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From the * Howard Hughes Medical Institute and
Department of Biochemistry and Biophysics,
Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel
Hill, Chapel Hill, North Carolina
Present address: Career-Path Promotion
Unit for Young Life Scientists and International Young Scientists Career
Development Organization (ICDO), Kyoto University, Yoshida Konoe-cho,
Sakyo-ku, Kyoto 606-8501, Japan.
|| Present address: Department of
Gastroenterology, University of Tokyo Hospital, 7-3-1 Hongo, Bunkyo-Ku, Tokyo
113-8655, Japan.
| Correspondence to: Yuki Okada, Yoshida Konoe-cho, Sakyo-ku, Kyoto 606-8501, Japan (e-mail: ytokada{at}cp.kyoto-u.ac.jp).. |
| Received for publication May 11, 2009; accepted for publication October 5, 2009. |
Recent studies indicate that histone lysine methylation is subject to
enzyme-catalyzed reversion, and jumonji C (JmjC) domain–containing
proteins have been identified as one of the members of histone demethylases.
Although an increasing number of histone demethylases have been identified and
biochemically characterized, their biological functions are poorly
characterized. To elucidate the physiological functions, we generated the
knockout mouse model of dimethylated or monomethylated histone 3 lysine 9
(H3K9me2/1)–specific JmjC domain–containing histone demethylase 2A
(JHDM2A; also known as JMJD1A and KDM3A) and showed that JHDM2A is essential
for spermatogenesis. Jhdm2a-deficient mice exhibited impaired
postmeiotic chromatin condensation, which caused infertility, even though the
hormonal levels were maintained. Further molecular and biochemical analysis
revealed that JHDM2A directly bound to the core promoter regions of transition
nuclear protein 1 (Tnp1) and protamine 1 (Prm1) genes, and
it induced the transcriptional activation of these genes by removing H3K9
methylation, which is known as a silencing marker of gene transcription. This
work uncovered a role for JHDM2A in spermatogenesis and identified 2
downstream genes that are critical for sperm nuclear condensation. In
addition, we also showed that JHDM2A plays a role in regulating fat metabolic
gene expression in muscle and brown fat tissue, and the knockout mice
exhibited obesity and hyperlipidemia. Thus, JHDM2A possesses
organ/tissue-specific target genes, and impairment of this molecule cannot be
compensated by other JmjC-containing histone demethylases, suggesting the
importance of this molecule in vivo.
Key words: Sperm, histone demethylation
Histone methylation is described as "cellular memory," because the modification is sometimes maintained over cell division (Peters and Schubeler, 2005). Therefore, the existence of histone demethylase(s) has been questioned until the first discovery of histone demethylase, lysine-specific demethylase 1 (LSD1), by Shi et al in 2004. The next identified histone demethylase, JmjC-containing histone demethylase/F-box and leucine-rich repeat protein 11 (JHDM1A/Fbxl11), consisted of a large family called JmjC domain–containing proteins (Tsukada et al, 2006). Because the JmjC domain is responsible for the catalytic activity, it was easy to predict that other JmjC domain–containing proteins are also histone demethylases. At present, nearly 20 JmjC family members have been identified as demethylases for distinct methylated lysine residues in H3 (Klose et al, 2006), and now the discovery of histone demethylases raises a new question: What is the physiological output to cancel "cellular memories"?
Histone 3 Lysine 9 Methylation and Spermatogenesis
During mammalian spermatogenesis, unique and dynamic genetic/epigenetic
changes are observed, such as establishment of imprinting information in
primordial germ cells (PGCs), meiotic chromosomal recombination and
segregation, and histone removal followed by chromatin condensation in
spermiogenesis. In these events, it has been elucidated that alteration of
chromatin structure by histone modifications plays an important role
(Rousseaux et al, 2005;
Godmann et al, 2009), and among
these modifications, dynamics of histone 3 lysine 9 (H3K9) methylation are one
of the best-characterized modifications in the study of germ cell
development.
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Spermiogenic Defect in the Jhdm2a Knockout Mouse
Before JmjC-containing histone demethylase 2a (JHDM2A), also known as
JMJD1A or KDM3A, was identified as an H3K9 demethylase (for monomethylation
and dimethylation) in 2005, it was originally cloned as a testis-specific gene
transcript (Hoog et al, 1991;
Yamane et al, 2006).
Consistent with this previous report, immunohistochemical analysis using
anti-JHDM2A antibody revealed an intense nuclear expression in round
spermatids and a subnuclear distribution that was merged with the expression
of RNA polymerase II, indicating that JHDM2A may contribute to transcriptional
activation (Figure 2;
Okada et al, 2007).
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However, as described above, the genome-wide H3K9 methylation level is continuously maintained during spermatogeneis. So, how does the demethylase play a role? To further elucidate the importance of JHDM2A during spermatogenesis, Jhdm2a-deficient mice were generated (Okada et al, 2007). Although the Jhdm2a knockout mice were viable, males exhibited smaller testes, and they were functionally infertile. Histologically, spermatids of the knockout mice failed to elongate because of impaired chromatin condensation (Figure 3). Unexpectedly, genome-wide H3K9 methylation was unaltered. In addition, levels of luteinizing hormone (LH), follicle-stimulating hormone (FSH), and testosterone were also maintained in the knockout mice, although JHMD2A was reported to function as a transcriptional coactivator of the androgen receptor in a human prostate cancer cell line (Yamane et al, 2006). If neither genome-wide hypomethylation of H3K9 nor impaired hormonal regulation is the cause, what is the underlying molecular mechanism of the infertility? According to the subnuclear distribution that was similar to that of RNA polymerase II, it was speculated that JHDM2A was involved in transcriptional activation of gene(s) that must be essential for sperm chromatin condensation. In fact, reverse transcription–quantitative polymerase chain reaction analysis revealed decreased expression of 2 testis-specific basic proteins, transition protein 1 (Tnp1) and protamine 1 (Prm1), in round spermatids of the knockout mice. Chromatin immunoprecipitation (ChIP) assays further demonstrated that JHDM2A was recruited to the core promoter regions of both Tnp1 and Prm1 in round spermatids, whereas the recruitment of JHDM2A was not observed in the knockout mice. In addition, the methylation levels of H3K9 in these promoter regions were significantly higher in round spermatids of Jhdm2a knockout mice compared with those of the wild-type control. Thus, we propose a model in which JHDM2A contributes to spermatogenesis by directly controlling expression of Tnp1 and Prm1, which are both essential for sperm chromatin condensation. One of the questions that remain is how JHDM2A is specifically recruited to the Tnp1 and Prm1 promoters but not to other genes. JHDM2A itself does not contain a defined DNA-binding motif, and it appears no consensus DNA sequence consistently exists in the promoter regions of the target genes, including not only Prm1 and Tnp1 but also peroxisome proliferator–activated receptor alpha (Ppara) and uncoupling protein 1 (Ucp1), which are described in the next paragraph. Therefore, it can be argued that other JHDM2A-binding proteins are responsible for the DNA targeting.
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(ER) target
genes pS2, suggesting that there might be a possibility that JHDM2A plays some
role in the ER pathway
(Garcia-Bassets et al, 2007).
Another possible factor(s) that can link these 2 phenotypes is expression of
glucose metabolism–related genes expressed in testis, especially because
the Jhdm2a knockout mice are diabetic. Furthermore, although the
possibility might be low, searching for genetic mutations in the
Jhdm2a gene has been under investigation in human patients
(summarized in Figure 5).
Acknowledgments
Generation of the knockout mice was performed by Dr Yuji Mishina and the Knockout Mouse Core Facility at National Institutes of Health, National Institute of Environmental Health Sciences.
Footnotes
Supported by the National Institutes of Health (Y.Z.). Y.Z. is an Investigator of the Howard Hughes Medical Institute.
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H2AX)-positive (red) spermatogonia (SG) and spermatocytes (SC).
JHDM2A-positive signals disappear in elongated spermatids (ES). Cytoplasmic
staining of JHDM2A is observed in Sertoli cells (S). Hoechst (DNA) staining is
shown as blue.
