Chromatin-associated protein kinase C-0 regulates an inducible gene expression program and microRNAs in human T lymphocytes. Homo sapiens

Studies in yeast have demonstrated that signalling kinases with well known cytoplasmic functions have a surprisingly active role in the nucleus, where they are tethered to chromatin and modulate gene expression programs. Here we provide evidence for a novel function of the signal transduction kinase, protein kinase C-theta (PKC-0) that physically associates with the proximal regulatory regions of key inducible immune response genes in human T cells. Chromatin-anchored PKC-q forms hitherto undescribed nuclear complexes by interacting with active RNA polymerase II, the histone kinase MSK-1 and the adaptor molecule 14-3-3z. ChIP-on-Chip analysis reveals that PKC-0 binds directly to both the promoter and transcribed regions of genes, as well as to the promoters of microRNA genes implicated in cell migration and invasion. Moreover, enforced expression of these microRNAs is associated with heightened production of mRNAs encoding a distinct subset of inducible immune response genes. Collectively, these data suggest that in addition to its well known role as a cytoplasmic signalling kinase, PKC-0 controls immune gene expression within the nucleus of T cells by participating in chromatin-associated signalling complexes Overall design: PKC-0 and Pol II ChIP DNA were pooled from five independent ChIP assay experiments that were first individually validated by real-time PCR. Pooled ChIP samples were subsequently amplified based on one round of the whole genome amplification method using the WGA2 kit (Sigma-Aldrich), as described previously (O'Geen, Hollenhorst, Dindot). An alternate random primed and linker-mediated PCR amplification protocol was compared with the WGA2 kit using quantitative real-time PCR, which generated similar yields of amplified material, but failed to preserve the degree of enrichment after the linker-mediated PCR amplification (data not shown). Labelling, hybridization and scanning were performed as described in the Mammalian ChIP-on-chip protocol (version 9.1; Agilent Technologies). Briefly, the control and PKC-0 or Pol II ChIP DNA were labelled with 5 ug cyanine-3 and 5 ug cyanine-5 (Invitrogen BioPrime CGH labeling kit), respectively. Samples were hybridised on each microarray slide for 40 h at 65°C. Agilent human promoter microarrays were utilised comprising of two slides per set defined to cover ~17,000 promoters of human transcripts from -5.5 to +2.5 Kb relative to transcriptional start site. The microarrays were scanned on an Agilent scanner (G2565BA) at 100% PMT gain. Two replicates of each ChIP-on-chip experiment were performed.

以酵母菌为模型的研究已证实，具备经典胞质功能的信号激酶在细胞核中具有出人意料的活跃作用：它们锚定在染色质上，并调控基因表达程序。本研究为信号转导激酶蛋白激酶C-θ（protein kinase C-theta, PKC-θ）的全新功能提供了实验证据——该激酶可在人类T细胞中与关键诱导型免疫应答基因的近端调控区域发生物理结合。
锚定在染色质上的PKC-θ可与活性RNA聚合酶II（RNA polymerase II）、组蛋白激酶MSK-1（histone kinase MSK-1）以及接头分子14-3-3ζ（adaptor molecule 14-3-3ζ）相互作用，形成此前未被报道过的核复合物。染色质免疫沉淀-芯片（ChIP-on-Chip）分析显示，PKC-θ可直接结合基因的启动子区与转录区，同时也可结合与细胞迁移、侵袭相关的microRNA基因（microRNA genes）的启动子区域。
此外，过表达这些microRNA基因与一类独特的诱导型免疫应答基因的编码mRNA表达量升高存在关联。综上，现有数据表明，除了其经典的胞质信号转导激酶功能外，PKC-θ还可通过参与染色质相关的信号复合物，在T细胞核内调控免疫基因的表达。
整体实验设计：将5次独立染色质免疫沉淀（Chromatin Immunoprecipitation, ChIP）实验所获得的PKC-θ与Pol II的ChIP DNA混合，上述每一次独立实验均通过实时荧光定量PCR（real-time PCR）进行了验证。随后，基于全基因组扩增方法（采用WGA2试剂盒（WGA2 kit），Sigma-Aldrich）对混合后的ChIP样品进行一轮扩增，具体操作如既往报道（O'Geen, Hollenhorst, Dindot）。我们通过实时荧光定量PCR（real-time PCR）比较了随机引物连接子介导PCR扩增方案与WGA2试剂盒的扩增效果，结果显示二者扩增产物得率相近，但连接子介导PCR扩增后无法保留原有的富集程度（数据未展示）。
标记、杂交与扫描操作均参照哺乳动物ChIP-on-Chip实验方案（版本9.1；安捷伦科技（Agilent Technologies））进行。简言之，对照组、PKC-θ组及Pol II组的ChIP DNA分别采用5 μg氰基3（cyanine-3, Cy3）与5 μg氰基5（cyanine-5, Cy5）进行标记（使用Invitrogen BioPrime CGH标记试剂盒（Invitrogen BioPrime CGH labeling kit））。样品在每张微阵列玻片上于65℃杂交40小时。本研究采用安捷伦人类启动子微阵列，每套包含两张玻片，可覆盖相对于转录起始位点（transcriptional start site）-5.5至+2.5 kb区域内的约17000个人类转录本启动子。使用安捷伦扫描仪（G2565BA）以100%光电倍增管（PMT）增益进行扫描。每项ChIP-on-Chip实验均设置两次生物学重复。