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Genet Mol Biol
2020 Mar 30;432:e20190017. doi: 10.1590/1678-4685-GMB-2019-0017.
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PACT/PRKRA and p53 regulate transcriptional activity of DMRT1.
Fujitani K, Otomo A, Nagayama Y, Tachibana T, Kato R, Kawashima Y, Kodera Y, Kato T, Takada S, Tamura K, Takamatsu N, Ito M.
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The transcription factor DMRT1 (doublesex and mab-3 related transcription factor) has two distinct functions, somatic-cell masculinization and germ-cell development in some vertebrate species, including mouse and the African clawed frog Xenopus laevis. However, its transcriptional regulation remains unclear. We tried to identify DMRT1-interacting proteins from X. laevis testes by immunoprecipitation with an anti-DMRT1 antibody and MS/MS analysis, and selected three proteins, including PACT/PRKRA (Interferon-inducible double-stranded RNA dependent protein kinase activator A) derived from testes. Next, we examined the effects of PACT/PRKRA and/or p53 on the transcriptional activity of DMRT1. In transfected 293T cells, PACT/PRKRA and p53 significantly enhanced and repressed DMRT1-driven luciferase activity, respectively. We also observed that the enhanced activity by PACT/PRKRA was strongly attenuated by p53. Moreover, in situ hybridization analysis of Pact/Prkra mRNA in tadpole gonads indicated high expression in female and male germline stem cells. Taken together, these findings suggest that PACT/PRKRA and p53 might positively and negatively regulate the activity of DMRT1, respectively, for germline stem cell fate.
Figure 1. Immunoreaction of an anti-DMRT1 monoclonal antibody 4F6 against
X. laevis DMRT1. (A) Immunoblot analysis using the
anti-FLAG (M5) or anti-DMRT1 (4F6) monoclonal antibodies. pcDNA3-FLAG or
pcDNA3-FLAG-DMRT1 was transiently transfected into 293T cells. Extracts
of 293T cells were examined by immunoblotting with each antibody
followed by a HRP conjugated anti-mouse IgG antibody. DMRT1 was detected
as a single band at the same size by both antibodies (arrowhead). (B)
Immunoprecipitation (IP) analysis with the anti-FLAG (M5) or anti-DMRT1
(4F6) monoclonal antibodies. pcDNA3-FLAG-DMRT1 was transiently
transfected into 293T cells. The cell lysate was mixed with each
antibody and pulled down with protein A/G agarose. IP extracts were
examined by immunoblotting using the anti-DMRT1 polyclonal antibody
followed by a HRP conjugated anti-rabbit IgG antibody. (C)
Immunohistochemical analysis using the anti-DMRT1 monoclonal antibody
4F6 (1/10) and anti-DMRT1 polyclonal antibodies (1/1000) . Frozen
sections of adult testis were stained with Hoechst 33258 for nuclei and
reacted with both the antibodies, followed by Alexa 594-conjugated
anti-mouse (red) and Alexa 488 anti-rabbit-IgG (green) antibodies. Both
signals showed the same staining patterns.
Figure 2. Silver staining of immunoprecipitates using the anti-DMRT1 monoclonal
antibody 4F6 from X. laevis adult testes. Testis
extracts were mixed with normal mouse IgG or the anti-DMRT1 antibody
4F6, and pulled down with protein A/G agarose. The immunoprecipitates
were examined by silver staining (the right and middle lanes) or
immunoblotted with an anti-DMRT1 polyclonal antibody (right lane). Seven
4F6-specific bands were excised, and examined by LC-MS.
Figure 3. Effects of PACT/PRKRA and/or p53 on transcriptional activity by DMRT1
using luciferase reporter assay. 150 ng of DMRT1-driven firefly
luciferase reporter plasmid (p4xDMRT1-luc), 3.3 ng of DMRT1 expression
plasmid (pcDNA3-FLAG-DMRT1), and 10 ng Renilla luciferase vector
(pRL-TK-luc) as transfection internal control in the presence or absence
of PACT/PRKRA and/or p53 expression plasmids (pcDNA3-FLAG-PACT/PRKRA
and/or -p53) were transiently co-transfected into 293T cells, using 1.2
μg PEI MAX. Total amount of DNA was kept at 250 ng per each transfection
with pcDNA3-FLAG empty vector. 24 hours after transfection, cell lysates
were used to measure luciferase activity. Relative activity is shown as
the fold increase compared with the value obtained with 250 ng of
pcDNA3-FLAG empty vector. The symbols -, +, and ++ indicate 0, 3.3, and
20 ng, respectively. Values are expressed as mean ± SE, n = 3. The
letters above the bars indicate the results of Tukey HSD test following
one-way ANOVA (p < 0.05).
Figure 4. Expression of PACT/PRKRA mRNA in developing ZW and
ZZ gonads. (A) Quantitative RT-PCR analysis of
Pact/Prkra mRNA during gonadal development of ZW
(red) and ZZ (blue) tadpoles and adults in X. laevis.
cDNAs were synthesized using total RNAs from ZW and ZZ gonads at various
stages of tadpoles after sex determination, and at 6 weeks, 1 year, and
2 years of frogs after metamorphosis, and then amplified by PCR using
specific primer pairs as described in Table
S1. W and Y show weeks and year(s),
respectively. EF1α was used for normalization. RT-qPCR
data represent the mean (n=3) and SD. Values are expressed as mean ± SE,
n = 3. Differences among stages were evaluated by one-way ANOVA followed
by the Tukey HSD test (p < 0.05). Mean values not
sharing the same letters are significantly different from each other.
Sexual differences between ZZ and ZW gonads at each stage were evaluated
or by Student’s t-test (*p < 0.05).
N.S., not significant. (B) Distribution of Pact/Prkra
mRNAs on transverse sections of ZW and ZZ tadpole gonads at stage 56.
Whole-mount in situ hybridization of the gonads with
the attached mesonephros was performed with the
Pact/Prkra sense or anti-sense RNA probe, followed
by 7-μm cryostat sectioning. The sections were treated with an anti-VASA
monoclonal antibody to identify germ cells (red) and Hoechst 33258 for
nuclei (blue). Note that the nuclei in germ cells were faintly stained
by Hoechst 33258. Arrowheads indicate
Pact/Prkra-expressing germ cells. Scale bars, 20
μm.
Bavelloni,
Prohibitin 2: At a communications crossroads.
2015, Pubmed
Bavelloni,
Prohibitin 2: At a communications crossroads.
2015,
Pubmed Bennett,
The RAX/PACT-PKR stress response pathway promotes p53 sumoylation and activation, leading to G₁ arrest.
2012,
Pubmed Cuddihy,
The double-stranded RNA activated protein kinase PKR physically associates with the tumor suppressor p53 protein and phosphorylates human p53 on serine 392 in vitro.
1999,
Pubmed Fujitani,
Sexually dimorphic expression of Dmrt1 and γH2AX in germ stem cells during gonadal development in Xenopus laevis.
2016,
Pubmed
,
Xenbase Gebel,
Control mechanisms in germ cells mediated by p53 family proteins.
2017,
Pubmed Huang,
The C-terminal, third conserved motif of the protein activator PACT plays an essential role in the activation of double-stranded-RNA-dependent protein kinase (PKR).
2002,
Pubmed Ito,
JSAP1, a novel jun N-terminal protein kinase (JNK)-binding protein that functions as a Scaffold factor in the JNK signaling pathway.
1999,
Pubmed Krentz,
DMRT1 promotes oogenesis by transcriptional activation of Stra8 in the mammalian fetal ovary.
2011,
Pubmed Li,
PACT is a negative regulator of p53 and essential for cell growth and embryonic development.
2007,
Pubmed Masuyama,
Dmrt1 mutation causes a male-to-female sex reversal after the sex determination by Dmy in the medaka.
2012,
Pubmed Matson,
DMRT1 prevents female reprogramming in the postnatal mammalian testis.
2011,
Pubmed Matson,
The mammalian doublesex homolog DMRT1 is a transcriptional gatekeeper that controls the mitosis versus meiosis decision in male germ cells.
2010,
Pubmed Matsuda,
DMY is a Y-specific DM-domain gene required for male development in the medaka fish.
2002,
Pubmed Mawaribuchi,
Sex chromosome differentiation and the W- and Z-specific loci in Xenopus laevis.
2017,
Pubmed
,
Xenbase Mawaribuchi,
Molecular Evolution of Two Distinct dmrt1 Promoters for Germ and Somatic Cells in Vertebrate Gonads.
2017,
Pubmed
,
Xenbase Mawaribuchi,
Molecular evolution of vertebrate sex-determining genes.
2012,
Pubmed
,
Xenbase Nanda,
A duplicated copy of DMRT1 in the sex-determining region of the Y chromosome of the medaka, Oryzias latipes.
2002,
Pubmed Okamoto,
Direct interaction of p53 with the Y-box binding protein, YB-1: a mechanism for regulation of human gene expression.
2000,
Pubmed Patel,
PACT, a protein activator of the interferon-induced protein kinase, PKR.
1998,
Pubmed Redfern,
RNA-induced silencing complex (RISC) Proteins PACT, TRBP, and Dicer are SRA binding nuclear receptor coregulators.
2013,
Pubmed Sado,
Lymphocytes from enlarged iliac lymph nodes as fusion partners for the production of monoclonal antibodies after a single tail base immunization attempt.
2006,
Pubmed Smith,
The avian Z-linked gene DMRT1 is required for male sex determination in the chicken.
2009,
Pubmed Takashima,
Regulation of pluripotency in male germline stem cells by Dmrt1.
2013,
Pubmed Wada,
Masculinization-Related Genes and Cell-Mass Structures During Early Gonadal Differentiation in the African Clawed Frog Xenopus laevis.
2017,
Pubmed
,
Xenbase Yoshimoto,
Expression and promoter analysis of Xenopus DMRT1 and functional characterization of the transactivation property of its protein.
2006,
Pubmed
,
Xenbase Yoshimoto,
Opposite roles of DMRT1 and its W-linked paralogue, DM-W, in sexual dimorphism of Xenopus laevis: implications of a ZZ/ZW-type sex-determining system.
2010,
Pubmed
,
Xenbase Yoshimoto,
A W-linked DM-domain gene, DM-W, participates in primary ovary development in Xenopus laevis.
2008,
Pubmed
,
Xenbase Yoshimoto,
A ZZ/ZW-type sex determination in Xenopus laevis.
2011,
Pubmed
,
Xenbase Zhao,
Female-to-male sex reversal in mice caused by transgenic overexpression of Dmrt1.
2015,
Pubmed
