University of Lille, CNRS, UMR 8576 UGSF, Lille, France.
Aix Marseille University, CNRS, LCB, Marseille, France.
Mol Microbiol. 2020 Jul;114(1):127-139. doi: 10.1111/mmi.14500. Epub 2020 Apr 10.
In Caulobacter crescentus the combined action of chromosome replication and the expression of DNA methyl-transferase CcrM at the end of S-phase maintains a cyclic alternation between a full- to hemi-methylated chromosome. This transition of the chromosomal methylation pattern affects the DNA-binding properties of the transcription factor GcrA that controls the several key cell cycle functions. However, the molecular mechanism by which GcrA and methylation are linked to transcription is not fully elucidated yet. Using a combination of cell biology, genetics, and in vitro analysis, we deciphered how GcrA integrates the methylation pattern of several S-phase expressed genes to their transcriptional output. We demonstrated in vitro that transcription of ctrA from the P1 promoter in its hemi-methylated state is activated by GcrA, while in its fully methylated state GcrA had no effect. Further, GcrA and methylation together influence a peculiar distribution of creS transcripts, encoding for crescentin, the protein responsible for the characteristic shape of Caulobacter cells. This gene is duplicated at the onset of chromosome replication and the two hemi-methylated copies are spatially segregated. Our results indicated that GcrA transcribed only the copy where coding strand is methylated. In vitro transcription assay further substantiated this finding. As several of the cell cycle-regulated genes are also under the influence of methylation and GcrA-dependent transcriptional regulation, this could be a mechanism responsible for maintaining the gene transcription dosage during the S-phase.
在新月柄杆菌中，染色体复制和 S 期结束时 DNA 甲基转移酶 CcrM 的表达的共同作用维持了全甲基化到半甲基化染色体的周期性交替。这种染色体甲基化模式的转变影响了转录因子 GcrA 的 DNA 结合特性，从而控制着几个关键的细胞周期功能。然而，GcrA 和甲基化与转录之间的分子机制尚未完全阐明。我们结合细胞生物学、遗传学和体外分析，揭示了 GcrA 如何将几个 S 期表达基因的甲基化模式与其转录输出联系起来。我们在体外证明，在其半甲基化状态下，P1 启动子转录的 ctrA 基因被 GcrA 激活，而在其完全甲基化状态下，GcrA 没有影响。此外，GcrA 和甲基化共同影响 crescentin 编码基因 creS 的转录物的特殊分布，creS 基因编码的 crescentin 蛋白负责新月柄杆菌细胞的特征形状。该基因在染色体复制开始时被复制，两个半甲基化的拷贝被空间分隔。我们的结果表明，GcrA 只转录编码链被甲基化的拷贝。体外转录实验进一步证实了这一发现。由于几个细胞周期调控基因也受到甲基化和 GcrA 依赖的转录调控的影响，这可能是一种在 S 期维持基因转录剂量的机制。
