Structural and functional study of Spindlin1/C11orf84 protein complex

Abstract

Histone methylation is critically important in transcriptional regulation and is involved in diverse biological processes. Aberrations in establishing or interpreting histone methylation marks may lead to disease. Spindlin1 is a histone methylation reader that recognizes H3K4me3 active transcription mark and H3K4me3R8me2a dual-methylation marks to regulate multiple cellular functions. However, it is challenging for a histone reader protein to complete such complicated functions. One intriguing question regarding Spindlin1 is whether it cooperates with other proteins to carry out its functions.

In this study, I identified and systematically characterized the structure and function of a novel protein complex formed by Spindlin1 and C11orf84. Chapter III describes the identification and structure determination of the Spindlin1/C11orf84 complex. C11orf84 binds to the Spindlin1 Tudor III domain but not Tudor I or Tudor II domains that are responsible for methylated histone H3 binding. Structural comparison of the Spindlin1/C11orf84 complex with the previously published Spindlin1 showed no obvious conformational change in the Spindlin1 Tudor I or Tudor II domains upon association with C11orf84, suggesting the Spindlin1/C11orf84 complex retains methylated H3 binding activity. Indeed, results from ITC and co-IP assays confirmed that the Spindlin1/C11orf84 complex binds to H3K4me3 or H3K4me3R8me2a peptides at a level comparable to that of Spindlin1 alone. To gain structural insight into the recognition of H3K4me3R8me2a dual-methylation marks by the Spindlin1/C11orf84 complex, I further crystallized and determined the high-resolution structure of the Spindlin1/C11orf84/H3K4me3R8me2a ternary complex. Comparison between the Spindlin1/C11orf84/H3K4me3R8me2a ternary complex structure and a previously published Spindlin1/H3K4me3R8me2a binary complex structure suggested that the H3R8me2a binding pocket of Spindlin1 may fit the H3K9me3 mark better. As predicted, the ITC assay showed that the Spindlin1/C11orf84 complex binds H3K4me3K9me3 more strongly than it binds H3K4me3R8me2a. I next determined the high-resolution structure of the Spindlin1/C11orf84/H3K4me3K9me3 ternary complex. This study, for the first time, reveals the molecular recognition of H3K4me3K9me3 bivalent marks by Spindlin1.

Chapter IV describes the characterization of the biological functions of the Spindlin1/C11orf84 complex. The genome-wide analysis demonstrated that C11orf84 and Spindlin1 co-occupy promoter regions of a large number of genes, in which a group of genes including an rRNA gene are co-regulated by both Spindlin1 and C11orf84. Spindlin1 and C11orf84 colocalize in nucleoli and active rDNA sites, reinforcing the regulatory role of the Spindlin1/C11orf84 complex in rRNA transcription. Depletion of Spindlin1 or C11orf84 represses the transcription of 45S pre-rRNA. Moreover, ectopic expression of wild type C11orf84 but not mutant C11orf84 that is unable to bind Spindlin1 could restore the 45S rRNA level, implying that C11orf84 cooperates with Spindlin1 in the activation of rRNA transcription. In addition to 45S rRNA transcription, C11orf84 is also involved in cell proliferation, similar to Spindlin1. Strikingly, C11orf84 stabilizes Spindlin1 in vivo. The functional studies suggest that C11orf84 stabilizes Spindlin1 and facilitates its functions in rRNA transcription.