School of Biological Sciences and Technology, Chonnam National University, Gwangju 61186, Korea.
Mol Cells. 2018 Sep 30;41(9):842-852. doi: 10.14348/molcells.2018.0196. Epub 2018 Aug 30.
Notch signaling is an evolutionarily conserved pathway and involves in the regulation of various cellular and developmental processes. Ligand binding releases the intracellular domain of Notch receptor (NICD), which interacts with DNA-bound CSL [CBF1/Su(H)/Lag-1] to activate transcription of target genes. In the absence of NICD binding, CSL down-regulates target gene expression through the recruitment of various corepressor proteins including SMRT/NCoR (silencing mediator of retinoid and thyroid receptors/nuclear receptor corepressor), SHARP (SMRT/HDAC1-associated repressor protein), and KyoT2. Structural and functional studies revealed the molecular basis of these interactions, in which NICD coactivator and corepressor proteins competitively bind to β-trefoil domain (BTD) of CSL using a conserved ϕWϕP motif (ϕ denotes any hydrophobic residues). To date, there are conflicting ideas regarding the molecular mechanism of SMRT-mediated repression of CSL as to whether CSL-SMRT interaction is direct or indirect (via the bridge factor SHARP). To solve this issue, we mapped the CSL-binding region of SMRT and employed a 'one- plus two-hybrid system' to obtain CSL interaction-defective mutants for this region. We identified the CSL-interaction module of SMRT (CIMS; amino acid 1816-1846) as the molecular determinant of its direct interaction with CSL. Notably, CIMS contains a canonical ϕWϕP sequence (AP, amino acids 1832-1837) and directly interacts with CSL-BTD in a mode similar to other BTD-binding corepressors. Finally, we showed that CSL-interaction motif, rather than SHARP-interaction motif, of SMRT is involved in transcriptional repression of NICD in a cell-based assay. These results strongly suggest that SMRT participates in CSL-mediated repression via direct binding to CSL.
Notch 信号通路是一条进化上保守的通路，参与调节各种细胞和发育过程。配体结合释放 Notch 受体（NICD）的细胞内结构域，该结构域与 DNA 结合的 CSL [CBF1/Su(H)/Lag-1]相互作用，激活靶基因的转录。在没有 NICD 结合的情况下，CSL 通过募集包括 SMRT/NCoR（视黄酸和甲状腺受体沉默调节剂/核受体共抑制因子）、SHARP（SMRT/HDAC1 相关抑制蛋白）和 KyoT2 在内的各种核心抑制蛋白来下调靶基因表达。结构和功能研究揭示了这些相互作用的分子基础，其中 NICD 共激活因子和核心抑制蛋白通过使用保守的 ϕWϕP 基序（ϕ 表示任何疏水残基）竞争结合 CSL 的 β-三叶因子（BTD）。迄今为止，关于 SMRT 介导的 CSL 抑制的分子机制存在相互矛盾的观点，即 CSL-SMRT 相互作用是直接的还是间接的（通过桥接因子 SHARP）。为了解决这个问题，我们绘制了 SMRT 与 CSL 结合的区域，并采用了“一加二杂交系统”来获得该区域的 CSL 相互作用缺陷突变体。我们确定了 SMRT 与 CSL 直接相互作用的分子决定簇，即 SMRT 的 CSL 相互作用模块（CIMS；氨基酸 1816-1846）。值得注意的是，CIMS 包含一个典型的 ϕWϕP 序列（AP，氨基酸 1832-1837），并以类似于其他 BTD 结合核心抑制因子的模式直接与 CSL-BTD 相互作用。最后，我们表明，在基于细胞的测定中，SMRT 的 CSL 相互作用基序而不是 SHARP 相互作用基序参与了 NICD 的转录抑制。这些结果强烈表明，SMRT 通过直接与 CSL 结合参与 CSL 介导的抑制。
