Nowak David E, Tian Bing, Jamaluddin Mohammad, Boldogh Istvan, Vergara Leoncio A, Choudhary Sanjeev, Brasier Allan R
Department of Medicine, The University of Texas Medical Branch, Galveston, TX 77555-1060, USA.
Mol Cell Biol. 2008 Jun;28(11):3623-38. doi: 10.1128/MCB.01152-07. Epub 2008 Mar 24.
NF-kappaB 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-kappaB/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-kappaB-dependent genes, serine 276 (Ser(276))-phosphorylated RelA binding is seen primarily on a subset of genes that are rapidly induced by tumor necrosis factor (TNF), including Gro-beta, interleukin-8 (IL-8), and IkappaBalpha. 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 IkappaBalpha 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 IkappaBalpha 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) RelA-independent activation of genes preloaded with Pol II, exemplified by the IkappaBalpha 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.
核因子-κB（NF-κB）在细胞因子诱导的炎症基因表达中起核心作用。此前，我们通过实验确定了直接受NF-κB/RelA控制的遗传网络中92个成员的身份，这些成员在转录诱导幅度和峰值激活动力学方面表现出显著的异质性。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分析方法，该方法能准确反映RelA与其染色质靶点的结合和解离。虽然诱导型RelA结合在所有NF-κB依赖基因上的动力学相似，但丝氨酸276（Ser(276)）磷酸化的RelA结合主要见于肿瘤坏死因子（TNF）快速诱导的一部分基因，包括Gro-β、白细胞介素-8（IL-8）和IκBα。先前的研究表明，TNF诱导的RelA Ser(276)磷酸化受活性氧（ROS）-蛋白激酶A信号通路控制。为了进一步了解磷酸化的Ser(276) RelA在靶基因表达中的作用，我们用ROS清除剂和抗氧化剂抑制其形成，这些处理会破坏磷酸化的Ser(276)形成，但不影响未磷酸化RelA的易位和DNA结合。在此我们发现，磷酸化的Ser(276) RelA仅对IL-8和Gro-β的激活是必需的，而IκBα不受影响。这些数据在用RelA Ser(276)Ala突变重建的RelA(-/-)小鼠胚胎成纤维细胞的实验中得到了证实。此外，我们观察到磷酸化的Ser(276) RelA与正转录延伸因子b（P-TEFb）结合，P-TEFb是一个包含细胞周期蛋白依赖性激酶9（CDK-9）和细胞周期蛋白T1亚基的复合物。通过短干扰RNA（siRNA）介导的敲低抑制P-TEFb活性表明，磷酸化的Ser(276) RelA-P-TEFb复合物对IL-8和Gro-β基因激活是必需的，但对IκBα基因激活不是必需的。这些研究表明，TNF通过异质性机制诱导靶基因表达。一种机制是由磷酸化的Ser(276) RelA形成以及P-TEFb的染色质靶向介导，P-TEFb控制IL-8和Gro-β基因上聚合酶II（Pol II）的募集和羧基末端结构域的磷酸化。第二种机制涉及由预先加载有Pol II的基因的磷酸化Ser(276) RelA非依赖性激活，以IκBα基因为例。总之，这些数据表明RelA的结合动力学、基因组靶点的选择以及启动子诱导机制受影响其与共激活因子和转录延伸因子相互作用的磷酸化密码控制。
