 1, white bars). Error bars represent s.e.m. (n = 4 to 5); * p < 0.05. (D) EMSA showing that RARG/RXRA heterodimers (Het) bind to the 32P-labelled IR1 of Sall4 (lane 4). Binding is competed when increasing amounts of unlabeled IR1 are added to reaction (lanes 5–7), but not when a mutated form IR1 is added (IR1m, lanes 8–10). 32P-IR1 probe indicates unbound DNA. (E) EMSA showing that RARG/RXRA heterodimers (Het) bound to the DR5 of Rarb (lane 3) are competed both when unlabeled DR5 (lane 4) or increasing amounts of IR1 (lanes 5–7) are added to reaction. 32P-DR5 probe indicates unbound DNA.
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pgen.1005501.g007: RARG/RXRA heterodimers bind to Sall4 in testis chromatin, on an IR1 motif located in the first intron.(A) UCSC Genome Browser snapshot of the Sall4 locus in NCBI37/mm9 assembly, including tracks for anti-RAR ChIP-seq [34], RefSeq genes and mammalian conservation (from top to bottom). RARE points to the RAR-binding region identified according to Moutier et al. [34]. (B) Alignments of the DNA sequences from the indicated species and corresponding to the region containing the RAR-binding region in mouse Sall4. The dotted arrows indicate orientations of the core motifs. Stars and grey boxes highlight the DNA residues and the RAR binding sequences that are conserved in all 6 species, respectively. (C) Left panel: relative expression of Sall4a mRNA quantified by RT-qPCR in whole testes from control (white bars) and Rarg−/− (grey bars) mice at PN5. Error bars represent s.e.m. (n = 9); * p < 0.05. Right panel: schematic representation of Sall4 locus and analysis of DNA recovered from testis chromatin immunoprecipitated using antibodies directed against RNApol2, all RAR or all RXR isotypes (RAR or RXR, respectively). The untranslated exon and the transcription start site (TSS) are depicted by an open box and a broken arrow, respectively. The locations of primers used are indicated at −11 kb and in the RAR-binding region (RARE). Mean fold enrichment of three experiments at RARE binding site (grey bars) is relative to the amount of DNA recovered at −11 kb (set at 1, white bars). Error bars represent s.e.m. (n = 4 to 5); * p < 0.05. (D) EMSA showing that RARG/RXRA heterodimers (Het) bind to the 32P-labelled IR1 of Sall4 (lane 4). Binding is competed when increasing amounts of unlabeled IR1 are added to reaction (lanes 5–7), but not when a mutated form IR1 is added (IR1m, lanes 8–10). 32P-IR1 probe indicates unbound DNA. (E) EMSA showing that RARG/RXRA heterodimers (Het) bound to the DR5 of Rarb (lane 3) are competed both when unlabeled DR5 (lane 4) or increasing amounts of IR1 (lanes 5–7) are added to reaction. 32P-DR5 probe indicates unbound DNA.

Mentions: Using data sets locating RAR-occupied sites genome-wide in several cell-types [33,34], we identified a 700 bp-long RAR-binding region located in the first intron of Sall4 (RARE, Fig 7A). This DNA fragment contained a RAR binding sequence called IR1, consisting of inverted repeats (two core motifs 5’-RGKTSA-3’ oriented head-to-tail) separated by 1 bp (Fig 7B), as well as two additional sites called DR1 and DR0 (direct repeats of the core motif separated by 1 and 0 bp, respectively). We performed triplicate IP experiments with anti-RAR and anti-RXR antibodies using chromatin extracted from PN5 wild-type mouse testes as substrate. At this developmental stage, Sall4a expression was dependent upon RARG (Fig 7C, left panel). We analyzed the immuno-precipitated chromatin fragments by qPCR and evidenced robust binding of both RAR and RXR in vivo, in a 106bp-long region restricted to chr2:168,591,142–168,591,247 (NCBI37/mm9) in Sall4 (RARE, Fig 7C, right panel). To further confirm interaction of IR1 with RAR/RXR heterodimers, we performed electrophoretic mobility shift assays (EMSA) (Fig 7D). They revealed that RARG isotype in combination with RXRA isotype (lane 4), but neither RARG nor RXRA alone (lanes 2 and 3, respectively), bound the radiolabelled IR1 sequence. Binding was competed by increasing amounts of unlabeled IR1 (lanes 5–7), but not by IR1m bearing point-mutations in the first core motif (lanes 8–10). They also showed that unlabeled IR1 efficiently competed binding of RARG/RXRA heterodimers to the radiolabeled, canonical, RAR binding site of Rarb gene (called DR5, Fig 7E). The data suggested therefore that RARG/RXRA heterodimers could enhance expression of SALL4A through binding to an IR1 motif located in Sall4 intron. This motif appeared moderately well-conserved in mouse, rat, human and primate genomes (Fig 7B), but single mismatches do not necessarily abrogate RAR/RXR binding, even when located at highly conserved positions [33]. The DR1 and DR0 were also able to bind RARG/RXRA heterodimers (S5 Fig). Their sequences were even well-conserved across the species than that of IR1 (Fig 7B). Thus the RAR binding region in Sall4 belongs to the category of “composite elements”, the functionality of which has already been demonstrated [34]. Interestingly, SALL4A is also expressed in human spermatogonia [35]. Moreover, Fertilysin (N,N’-1,8-octanediylbis[2,2-dichloro-acetamide], also called WIN 18,446), which acts by inhibiting ATRA synthesis [36], reversibly inhibits spermatogenesis in men by inducing an arrest of germ cell differentiation at the spermatogonia stage [37,38], which resembles the phenotype we describe here in the mouse. Thus it is possible that RAR/RXR heterodimers also drive SALL4A expression in human spermatogonia.


Retinoic Acid Receptors Control Spermatogonia Cell-Fate and Induce Expression of the SALL4A Transcription Factor.

Gely-Pernot A, Raverdeau M, Teletin M, Vernet N, Féret B, Klopfenstein M, Dennefeld C, Davidson I, Benoit G, Mark M, Ghyselinck NB - PLoS Genet. (2015)

RARG/RXRA heterodimers bind to Sall4 in testis chromatin, on an IR1 motif located in the first intron.(A) UCSC Genome Browser snapshot of the Sall4 locus in NCBI37/mm9 assembly, including tracks for anti-RAR ChIP-seq [34], RefSeq genes and mammalian conservation (from top to bottom). RARE points to the RAR-binding region identified according to Moutier et al. [34]. (B) Alignments of the DNA sequences from the indicated species and corresponding to the region containing the RAR-binding region in mouse Sall4. The dotted arrows indicate orientations of the core motifs. Stars and grey boxes highlight the DNA residues and the RAR binding sequences that are conserved in all 6 species, respectively. (C) Left panel: relative expression of Sall4a mRNA quantified by RT-qPCR in whole testes from control (white bars) and Rarg−/− (grey bars) mice at PN5. Error bars represent s.e.m. (n = 9); * p < 0.05. Right panel: schematic representation of Sall4 locus and analysis of DNA recovered from testis chromatin immunoprecipitated using antibodies directed against RNApol2, all RAR or all RXR isotypes (RAR or RXR, respectively). The untranslated exon and the transcription start site (TSS) are depicted by an open box and a broken arrow, respectively. The locations of primers used are indicated at −11 kb and in the RAR-binding region (RARE). Mean fold enrichment of three experiments at RARE binding site (grey bars) is relative to the amount of DNA recovered at −11 kb (set at 1, white bars). Error bars represent s.e.m. (n = 4 to 5); * p < 0.05. (D) EMSA showing that RARG/RXRA heterodimers (Het) bind to the 32P-labelled IR1 of Sall4 (lane 4). Binding is competed when increasing amounts of unlabeled IR1 are added to reaction (lanes 5–7), but not when a mutated form IR1 is added (IR1m, lanes 8–10). 32P-IR1 probe indicates unbound DNA. (E) EMSA showing that RARG/RXRA heterodimers (Het) bound to the DR5 of Rarb (lane 3) are competed both when unlabeled DR5 (lane 4) or increasing amounts of IR1 (lanes 5–7) are added to reaction. 32P-DR5 probe indicates unbound DNA.
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Show All Figures
getmorefigures.php?uid=PMC4591280&req=5

pgen.1005501.g007: RARG/RXRA heterodimers bind to Sall4 in testis chromatin, on an IR1 motif located in the first intron.(A) UCSC Genome Browser snapshot of the Sall4 locus in NCBI37/mm9 assembly, including tracks for anti-RAR ChIP-seq [34], RefSeq genes and mammalian conservation (from top to bottom). RARE points to the RAR-binding region identified according to Moutier et al. [34]. (B) Alignments of the DNA sequences from the indicated species and corresponding to the region containing the RAR-binding region in mouse Sall4. The dotted arrows indicate orientations of the core motifs. Stars and grey boxes highlight the DNA residues and the RAR binding sequences that are conserved in all 6 species, respectively. (C) Left panel: relative expression of Sall4a mRNA quantified by RT-qPCR in whole testes from control (white bars) and Rarg−/− (grey bars) mice at PN5. Error bars represent s.e.m. (n = 9); * p < 0.05. Right panel: schematic representation of Sall4 locus and analysis of DNA recovered from testis chromatin immunoprecipitated using antibodies directed against RNApol2, all RAR or all RXR isotypes (RAR or RXR, respectively). The untranslated exon and the transcription start site (TSS) are depicted by an open box and a broken arrow, respectively. The locations of primers used are indicated at −11 kb and in the RAR-binding region (RARE). Mean fold enrichment of three experiments at RARE binding site (grey bars) is relative to the amount of DNA recovered at −11 kb (set at 1, white bars). Error bars represent s.e.m. (n = 4 to 5); * p < 0.05. (D) EMSA showing that RARG/RXRA heterodimers (Het) bind to the 32P-labelled IR1 of Sall4 (lane 4). Binding is competed when increasing amounts of unlabeled IR1 are added to reaction (lanes 5–7), but not when a mutated form IR1 is added (IR1m, lanes 8–10). 32P-IR1 probe indicates unbound DNA. (E) EMSA showing that RARG/RXRA heterodimers (Het) bound to the DR5 of Rarb (lane 3) are competed both when unlabeled DR5 (lane 4) or increasing amounts of IR1 (lanes 5–7) are added to reaction. 32P-DR5 probe indicates unbound DNA.
Mentions: Using data sets locating RAR-occupied sites genome-wide in several cell-types [33,34], we identified a 700 bp-long RAR-binding region located in the first intron of Sall4 (RARE, Fig 7A). This DNA fragment contained a RAR binding sequence called IR1, consisting of inverted repeats (two core motifs 5’-RGKTSA-3’ oriented head-to-tail) separated by 1 bp (Fig 7B), as well as two additional sites called DR1 and DR0 (direct repeats of the core motif separated by 1 and 0 bp, respectively). We performed triplicate IP experiments with anti-RAR and anti-RXR antibodies using chromatin extracted from PN5 wild-type mouse testes as substrate. At this developmental stage, Sall4a expression was dependent upon RARG (Fig 7C, left panel). We analyzed the immuno-precipitated chromatin fragments by qPCR and evidenced robust binding of both RAR and RXR in vivo, in a 106bp-long region restricted to chr2:168,591,142–168,591,247 (NCBI37/mm9) in Sall4 (RARE, Fig 7C, right panel). To further confirm interaction of IR1 with RAR/RXR heterodimers, we performed electrophoretic mobility shift assays (EMSA) (Fig 7D). They revealed that RARG isotype in combination with RXRA isotype (lane 4), but neither RARG nor RXRA alone (lanes 2 and 3, respectively), bound the radiolabelled IR1 sequence. Binding was competed by increasing amounts of unlabeled IR1 (lanes 5–7), but not by IR1m bearing point-mutations in the first core motif (lanes 8–10). They also showed that unlabeled IR1 efficiently competed binding of RARG/RXRA heterodimers to the radiolabeled, canonical, RAR binding site of Rarb gene (called DR5, Fig 7E). The data suggested therefore that RARG/RXRA heterodimers could enhance expression of SALL4A through binding to an IR1 motif located in Sall4 intron. This motif appeared moderately well-conserved in mouse, rat, human and primate genomes (Fig 7B), but single mismatches do not necessarily abrogate RAR/RXR binding, even when located at highly conserved positions [33]. The DR1 and DR0 were also able to bind RARG/RXRA heterodimers (S5 Fig). Their sequences were even well-conserved across the species than that of IR1 (Fig 7B). Thus the RAR binding region in Sall4 belongs to the category of “composite elements”, the functionality of which has already been demonstrated [34]. Interestingly, SALL4A is also expressed in human spermatogonia [35]. Moreover, Fertilysin (N,N’-1,8-octanediylbis[2,2-dichloro-acetamide], also called WIN 18,446), which acts by inhibiting ATRA synthesis [36], reversibly inhibits spermatogenesis in men by inducing an arrest of germ cell differentiation at the spermatogonia stage [37,38], which resembles the phenotype we describe here in the mouse. Thus it is possible that RAR/RXR heterodimers also drive SALL4A expression in human spermatogonia.

Bottom Line: We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia.Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia.They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation.

View Article: PubMed Central - PubMed

Affiliation: Institut de Génétique et de Biologie Moléculaire et Cellulaire (IGBMC), Département de Génétique Fonctionnelle et Cancer, Illkirch, France; Centre National de la Recherche Scientifique (CNRS), UMR7104, Illkirch, France; Institut National de la Santé et de la Recherche Médicale (INSERM), U964, Illkirch, France; Université de Strasbourg (UNISTRA), Illkirch Cedex, France.

ABSTRACT
All-trans retinoic acid (ATRA) is instrumental to male germ cell differentiation, but its mechanism of action remains elusive. To address this question, we have analyzed the phenotypes of mice lacking, in spermatogonia, all rexinoid receptors (RXRA, RXRB and RXRG) or all ATRA receptors (RARA, RARB and RARG). We demonstrate that the combined ablation of RXRA and RXRB in spermatogonia recapitulates the set of defects observed both upon ablation of RAR in spermatogonia. We also show that ATRA activates RAR and RXR bound to a conserved regulatory region to increase expression of the SALL4A transcription factor in spermatogonia. Our results reveal that this major pluripotency gene is a target of ATRA signaling and that RAR/RXR heterodimers are the functional units driving its expression in spermatogonia. They add to the mechanisms through which ATRA promote expression of the KIT tyrosine kinase receptor to trigger a critical step in spermatogonia differentiation. Importantly, they indicate also that meiosis eventually occurs in the absence of a RAR/RXR pathway within germ cells and suggest that instructing this process is either ATRA-independent or requires an ATRA signal originating from Sertoli cells.

No MeSH data available.


Related in: MedlinePlus