Methylation of histone H3 lysine 36 is required for normal development in Neurospora crassa
Methylation of histone H3 lysine 36 is required for normal development in Neurospora crassa
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DOI：
10.1128/ec.4.8.1455-1464.2005
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发表时间：
2005-08-01
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影响因子：
--
通讯作者：
Selker, EU
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文献类型：
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作者：
Adhvaryu, KK;Morris, SA;Selker, EU
SET结构域是主要在组蛋白甲基转移酶（HMT）中发现的进化上保守的结构域。粗糙脉孢菌基因组包括9个SET结构域基因（set-1至set-9）以及dim-5，其编码DNA甲基化所需的组蛋白H3赖氨酸9 HMT。我们证明，脉孢菌set-2编码组蛋白H3赖氨酸36（K36）甲基转移酶，它是必不可少的正常生长和发育。我们使用重复诱导的点突变来产生具有多个无义突变的set-2突变体（set-2（RIP 1））。Western分析显示突变体缺乏SET-2蛋白和K36甲基化。一个氨基末端片段，包括AWS，SET，和后SET结构域的SET-2证明足够的K36 HMT活性在体外。核小体是比游离组蛋白更好的底物。set-2（RIP 1）突变体生长缓慢，分生孢子差，雌性不育。将野生型基因引入突变体补充了这些缺陷，证实它们是由于set-2功能丧失所致。我们将野生型组蛋白H3基因（hH 3）替换为在36位产生Lys至Leu取代的等位基因，并发现该hH 3（K36 L）突变体表型模仿set-2（RIP 1）突变体，证实观察到的生长和发育缺陷是由于不能甲基化H3的K36所致。最后，我们使用染色质免疫沉淀证明，活跃的粗糙脉孢菌转录基因富含H3甲基化的赖氨酸4和36。综上所述，我们的研究结果表明，K36在粗糙脉孢菌的甲基化是必不可少的正常生长和发育。
The SET domain is an evolutionarily conserved domain found predominantly in histone methyltransferases (HMTs). The Neurospora crassa genome includes nine SET domain genes (set-1 through set-9) in addition to dim-5, which encodes a histone H3 lysine 9 HMT required for DNA methylation. We demonstrate that Neurospora set-2 encodes a histone H3 lysine 36 (K36) methyltransferase and that it is essential for normal growth and development. We used repeat induced point mutation to make a set-2 mutant (set-2(RIP1)) with multiple nonsense mutations. Western analyses revealed that the mutant lacks SET-2 protein and K36 methylation. An amino-terminal fragment that includes the AWS, SET, and post-SET domains of SET-2 proved sufficient for K36 HMT activity in vitro. Nucleosomes were better substrates than free histones. The set-2(RIP1) mutant grows slowly, conidiates poorly, and is female sterile. Introducing the wild-type gene into the mutant complemented the defects, confirming that they resulted from loss of set-2 function. We replaced the wild-type histone H3 gene (hH3) with an allele producing a Lys to Leu substitution at position 36 and found that this hH3(K36L) mutant phenocopied the set-2(RIP1) mutant, confirming that the observed defects in growth and development result from inability to methylate K36 of H3. Finally, we used chromatin immunoprecipitation to demonstrate that actively transcribed genes in Neurospora crassa are enriched for H3 methylated at lysines 4 and 36. Taken together, our results suggest that methylation of K36 in Neurospora crassa is essential for normal growth and development.