Histone H4K20 Demethylation by Two hHR23 Proteins

Histone methyl groups can be removed by demethylases. Although LSD1 and JmjC domain-containing proteins have been identified as histone demethylases, enzymes for many histone methylation states or sites are still unknown. Here, we perform a screening of a cDNA library containing 2,500 nuclear proteins and identify hHR23A as a histone H4K20 demethylase. Overexpression of hHR23A reduces the levels of H4K20me1/2/3 in cells. In vitro, hHR23A specifically demethylates H4K20me1/2/3 and generates formaldehyde. The enzymatic activity requires Fe(II) and alpha-ketoglutarate as cofactors and the UBA domains of hHR23A. hHR23B, a protein homologous to hHR23A, also demethylates H4K20me1/2/3 in vitro and in vivo. We further demonstrate that hHR23A/B activate the transcription of coding genes by demethylating H4K20me1 and the transcription of repetitive elements by demethylating H4K20me3. Nuclear magnetic resonance (NMR) analyses demonstrate that an HxxxE motif in the UBA1 domain is crucial for iron binding and demethylase activity. Thus, we identify two hHR23 proteins as histone demethylases.

组蛋白甲基基团可被去甲基化酶去除。尽管LSD1和含JmjC结构域的蛋白质已被确定为组蛋白去甲基化酶，但许多组蛋白甲基化状态或位点所对应的酶仍不为人知。在此，我们对一个包含2500种核蛋白的cDNA文库进行了筛选，并确定hHR23A是一种组蛋白H4K20去甲基化酶。hHR23A的过表达会降低细胞中H4K20me1/2/3的水平。在体外，hHR23A特异性地使H4K20me1/2/3去甲基化并产生甲醛。该酶活性需要Fe(II)和α-酮戊二酸作为辅因子以及hHR23A的UBA结构域。hHR23B是一种与hHR23A同源的蛋白质，它在体外和体内也能使H4K20me1/2/3去甲基化。我们进一步证明hHR23A/B通过使H4K20me1去甲基化激活编码基因的转录，并通过使H4K20me3去甲基化激活重复元件的转录。核磁共振（NMR）分析表明，UBA1结构域中的一个HxxxE基序对铁结合和去甲基化酶活性至关重要。因此，我们确定了两种hHR23蛋白质为组蛋白去甲基化酶。