Department of Cell Biology and Genetics, Erasmus MC, The Netherlands.
Epigenetics Chromatin. 2010 Nov 8;3(1):19. doi: 10.1186/1756-8935-3-19.
CCCTC binding factor (CTCF) is a highly conserved zinc finger protein, which is involved in chromatin organization, local histone modifications, and RNA polymerase II-mediated gene transcription. CTCF may act by binding tightly to DNA and recruiting other proteins to mediate its various functions in the nucleus. To further explore the role of this essential factor, we used a mass spectrometry-based approach to screen for novel CTCF-interacting partners.
Using biotinylated CTCF as bait, we identified upstream binding factor (UBF) and multiple other components of the RNA polymerase I complex as potential CTCF-interacting partners. Interestingly, CTCFL, the testis-specific paralog of CTCF, also binds UBF. The interaction between CTCF(L) and UBF is direct, and requires the zinc finger domain of CTCF(L) and the high mobility group (HMG)-box 1 and dimerization domain of UBF. Because UBF is involved in RNA polymerase I-mediated ribosomal (r)RNA transcription, we analyzed CTCF binding to the rDNA repeat. We found that CTCF bound to a site upstream of the rDNA spacer promoter and preferred non-methylated over methylated rDNA. DNA binding by CTCF in turn stimulated binding of UBF. Absence of CTCF in cultured cells resulted in decreased association of UBF with rDNA and in nucleolar fusion. Furthermore, lack of CTCF led to reduced binding of RNA polymerase I and variant histone H2A.Z near the rDNA spacer promoter, a loss of specific histone modifications, and diminished transcription of non-coding RNA from the spacer promoter.
UBF is the first common interaction partner of CTCF and CTCFL, suggesting a role for these proteins in chromatin organization of the rDNA repeats. We propose that CTCF affects RNA polymerase I-mediated events globally by controlling nucleolar number, and locally by regulating chromatin at the rDNA spacer promoter, similar to RNA polymerase II promoters. CTCF may load UBF onto rDNA, thereby forming part of a network that maintains rDNA genes poised for transcription.
CCCTC 结合因子（CTCF）是一种高度保守的锌指蛋白，参与染色质组织、局部组蛋白修饰和 RNA 聚合酶 II 介导的基因转录。CTCF 可能通过紧密结合 DNA 并募集其他蛋白质来发挥其在核内的各种功能。为了进一步探索这个必需因子的作用，我们使用基于质谱的方法筛选新的 CTCF 相互作用伙伴。
使用生物素化的 CTCF 作为诱饵，我们鉴定出上游结合因子（UBF）和 RNA 聚合酶 I 复合物的多个其他成分是潜在的 CTCF 相互作用伙伴。有趣的是，CTCF 的睾丸特异性同源物 CTCFL 也与 UBF 结合。CTCF(L)与 UBF 的相互作用是直接的，需要 CTCF(L)的锌指结构域和 UBF 的高迁移率族框 1 和二聚化结构域。由于 UBF 参与 RNA 聚合酶 I 介导的核糖体（r）RNA 转录，我们分析了 CTCF 与 rDNA 重复序列的结合。我们发现 CTCF 结合在 rDNA 间隔启动子的上游位点，并且偏爱非甲基化的 rDNA 而不是甲基化的 rDNA。CTCF 的 DNA 结合反过来刺激 UBF 的结合。在培养细胞中缺乏 CTCF 导致 UBF 与 rDNA 的结合减少，核仁融合。此外，缺乏 CTCF 导致 rDNA 间隔启动子附近 RNA 聚合酶 I 和变体组蛋白 H2A.Z 的结合减少、特定组蛋白修饰的丧失以及非编码 RNA 从间隔启动子的转录减少。
UBF 是 CTCF 和 CTCFL 的第一个共同相互作用伙伴，表明这些蛋白质在 rDNA 重复序列的染色质组织中发挥作用。我们提出 CTCF 通过控制核仁数量全局影响 RNA 聚合酶 I 介导的事件，并且通过调节 rDNA 间隔启动子的染色质局部影响 RNA 聚合酶 I 介导的事件，类似于 RNA 聚合酶 II 启动子。CTCF 可以将 UBF 加载到 rDNA 上，从而形成维持 rDNA 基因转录状态的网络的一部分。
