The mental retardation gene PHF8 mediates histone H4K20/H3K9 demethylation and regulates zebrafish brain apoptosis and craniofacial development: expression analysis

PHF8 (PHD Finger 8) mutations have been found in patients with X-linked mental retardation (XLMR) and craniofacial deformities. Here we identify PHF8 as the first enzyme that mediates demethylation of mono-methylated histone H4 lysine (K) 20 (H4K20me1), with additional activities towards H3K9me2/1 and H3K27me2. Patient mutations significantly compromise the demethylase activity, indicating functional importance. ChIP-seq identified PHF8 near the transcription start sites (TSS) of over 7000 target genes as well as in gene bodies and intergenic regions. PHF8 depletion resulted in up-regulation of H4K20me1 and H3K9me1 at the TSS-associated sites, and H3K9me2 in the gene bodies and intergenic regions, respectively, demonstrating differential substrate specificities at different target genomic locations. PHF8 depletion at the TSS results in decreased target gene expression, which is coincident with increased occupancy of the protein L3MBTL1 previously shown to induce chromatin compaction via binding to lower methyl states including H4K20me1 and H3K9me1. PHF8 RNAi and control RNAi stable HeLa cell lines were established. RNA were isolated from triplicate cell cultures. RNAs were subject to microarray expression analysis. Hybridizations were performed in duplicate.

PHF8（PHD Finger 8）突变已在X连锁智力障碍（X-linked mental retardation, XLMR）与颅面部畸形患者中被检出。本研究首次鉴定PHF8为首个介导单甲基化组蛋白H4赖氨酸20（H4K20me1）去甲基化的酶，同时对H3K9me2/1与H3K27me2具备额外的去甲基化活性。患者携带的PHF8突变可显著削弱其去甲基化酶活性，提示该蛋白具有关键生物学功能。染色质免疫共沉淀测序（ChIP-seq）分析显示，PHF8富集于超过7000个靶基因的转录起始位点（transcription start sites, TSS）附近，同时也分布于基因本体区域与基因间区。敲低PHF8可分别导致转录起始位点关联区域的H4K20me1与H3K9me1水平上调，以及基因本体区域与基因间区的H3K9me2水平升高，表明PHF8在不同基因组靶位点存在差异化的底物特异性。在转录起始位点处敲低PHF8会导致靶基因表达量下降，这与L3MBTL1蛋白的结合占有率升高相一致；既往研究表明，L3MBTL1可通过结合包括H4K20me1与H3K9me1在内的低甲基化状态组蛋白，诱导染色质浓缩。本研究构建了稳定转染靶向PHF8的RNA干扰（RNA interference, RNAi）与对照RNAi的HeLa细胞系。从三份生物学重复的细胞培养物中提取总RNA，随后进行微阵列表达谱分析。杂交实验重复进行了两次。