As A Histone Demethylase PHF8 Involved In Neural Differentiation And The Function Of UHRF1 And UHRF2 In The Regulation Of Denovo DNA Methylation

Epigenetics is the study of heritable changes in gene expression or cellular phenotype caused by mechanisms other than changes in the underlying DNA sequence. DNA methylation and histone modifications are the two major mechanisms of epigenetic regulations. They both play critical roles in transcriptional regulation, X-chromosome inactivation, cell regulation, embryonic development, tumorigenesis, etc.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 and H3K9me2/l 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. 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 RARa 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.UHRF1 has emerged as a key epigenetic regulator due to its important role in DNA methylation maintenance. UHRF1 is able to bind to DNA replication forks through binding both hemi-methylated DNA and H3K9me2/3 and recruits DNMT1, which in turn carries out methylation of the new-synthesized DNA using the parent DNA as template, thus guaranteeing the correct transmission of DNA methylation to offspring. As another member of the UHRF protein family proteins, UHRF2 is highly similar to UHRF1 in amino acid sequence and protein structure.Although previous studies indicated that UHRF2 cannot substitute for UHRF1 and plays no role in DNA maintenance methylation. We have demonstrated in this study that UHRF2 is a negative regulator of DNA methylation and does so by inhibiting de novo DNA methylation via inducing DNMT3A degradation. Remarkably, we found that like UHRF2, UHRF1 was also capable of inducing ubiquitin-dependent protein degradation of DNMT3A, indicating that UHRF1 is not only required for DNA maintenance methylation but also plays a role in control of de novo DNA methylation. Subsequent analysis of lung cancer specimens with paired normal and tumor tissues revealed a general inverse correlation between UHRF1/2 and DNMT3A in tumors. We observed that in lung cancer A549 cell line knockdown of UHRF2 led to increased level of DNMT3A and impaired tumorigenesis in nude mice. Similarly, overexpression of the wild-type but not enzymatic deficient mutant DNMT3A in A549 cells resulted in impaired tumorigenesis in nude mice. Together these results indicate that both UHRF1 and UHRF2 negatively regulate de novo DNA methylation and that DNMT3A degradation induced by overexpression of UHRF1 and/or UHRF2 is a potential therapeutic targets for cancer.