Estève Pierre-Olivier, Chin Hang Gyeong, Smallwood Andrea, Feehery George R, Gangisetty Omkaram, Karpf Adam R, Carey Michael F, Pradhan Sriharsa
New England Biolabs, Ipswich, Massachusetts 01938, USA.
Genes Dev. 2006 Nov 15;20(22):3089-103. doi: 10.1101/gad.1463706. Epub 2006 Nov 3.
Chromatin methylation is necessary for stable repression of gene expression during mammalian development. During cell division, DNMT1 maintains the DNA methylation pattern of the newly synthesized daughter strand, while G9a methylates H3K9. Here, DNMT1 is shown to directly bind G9a both in vivo and in vitro and to colocalize in the nucleus during DNA replication. The complex of DNMT1 and G9a colocalizes with dimethylated H3K9 (H3K9me2) at replication foci. Similarly, another H3K9 histone methyltransferase, SUV39H1, colocalizes with DNMT1 on heterochromatic regions of the nucleoli exclusively before cell division. Both DNMT1 and G9a are loaded onto the chromatin simultaneously in a ternary complex with loading factor PCNA during chromatin replication. Small interfering RNA (siRNA) knockdown of DNMT1 impairs DNA methylation, G9a loading, and H3K9 methylation on chromatin and rDNA repeats, confirming DNMT1 as the primary loading factor. Additionally, the complex of DNMT1 and G9a led to enhanced DNA and histone methylation of in vitro assembled chromatin substrates. Thus, direct cooperation between DNMT1 and G9a provides a mechanism of coordinated DNA and H3K9 methylation during cell division.
染色质甲基化对于哺乳动物发育过程中基因表达的稳定抑制是必需的。在细胞分裂过程中，DNMT1维持新合成子链的DNA甲基化模式，而G9a使H3K9甲基化。在此，研究表明DNMT1在体内和体外均直接与G9a结合，并在DNA复制期间于细胞核中共定位。DNMT1与G9a的复合物在复制位点与二甲基化的H3K9（H3K9me2）共定位。同样，另一种H3K9组蛋白甲基转移酶SUV39H1仅在细胞分裂前在核仁的异染色质区域与DNMT1共定位。在染色质复制期间，DNMT1和G9a与装载因子PCNA以三元复合物的形式同时加载到染色质上。用小干扰RNA（siRNA）敲低DNMT1会损害染色质和rDNA重复序列上的DNA甲基化、G9a加载以及H3K9甲基化，证实DNMT1是主要的装载因子。此外，DNMT1与G9a的复合物导致体外组装的染色质底物的DNA和组蛋白甲基化增强。因此，DNMT1与G9a之间的直接合作提供了一种在细胞分裂期间协调DNA和H3K9甲基化的机制。
