Recent studies have identified mutations in PHF8, an X-linked gene encoding a JmjC domain-containing protein, as a causal factor for X-linked mental retardation （XLMR） and cleft lip/cleft palate. However, the underl...Recent studies have identified mutations in PHF8, an X-linked gene encoding a JmjC domain-containing protein, as a causal factor for X-linked mental retardation （XLMR） and cleft lip/cleft palate. However, the underlying mechanism is unknown. Here we show that PHF8 is a histone demethylase and coactivator for retinoic acid receptor （RAR）. Although activities for both H3K4me3/2/1 and H3K9me2/1 demethylation were detected in cellularbased assays, reeombinant PHF8 exhibited only H3K9me2/1 demethylase activity in vitro, suggesting that PHF8 is an H3K9me2/1 demethylase whose specificity may be modulated in vivo. Importantly, a mutant PHF8 （phenylalanine at position 279 to serine） identified in the XLMR patients is defective in enzymatie activity, indicating that the loss of histone demethylase activity is causally linked with the onset of disease. In addition, we show that PHF8 binds specifically to H3K4me3/2 peptides via an N-terminal PHD finger domain. Consistent with a role for PHF8 in neuronal differentiation, knockdown of PHF8 in mouse embryonic carcinoma P19 cells impairs RA-induced neuronal differentiation, whereas overexpression of the wild-type but not the F279S mutant PHF8 drives PI9 cells toward neuronal differentiation. Furthermore, we show that PHF8 interacts with RAR~ and functions as a coactivator for RARa. Taken together, our results suggest that histone methylation modulated by PHF8 plays a critical role in neuronal differentiation.展开更多
Dynamic regulation of histone methylation/demethylation plays an important role during development. Mutations and truncations in human plant homeodomain （PHD） finger protein 8 （PHF8） are associated with X-linked m...Dynamic regulation of histone methylation/demethylation plays an important role during development. Mutations and truncations in human plant homeodomain （PHD） finger protein 8 （PHF8） are associated with X-linked mental retardation and facial anomalies, such as a long face, broad nasal tip, cleft lip/cleft palate and large hands, yet its molecular function and structural basis remain unclear. Here, we report the crystal structures of the catalytic core of PHF8 with or without α-ketoglutarate （α-KG） at high resolution. Biochemical and structural studies reveal that PHF8 is a novel histone demethylase specific for di- and mono-methylated histone H3 lysine 9 （H3K9me2/1）, but not for H3K9me3. Our analyses also reveal how human PHF8 discriminates between methylation states and achieves sequence specificity for methylated H3K9. The in vitro demethylation assay also showed that the F279S mutant observed in clinical patients possesses no demethylation activity, suggesting that loss of enzymatic activity is crucial for pathogenesis of PHF8 patients. Taken together, these results will shed light on the molecular mechanism underlying PHF8-associated developmental and neurological diseases.展开更多
文摘Recent studies have identified mutations in PHF8, an X-linked gene encoding a JmjC domain-containing protein, as a causal factor for X-linked mental retardation （XLMR） and cleft lip/cleft palate. However, the underlying mechanism is unknown. Here we show that PHF8 is a histone demethylase and coactivator for retinoic acid receptor （RAR）. Although activities for both H3K4me3/2/1 and H3K9me2/1 demethylation were detected in cellularbased assays, reeombinant PHF8 exhibited only H3K9me2/1 demethylase activity in vitro, suggesting that PHF8 is an H3K9me2/1 demethylase whose specificity may be modulated in vivo. Importantly, a mutant PHF8 （phenylalanine at position 279 to serine） identified in the XLMR patients is defective in enzymatie activity, indicating that the loss of histone demethylase activity is causally linked with the onset of disease. In addition, we show that PHF8 binds specifically to H3K4me3/2 peptides via an N-terminal PHD finger domain. Consistent with a role for PHF8 in neuronal differentiation, knockdown of PHF8 in mouse embryonic carcinoma P19 cells impairs RA-induced neuronal differentiation, whereas overexpression of the wild-type but not the F279S mutant PHF8 drives PI9 cells toward neuronal differentiation. Furthermore, we show that PHF8 interacts with RAR~ and functions as a coactivator for RARa. Taken together, our results suggest that histone methylation modulated by PHF8 plays a critical role in neuronal differentiation.
基金Supplementary information is linked to the online version of the paper on the Cell Research website.Acknowledgments We thank Dr Dawei Li （China Agricultural University） for generously providing us with the experimental conditions during the early stages of this project. We thank Dr Ruiming Xu （Institute of Biophysics, Chinese Academy of Sciences） for critical reading of this manuscript and advice. We thank Dr Pinchao Mei （Chinese Academy of Medical Sciences and Peking Union Medical College）, Xinqi Liu （Nankai University） and Jiemin Wong （East China Normal University） for discussions and advice. The synchrotronradiation experiments were performed at Shanghai Synchrotron Radiation Facility （SSRF） and NE3A in the Photon Factory. Z.C. is supported by the National Basic Research Program of China （973 Program, 2009CB825501）, the National Natural Science Foundation of China （30870494 and 90919043）, the New Century Excellent Talents in University （NCET-07-0808） and the Innovative Project of SKLAB. S. H. is supported by the National Key Laboratory Special Fund 2060204. Z. D. is supported by the National Natural Science Foundation of China （J0730639）.
文摘Dynamic regulation of histone methylation/demethylation plays an important role during development. Mutations and truncations in human plant homeodomain （PHD） finger protein 8 （PHF8） are associated with X-linked mental retardation and facial anomalies, such as a long face, broad nasal tip, cleft lip/cleft palate and large hands, yet its molecular function and structural basis remain unclear. Here, we report the crystal structures of the catalytic core of PHF8 with or without α-ketoglutarate （α-KG） at high resolution. Biochemical and structural studies reveal that PHF8 is a novel histone demethylase specific for di- and mono-methylated histone H3 lysine 9 （H3K9me2/1）, but not for H3K9me3. Our analyses also reveal how human PHF8 discriminates between methylation states and achieves sequence specificity for methylated H3K9. The in vitro demethylation assay also showed that the F279S mutant observed in clinical patients possesses no demethylation activity, suggesting that loss of enzymatic activity is crucial for pathogenesis of PHF8 patients. Taken together, these results will shed light on the molecular mechanism underlying PHF8-associated developmental and neurological diseases.
