Völkel Sara, Stielow Bastian, Finkernagel Florian, Stiewe Thorsten, Nist Andrea, Suske Guntram
Institute of Molecular Biology and Tumor Research (IMT), Philipps-University of Marburg, Marburg, Germany.
Genomics Core Facility, Zentrum für Tumor- und Immunbiologie (ZTI), Philipps-University of Marburg, Marburg, Germany.
PLoS Genet. 2015 Mar 20;11(3):e1005102. doi: 10.1371/journal.pgen.1005102. eCollection 2015 Mar.
Transcription factors are grouped into families based on sequence similarity within functional domains, particularly DNA-binding domains. The Specificity proteins Sp1, Sp2 and Sp3 are paradigmatic of closely related transcription factors. They share amino-terminal glutamine-rich regions and a conserved carboxy-terminal zinc finger domain that can bind to GC rich motifs in vitro. All three Sp proteins are ubiquitously expressed; yet they carry out unique functions in vivo raising the question of how specificity is achieved. Crucially, it is unknown whether they bind to distinct genomic sites and, if so, how binding site selection is accomplished. In this study, we have examined the genomic binding patterns of Sp1, Sp2 and Sp3 in mouse embryonic fibroblasts by ChIP-seq. Sp1 and Sp3 essentially occupy the same promoters and localize to GC boxes. The genomic binding pattern of Sp2 is different; Sp2 primarily localizes at CCAAT motifs. Consistently, re-expression of Sp2 and Sp3 mutants in corresponding knockout MEFs revealed strikingly different modes of genomic binding site selection. Most significantly, while the zinc fingers dictate genomic binding of Sp3, they are completely dispensable for binding of Sp2. Instead, the glutamine-rich amino-terminal region is sufficient for recruitment of Sp2 to its target promoters in vivo. We have identified the trimeric histone-fold CCAAT box binding transcription factor Nf-y as the major partner for Sp2-chromatin interaction. Nf-y is critical for recruitment of Sp2 to co-occupied regulatory elements. Equally, Sp2 potentiates binding of Nf-y to shared sites indicating the existence of an extensive Sp2-Nf-y interaction network. Our results unveil strikingly different recruitment mechanisms of Sp1/Sp2/Sp3 transcription factor members uncovering an unexpected layer of complexity in their binding to chromatin in vivo.
转录因子根据功能域内的序列相似性被分为不同家族，尤其是DNA结合域。特异性蛋白Sp1、Sp2和Sp3是密切相关转录因子的典型代表。它们共享富含谷氨酰胺的氨基末端区域和保守的羧基末端锌指结构域，该结构域在体外可与富含GC的基序结合。所有三种Sp蛋白均在全身表达；然而，它们在体内发挥独特功能，这就引发了特异性是如何实现的问题。至关重要的是，尚不清楚它们是否结合不同的基因组位点，如果是，那么结合位点的选择是如何完成的。在本研究中，我们通过ChIP-seq检测了小鼠胚胎成纤维细胞中Sp1、Sp2和Sp3的基因组结合模式。Sp1和Sp3基本上占据相同的启动子并定位于GC盒。Sp2的基因组结合模式不同；Sp2主要定位于CCAAT基序。一致地，在相应的基因敲除MEF中重新表达Sp2和Sp3突变体揭示了截然不同的基因组结合位点选择模式。最显著的是，虽然锌指决定了Sp3的基因组结合，但它们对于Sp2的结合完全是可有可无的。相反，富含谷氨酰胺的氨基末端区域足以在体内将Sp2募集到其靶启动子。我们已经确定三聚体组蛋白折叠CCAAT盒结合转录因子Nf-y是Sp2-染色质相互作用的主要伙伴。Nf-y对于将Sp2募集到共同占据的调控元件至关重要。同样，Sp2增强了Nf-y与共享位点的结合，表明存在广泛的Sp2-Nf-y相互作用网络。我们的结果揭示了Sp1/Sp2/Sp3转录因子成员截然不同的募集机制，揭示了它们在体内与染色质结合中意想不到的复杂层面。
