Abstract
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 cellular-based assays, recombinant 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 enzymatic 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 P19 cells toward neuronal differentiation. Furthermore, we show that PHF8 interacts with RARĪ± and functions as a coactivator for RARĪ±. Taken together, our results suggest that histone methylation modulated by PHF8 plays a critical role in neuronal differentiation.
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Acknowledgements
We thank Drs Paul Wade (National Institute of Environmental Health Sciences, USA) and David Stewart (University of Houston, USA) for their critical reading of the manuscript. We also thank Dr Naihe Jing (Institute of Biochemistry and Cell Biology, SIBS, China) for assistance in RA-induced neuronal differentiation of P19 cells. This study was supported by grants from the National Natural Science Foundation of China (90919025, 30871381), the Ministry of Science and Technology of China (2009CB918402, 2009CB825601), and by funds from Ladies Leukemia League (LLL) (to SD), DLDCC Cancer Biology Pilot Project Award (to SD) and the Albert and Margaret Alkek Foundation (to SD).
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(Supplementary information is linked to the online version of the paper on Cell Research website.)
Supplementary information
Supplementary information, Figure S1
PHF8 did not appear to affect H3K27me1/2/3 and H3K36me2/3 in immunofluorescence assays. (PDF 60 kb)
Supplementary information, Figure S2
PHF8 exhibited no demethylase activity toward H3K4me3/2/1 in vitro. (PDF 44 kb)
Supplementary information, Figure S3
The PHF8 is associated with mitotic chromosomes in mitotic phase of cell cycle. (PDF 39 kb)
Supplementary information, Figure S4
Knockdown of PHF8 in P19 cells did not significantly affect RA-induced reduction of H3K9me2. (PDF 49 kb)
Supplementary information, Figure S5
The western blot analysis of siRNA knockdown of PHF8. (PDF 25 kb)
Supplementary information, Figure S6
PHF8 interacts with RARĪ± in vitro. (PDF 41 kb)
Supplementary information, Table S1
Antibodies against H3K4me2 (07-030), H3K4me3 (05-745), H3K9me2 (07-212), H3K9me3 (07-523), H3K27me3 (07-449) and H3K36me2 (07-274) were from Upstate Biotechnology. (PDF 54 kb)
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Qiu, J., Shi, G., Jia, Y. et al. The X-linked mental retardation gene PHF8 is a histone demethylase involved in neuronal differentiation. Cell Res 20, 908ā918 (2010). https://doi.org/10.1038/cr.2010.81
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DOI: https://doi.org/10.1038/cr.2010.81
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