Beatson Institute for Cancer Research, Garscube Estate, Switchback Road, Bearsden, Glasgow G61 1BD, UK.
BMC Mol Biol. 2010 Mar 12;11:21. doi: 10.1186/1471-2199-11-21.
Differentiation of F9 embryonal carcinoma (EC) cells into parietal endoderm (PE) provides a tractable model system for studying molecular events during early and inaccessible stages of murine development. PE formation is accompanied by extensive changes in gene expression both in vivo and in culture. One of the most dramatic is the ~10-fold decrease in transcriptional output by RNA polymerase (pol) III. This has been attributed to changes in activity of TFIIIB, a factor that is necessary and sufficient to recruit pol III to promoters. The goal of this study was to identify molecular changes that can account for the low activity of TFIIIB following F9 cell differentiation.
Three essential subunits of TFIIIB decrease in abundance as F9 cells differentiate; these are Brf1 and Bdp1, which are pol III-specific, and TBP, which is also used by pols I and II. The decreased levels of Brf1 and Bdp1 proteins can be explained by reduced expression of the corresponding mRNAs. However, this is not the case for TBP, which is regulated post-transcriptionally. In proliferating cells, pol III transcription is stimulated by the proto-oncogene product c-Myc and the mitogen-activated protein kinase Erk, both of which bind to TFIIIB. However, c-Myc levels fall during differentiation and Erk becomes inactive through dephosphorylation. The diminished abundance of TFIIIB is therefore likely to be compounded by changes to these positive regulators that are required for its full activity. In addition, PE cells have elevated levels of the retinoblastoma protein RB, which is known to bind and repress TFIIIB.
The low activity of TFIIIB in PE can be attributed to a combination of changes, any one of which could be sufficient to inhibit pol III transcription. Declining levels of essential TFIIIB subunits and of activators that are required for maximal TFIIIB activity are accompanied by an increase in a potent repressor of TFIIIB. These events provide fail-safe guarantees to ensure that pol III output is appropriate to the diminished metabolic requirements of terminally differentiated cells.
F9 胚胎癌细胞（EC）向滋养外胚层（PE）的分化为研究小鼠发育早期和难以接近阶段的分子事件提供了一个易于处理的模型系统。PE 的形成伴随着体内和体外基因表达的广泛变化。其中最显著的变化之一是 RNA 聚合酶（pol）III 的转录输出减少了约 10 倍。这归因于 TFIIIB 活性的变化，TFIIIB 是招募 pol III 到启动子所必需和充分的因素。本研究的目的是鉴定可以解释 F9 细胞分化后 TFIIIB 低活性的分子变化。
TFIIIB 的三个必需亚基在 F9 细胞分化时丰度降低；这些亚基是 pol III 特异性的 Brf1 和 Bdp1，以及也被 pols I 和 II 使用的 TBP。Brf1 和 Bdp1 蛋白水平的降低可以用相应 mRNA 的表达减少来解释。然而，TBP 情况并非如此，TBP 是受转录后调控的。在增殖细胞中，pol III 转录受到原癌基因产物 c-Myc 和丝裂原活化蛋白激酶 Erk 的刺激，两者都与 TFIIIB 结合。然而，c-Myc 水平在分化过程中下降，Erk 通过去磷酸化而失活。TFIIIB 的丰度降低因此可能与这些正调控因子的变化有关，这些因子是其完全活性所必需的。此外，PE 细胞中视网膜母细胞瘤蛋白 RB 的水平升高，已知 RB 与 TFIIIB 结合并抑制 TFIIIB。
PE 中 TFIIIB 的低活性可以归因于多种变化的组合，其中任何一种变化都足以抑制 pol III 转录。必需的 TFIIIB 亚基和最大 TFIIIB 活性所需的激活剂的水平下降，伴随着 TFIIIB 强有力的抑制剂的增加。这些事件提供了可靠的保障，以确保 pol III 的输出与终末分化细胞降低的代谢需求相适应。
Zotero 插件
