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

SATB1 （special AT-rich sequence binding protein 1）是胸腺中调节免疫t细胞成熟的转录因子。它通过结合靶基因启动子DNA的基质附着区（MAR）和将组蛋白去乙酰化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SATB1对MAR-DNA的识别机制。我们首先通过核磁共振光谱测定了SATB1的MAR结合域（MBD）的结构。该结构具有以新颖的拓扑结构排列的五个a型螺旋。通过核磁共振滴定实验和表面等离子体共振分析突变蛋白的DNA结合，确定了与DNA结合相关的蛋白质区域。此外，通过与DNA的表面等离子体共振实验，包括修饰碱基和凹槽特异性结合药物，发现SATB1-MBD从主凹槽侧结合DNA，这与之前的预期不同。接下来，我们结晶了SATB1-MBD和MAR-DNA的复合物，在实验室衍射仪中反射高达2.0 Å，在同步加速器中反射高达1.75 Å。数据揭示了SATB1-MBD与DNA主槽结合的复合物结构。分子间单个氨基酸和单个碱基之间存在直接氢键，水介导的氢键和疏水相互作用在序列特异性识别中起主要作用。SATB1-MBD属于CUT结构域，这是关于CUT结构域识别DNA机制的首次报道。

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.