Structural biology of transcriptional regulation by SATB1 that binds to matrix attachment region of DNA

SATB 1（specific AT-rich sequence binding protein 1）是一种转录因子，调节胸腺中免疫T细胞的成熟。它通过与靶基因启动子DNA的基质附着区（MAR）结合并将组蛋白脱乙酰酶募集到结合位点来抑制包括白细胞介素2受体在内的几种基因的转录。本研究从结构生物学的角度揭示了SATB 1识别MAR-DNA的机制，首先用核磁共振波谱法确定了SATB 1的MAR结合域（MBD）的结构。该结构具有五个α-螺旋排列在一个新的拓扑结构。通过NMR滴定实验和表面等离子体共振分析，确定了突变蛋白与DNA结合的相关区域。此外，通过对修饰碱基和沟特异性结合药物的DNA进行表面等离子体共振实验，发现SATB 1-MBD与DNA的结合是从大沟侧进行的，这与以往的推测不同。接下来，我们对SATB 1-MBD与MAR-DNA的复合物进行了结晶，在实验室衍射仪中的反射率高达2.0 nm，在同步加速器中的反射率高达1.75 nm。数据揭示了SATB 1-MBD与DNA大沟结合的复合物的结构。分子之间的直接氢键之间观察到一个单一的氨基酸和一个单一的碱基，和水介导的氢键和疏水相互作用发挥了重要作用的序列特异性识别。SATB 1-MBD属于CUT结构域，这是首次报道CUT结构域对DNA的识别机制。

SATB1 (special AT-rich sequence binding protein 1) is a transcription factor that regulates maturation of immune T-cells in thymus. It represses transcription of several genes including interleukin 2 receptor by binding to the matrix attachment region (MAR) of DNA of the target gene promoters and by recruiting histone deacetylase to the binding site. In this study we revealed the mechanism of recognition of MAR-DNA by SATB1 by means of structural biology.We first determined the structure of MAR binding domain (MBD) of SATB1 by NMR spectroscopy. The structure possesses five a-helices arranged in a novel topology. By NMR titration experiments and surface plasmon resonance analyses of DNA binding of mutant proteins, the region of the protein relevant to DNA binding was identified. Also, by surface plasmon resonance experiments with DNA including modified bases and groove-specific binding drugs, it was revealed that SATB1-MBD binds to DNA from the major groove side, which is different from what was expected previously.Next, we crystallized the complex of SATB1-MBD and MAR-DNA reflecting up to 2.0 Å in the laboratory diffractometer or 1.75 Å at synchrotron. Data revealed a structure of the complex in which SATB1-MBD binds to the major groove of DNA. Between the molecules direct hydrogen bonds are observed between a single amino acid and a single base, and water-mediated hydrogen bonds and hydrophobic interactions play a major role in the sequence-specific recognition. SATB1-MBD belongs to CUT domain and this is the first report on the mechanism of DNA recognition by CUT domains.