Molecular basis for SPINDOC-Spindlin1 engagement and its role in transcriptional inhibition

Spindlin1 is a transcriptional coactivator with three Tudor-like domains, of which the first and second Tudors are engaged in histone methylation readout, while the function of the third Tudor is largely unknown. Recent studies revealed that the transcriptional co-activator activity of Spindlin1 could be attenuated by SPIN*DOC. Here we solved the crystal structure of SPIN*DOC-Spindlin1 complex, revealing that a hydrophobic motif, DOCpep3 (256-281), of SPIN*DOC interacts with Tudor 3 of Spindlin1 and completes its {beta}-barrel fold. Massive hydrophobic contacts and hydrogen bonding interactions ensure a high affinity DOCpep3-Spindlin1 engagement with a binding Kd of 30 nM. Interestingly, we characterized two more K/R-rich motifs of SPIN*DOC, DOCpep1 (187-195) and DOCpep2 (228-239), which bind to Spindlin1 at lower affinities with Kd values of 78 M and 31 M, respectively. Structural and binding studies revealed that DOCpep1 and DOCpep2 competitively bind to the aromatic cage of Spindlin1 Tudor 2 that is responsible for H3K4me3 readout. Although DOCpep3-Spindlin1 engagement is compatible with histone readout, an extended SPIN*DOC fragment containing DOCpep1 and DOCpep2 inhibits histone or TCF4 binding by Spindin1 due to introduced competition. This inhibitory effect is more pronounced for weaker binding targets but not for strong ones such as H3 "K4me3-K9me3" bivalent mark. Our RT-qPCR experiment showed that the removal of the hydrophobic motif or the K/R-rich region compromised the inhibitory effects of SPIN*DOC on Spindlin1-mediated transcriptional activation. In sum, here we revealed multivalent engagement between SPIN*DOC and Spindlin1, in which a hydrophobic motif acts as the primary binding site for stable SPIN*DOC-Spindlin1 association, while two more neighboring K/R-rich motifs further modulate the target selectivity of Spindlin1 via competitive inhibition, therefore attenuating the transcriptional co-activator activities of Spindlin1 through affecting its chromatin association.