DNA binding of the cell cycle transcriptional regulator GcrA depends on N6-adenosine methylation in Caulobacter crescentus and other Alphaproteobacteria.
DNA binding of the cell cycle transcriptional regulator GcrA depends on N6-adenosine methylation in Caulobacter crescentus and other Alphaproteobacteria.
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DOI：
10.1371/journal.pgen.1003541
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发表时间：
2013-05
期刊：
影响因子：
4.5
通讯作者：
Biondi EG
中科院分区：
文献类型：
--
作者：
Fioravanti A;Fumeaux C;Mohapatra SS;Bompard C;Brilli M;Frandi A;Castric V;Villeret V;Viollier PH;Biondi EG
在细菌模式系统新月形茎杆菌中，几种调节因子参与细胞周期进程的控制，该系统不对称地分为营养g1期（蜂拥）细胞和复制s期（跟踪）细胞。在这里，我们报道了谜一样的细胞周期调节因子GcrA和n6 -腺苷甲基转移酶CcrM之间的一种新的功能相互作用，这两种蛋白在α变形菌中都是高度保守的，分别在s期早期和末期被激活。由于GcrA和CcrM之间没有直接的生化和调控关系，我们使用ChIP（染色质免疫沉淀），生化和生物物理实验以及遗传学相结合的方法来证明GcrA是一种二聚体dna结合蛋白，优先靶向含有CcrM甲基化位点的启动子。在全基因组范围内追踪ccrm依赖性n6 -甲基腺苷启动子标记后，我们发现这些标记在体外和体内招募GcrA。此外，我们发现，在甲基化靶标存在的情况下，GcrA将RNA聚合酶招募到启动子上，这与其在转录激活中的作用一致。由于甲基化依赖的DNA结合也被观察到与其他α变形菌的GcrA同源物结合，我们得出结论，GcrA是一类新的和保守的转录调节因子的创始成员，这些转录调节因子作为甲基化依赖（不可遗传）表观遗传开关的分子效应物，在细胞周期中调节基因表达。基因组DNA在特定调控位点的甲基化可以影响真核细胞中无数的过程。在细菌中，已知腺苷（m6A） N6位置的甲基化可介导非适应性免疫反应，以保护细胞免受外来DNA的侵害。虽然目前还不知道m6A标记在γ变形菌中调控细胞周期基因的表达，但在模型α变形菌Caulobacter crescentus中，细胞周期转录需要m6A甲基转移酶CcrM，该酶在GAnTC序列中引入m6A标记和神秘因子GcrA。在研究CcrM和GcrA之间是否存在功能和生化关系时，我们发现CcrM依赖的m6A标记在体外和体内将GcrA招募到细胞周期基因的启动子中，这是高效转录所必需的。GcrA与RNA聚合酶相互作用，解释了细胞周期转录如何受到影响。重要的是，m6a依赖性结合也在GcrA同源物中发现，这表明CcrM和GcrA的这种转录调控机制在Alphaproteobacteria中是保守的。
Several regulators are involved in the control of cell cycle progression in the bacterial model system Caulobacter crescentus, which divides asymmetrically into a vegetative G1-phase (swarmer) cell and a replicative S-phase (stalked) cell. Here we report a novel functional interaction between the enigmatic cell cycle regulator GcrA and the N6-adenosine methyltransferase CcrM, both highly conserved proteins among Alphaproteobacteria, that are activated early and at the end of S-phase, respectively. As no direct biochemical and regulatory relationship between GcrA and CcrM were known, we used a combination of ChIP (chromatin-immunoprecipitation), biochemical and biophysical experimentation, and genetics to show that GcrA is a dimeric DNA–binding protein that preferentially targets promoters harbouring CcrM methylation sites. After tracing CcrM-dependent N6-methyl-adenosine promoter marks at a genome-wide scale, we show that these marks recruit GcrA in vitro and in vivo. Moreover, we found that, in the presence of a methylated target, GcrA recruits the RNA polymerase to the promoter, consistent with its role in transcriptional activation. Since methylation-dependent DNA binding is also observed with GcrA orthologs from other Alphaproteobacteria, we conclude that GcrA is the founding member of a new and conserved class of transcriptional regulators that function as molecular effectors of a methylation-dependent (non-heritable) epigenetic switch that regulates gene expression during the cell cycle. Methylation of genomic DNA at a specific regulatory site can impact a myriad of processes in eukaryotic cells. In bacteria, methylation at the N6 position of adenosine (m6A) is known to mediate a non-adaptive immunity response to protect cells from foreign DNA. While m6A marks are not known to govern expression of cell cycle genes in Gammaproteobacteria, cell cycle transcription in the model alphaproteobacterium Caulobacter crescentus requires the m6A methyltransferase CcrM that introduces m6A marks at GAnTC sequences and the enigmatic factor GcrA. Investigating if a functional and biochemical relationship exists between CcrM and GcrA, we found that CcrM-dependent m6A marks recruit GcrA to the promoters of cell cycle genes in vitro and in vivo and is required for efficient transcription. GcrA interacts with RNA polymerase, explaining how cell cycle transcription is affected. Importantly, m6A-dependent binding is also seen in GcrA orthologs, indicating that this transcriptional regulatory mechanism by CcrM and GcrA is conserved in Alphaproteobacteria.
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影响因子：
14.9
作者：
Goujon M;McWilliam H;Li W;Valentin F;Squizzato S;Paern J;Lopez R
通讯作者：
Lopez R
影响因子：
4.5
作者：
Brunet YR;Bernard CS;Gavioli M;Lloubès R;Cascales E
通讯作者：
Cascales E
影响因子：
4.5
作者：
Demarre G;Chattoraj DK
通讯作者：
Chattoraj DK
DOI：
10.1073/pnas.93.3.1210
发表时间：
1996-02-06
影响因子：
11.1
作者：
Stephens, C;Reisenauer, A;Shapiro, L
通讯作者：
Shapiro, L
影响因子：
64.5
作者：
Thanbichler, Martin;Shapiro, Lucy
通讯作者：
Shapiro, Lucy