RelA Ser276 phosphorylation is required for activation of a subset of NF-κB-dependent genes by recruiting cyclin-dependent kinase 9/cyclin T1 complexes
RelA Ser276 phosphorylation is required for activation of a subset of NF-κB-dependent genes by recruiting cyclin-dependent kinase 9/cyclin T1 complexes
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DOI：
10.1128/mcb.01152-07
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发表时间：
2008-06-01
影响因子：
5.3
通讯作者：
Brasier, Allan R.
中科院分区：
文献类型：
--
作者：
Nowak, David E.;Tian, Bing;Brasier, Allan R.
NF-κ B在尼古丁诱导的炎症基因表达中起着重要作用。以前，我们凭经验确定了直接NF-κ B/RelA控制下的遗传网络的92个成员的身份，这些成员在转录诱导和峰值激活动力学的幅度上显示出显著的异质性。为了进一步研究这个网络，我们应用了最近开发的两步染色质免疫沉淀试验，准确地反映了RelA结合到其染色质靶点的关联和解离。尽管诱导型RelA结合在所有NF-κ B依赖性基因上以相似的动力学发生，但丝氨酸276（Ser（271））-磷酸化RelA结合主要见于由肿瘤坏死因子（TNF）快速诱导的基因子集上，包括Gro-β、白细胞介素-8（IL-8）和I κ B α。以前的工作表明，TNF诱导的RelA Ser（276）磷酸化是由活性氧（ROS）-蛋白激酶A信号通路控制的。为了进一步了解磷酸化Ser（276）RelA在靶基因表达中的作用，我们通过ROS清除剂和抗氧化剂抑制其形成，这些处理破坏磷酸化Ser（276）形成，但不破坏非磷酸化RelA的易位和DNA结合。我们发现磷酸化Ser（276）RelA仅是激活IL-8和Gro-β所必需的，而I κ B α不受影响。这些数据在使用用RelA Ser（276）Ala突变重建的RelA（-/-）鼠胚胎成纤维细胞的实验中得到证实。此外，我们观察到磷酸化Ser（276）RelA结合正性转录延伸因子B（P-TEF B），该复合物含有细胞周期蛋白依赖性激酶9（CDK-9）和细胞周期蛋白T1亚基。通过短干扰RNA（siRNA）介导的敲低抑制P-TEFb活性显示磷酸-Ser（276）RelA-P-TEFb复合物是IL-8和Gro-β基因激活所需的，但不是IKBot基因激活所需的。这些研究表明，TNF诱导靶基因表达的异质性机制。一种是由磷酸化-Ser（276）RelA形成和P-TEFb的染色质靶向介导的，所述P-TEFb控制聚合酶II（Pol II）募集和IL-8和Gro-β基因上的羧基末端结构域磷酸化。第二个涉及与Pol II预载的基因的磷酸-Ser（276）RetA非依赖性激活，例如I κ B α基因。总之，这些数据表明，结合动力学，基因组靶点的选择，和启动子诱导RelA的机制是由磷酸化密码影响其与辅激活因子和转录延伸因子的相互作用。
NF-kappa B plays a central role in cytokine-inducible inflammatory gene expression. Previously we empirically determined the identity of 92 members of the genetic network under direct NF-kappa B/RelA control that show marked heterogeneity in magnitude of transcriptional induction and kinetics of peak activation. To investigate this network further, we have applied a recently developed two-step chromatin immunoprecipitation assay that accurately reflects association and disassociation of RelA binding to its chromatin targets. Although inducible RelA binding occurs with similar kinetics on all NF-kappa B-dependent genes, serine 276 (Ser(271))-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-beta, interieukin-8 (IL-8), and I kappa B alpha. Previous work has shown that TNF-inducible RelA Ser(276) phosphorylation is controlled by a reactive oxygen species (ROS)-protein kinase A signaling pathway. To further understand the role of phospho-Ser(276) RelA in target gene expression, we inhibited its formation by ROS scavengers and antioxidants, treatments that disrupt phospho-Ser(276) formation but not the translocation and DNA binding of nonphosphorylated RelA. Here we find that phospho-Ser(276) RelA is required only for activation of IL-8 and Gro-beta, with I kappa B alpha being unaffected. These data were confirmed in experiments using RelA(-/-) murine embryonic fibroblasts reconstituted with a RelA Ser(276) Ala mutation. In addition, we observe that phospho-Ser(276) RelA binds the positive transcription elongation factor b (P-TEFb), a complex containing the cyclin-dependent kinase 9 (CDK-9) and cyclin T1 subunits. Inhibition of P-TEFb activity by short interfering RNA (siRNA)-mediated knockdown shows that the phospho-Ser(276) RelA-P-TEFb complex is required for IL-8 and Gro-beta gene activation but not for IKBot gene activation. These studies indicate that TNF induces target gene expression by heterogeneous mechanisms. One is mediated by phospho-Ser(276) RelA formation and chromatin targeting of P-TEFb controlling polymerase II (Pol II) recruitment and carboxy-terminal domain phosphorylation on the IL-8 and Gro-beta genes. The second involves a phospho-Ser(276) RetA-independent activation of genes preloaded with Pol II, exemplified by the I kappa B alpha gene. Together, these data suggest that the binding kinetics, selection of genomic targets, and mechanisms of promoter induction by RelA are controlled by a phosphorylation code influencing its interactions with coactivators and transcriptional elongation factors.