Nucleolar protein Spindlin1 recognizes H3K4 methylation and stimulates the expression of rRNA genes.
ABSTRACT: The tandem Tudor-like domain-containing protein Spindlin1 has been reported to be a meiotic spindle-associated protein. Here we report that Spindlin1 is not associated with the spindle in mouse embryonic fibroblast cells during mitotic divisions. In interphase cells, Spindlin1 specifically localizes to the nucleoli. Moreover, Spindlin1 is a histone methylation effector protein that specifically recognizes H3K4 methylation. Finally, Spindlin1 localizes to the active ribosomal DNA (rDNA) repeats, and Spindlin1 facilitates the expression of rRNA genes.
Project description:Histone modification patterns and their combinatorial readout have emerged as a fundamental mechanism for epigenetic regulation. Here we characterized Spindlin1 as a histone effector that senses a cis-tail histone H3 methylation pattern involving trimethyllysine 4 (H3K4me3) and asymmetric dimethylarginine 8 (H3R8me2a) marks. Spindlin1 consists of triple tudor-like Spin/Ssty repeats. Cocrystal structure determination established concurrent recognition of H3K4me3 and H3R8me2a by Spin/Ssty repeats 2 and 1, respectively. Both H3K4me3 and H3R8me2a are recognized using an "insertion cavity" recognition mode, contributing to a methylation state-specific layer of regulation. In vivo functional studies suggest that Spindlin1 activates Wnt/?-catenin signaling downstream from protein arginine methyltransferase 2 (PRMT2) and the MLL complex, which together are capable of generating a specific H3 "K4me3-R8me2a" pattern. Mutagenesis of Spindlin1 reader pockets impairs activation of Wnt target genes. Taken together, our work connects a histone "lysine-arginine" methylation pattern readout by Spindlin1-to-Wnt signaling at the transcriptional level.
Project description:Recognition of methylated histone tail lysine residues by tudor domains plays important roles in epigenetic control of gene expression and DNA damage response. Previous studies revealed the binding of methyllysine in a cage of aromatic residues, but the molecular mechanism by which the sequence specificity for surrounding histone tail residues is achieved remains poorly understood. In the crystal structure of a trimethylated histone H3 lysine 4 (H3K4) peptide bound to the tudor-like domains of Spindlin1 presented here, an atypical mode of methyllysine recognition by an aromatic pocket of Spindlin1 is observed. Furthermore, the histone sequence is recognized in a distinct manner involving the amino terminus and a pair of arginine residues of histone H3, and disruption of the binding impaired stimulation of pre-RNA expression by Spindlin1. Our analysis demonstrates considerable diversities of methyllysine recognition and sequence-specific binding of histone tails by tudor domains, and the revelation furthers the understanding of tudor domain proteins in deciphering epigenetic marks on histone tails.
Project description:Epigenetic modifications of histone tails play an essential role in the regulation of eukaryotic transcription. Writer and eraser enzymes establish and maintain the epigenetic code by creating or removing posttranslational marks. Specific binding proteins, called readers, recognize the modifications and mediate epigenetic signalling. Here, we present a versatile assay platform for the investigation of the interaction between methyl lysine readers and their ligands. This can be utilized for the screening of small-molecule inhibitors of such protein-protein interactions and the detailed characterization of the inhibition. Our platform is constructed in a modular way consisting of orthogonal in vitro binding assays for ligand screening and verification of initial hits and biophysical, label-free techniques for further kinetic characterization of confirmed ligands. A stability assay for the investigation of target engagement in a cellular context complements the platform. We applied the complete evaluation chain to the Tudor domain containing protein Spindlin1 and established the in vitro test systems for the double Tudor domain of the histone demethylase JMJD2C. We finally conducted an exploratory screen for inhibitors of the interaction between Spindlin1 and H3K4me3 and identified A366 as the first nanomolar small-molecule ligand of a Tudor domain containing methyl lysine reader.
Project description:Hepatitis B virus infection (HBV) is a major risk factor for the development of hepatocellular carcinoma. HBV replicates from a covalently closed circular DNA (cccDNA) that remains as an episome within the nucleus of infected cells and serves as a template for the transcription of HBV RNAs. The regulatory protein HBx has been shown to be essential for cccDNA transcription in the context of infection. Here we identified Spindlin1, a cellular Tudor-domain protein, as an HBx interacting partner. We further demonstrated that Spindlin1 is recruited to the cccDNA and inhibits its transcription in the context of infection. Spindlin1 knockdown induced an increase in HBV transcription and in histone H4K4 trimethylation at the cccDNA, suggesting that Spindlin1 impacts on epigenetic regulation. Spindlin1-induced transcriptional inhibition was greater for the HBV virus deficient for the expression of HBx than for the HBV WT virus, suggesting that HBx counteracts Spindlin1 repression. Importantly, we showed that the repressive role of Spindlin1 is not limited to HBV transcription but also extends to other DNA virus that replicate within the nucleus such as Herpes Simplex Virus type 1 (HSV-1). Taken together our results identify Spindlin1 as a critical component of the intrinsic antiviral defense and shed new light on the function of HBx in HBV infection.
Project description:The discovery of inhibitors of methyl- and acetyl-binding domains has provided evidence for the 'druggability' of epigenetic effector molecules. The small-molecule probe UNC1215 prevents methyl-dependent protein-protein interactions by engaging the aromatic cage of MBT domains and, with lower affinity, Tudor domains. Using a library of tagged UNC1215 analogs, we screened a protein-domain microarray of human methyllysine effector molecules to rapidly detect compounds with new binding profiles with either increased or decreased specificity. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor-domain-containing protein Spindlin1 (SPIN1). Structural studies facilitated the rational synthesis of SPIN1 inhibitors with increased selectivity (EML631-633), which engage SPIN1 in cells, block its ability to 'read' H3K4me3 marks and inhibit its transcriptional-coactivator activity. Protein microarrays can thus be used as a platform to 'target-hop' and identify small molecules that bind and compete with domain-motif interactions.
Project description:Histone recognition by "reader" modules serves as a fundamental mechanism in epigenetic regulation. Previous studies have shown that Spindlin1 is a reader of histone H3K4me3 as well as "K4me3-R8me2a" and promotes transcription of rDNA or Wnt/TCF4 target genes. Here we show that Spindlin1 also acts as a potent reader of histone H3 "K4me3-K9me3/2" bivalent methylation pattern. Calorimetric titration revealed a binding affinity of 16 nm between Spindlin1 and H3 "K4me3-K9me3" peptide, which is one to three orders of magnitude stronger than most other histone readout events at peptide level. Structural studies revealed concurrent recognition of H3K4me3 and H3K9me3/2 by aromatic pockets 2 and 1 of Spindlin1, respectively. Epigenomic profiling studies showed that Spindlin1 colocalizes with both H3K4me3 and H3K9me3 peaks in a subset of genes enriched in biological processes of transcription and its regulation. Moreover, the distribution of Spindlin1 peaks is primarily associated with H3K4me3 but not H3K9me3, which suggests that Spindlin1 is a downstream effector of H3K4me3 generated in heterochromatic regions. Collectively, our work calls attention to an intriguing function of Spindlin1 as a potent H3 "K4me3-K9me3/2" bivalent mark reader, thereby balancing gene expression and silencing in H3K9me3/2-enriched regions.
Project description:Mammalian oocytes are arrested at prophase I of meiosis, and resume meiosis prior to ovulation. Coordination of meiotic arrest and resumption is partly dependent on the post-transcriptional regulation of maternal transcripts. Here, we report that, SPINDLIN1 (SPIN1), a maternal protein containing Tudor-like domains, interacts with a known mRNA-binding protein SERBP1, and is involved in regulating maternal transcripts to control meiotic resumption. Mouse oocytes deficient for Spin1 undergo normal folliculogenesis, but are defective in resuming meiosis. SPIN1, via its Tudor-like domain, forms a ribonucleoprotein complex with SERBP1, and regulating mRNA stability and/or translation. The mRNA for the cAMP-degrading enzyme, PDE3A, is reduced in Spin1 mutant oocytes, possibly contributing to meiotic arrest. Our study demonstrates that Spin1 regulates maternal transcripts post-transcriptionally and is involved in meiotic resumption.
Project description:The Tudor domain comprises a family of motifs that mediate protein-protein interactions required for various DNA-templated biological processes. Emerging evidence demonstrates a versatility of the Tudor family domains by identifying their specific interactions to a wide variety of histone methylation marks. Here, we discuss novel functions of a number of Tudor-containing proteins [including Jumonji domain-containing 2A (JMJD2A), p53-binding protein 1 (53BP1), SAGA-associated factor 29 (SGF29), Spindlin1, ubiquitin-like with PHD and RING finger domains 1 (UHRF1), PHD finger protein 1 (PHF1), PHD finger protein 19 (PHF19), and SAWADEE homeodomain homolog 1 (SHH1)] in 'reading' unique methylation events on histones in order to facilitate DNA damage repair or regulate transcription. This review covers our recent understanding of the molecular bases for histone-Tudor interactions and their biological outcomes. As deregulation of Tudor-containing proteins is associated with certain human disorders, pharmacological targeting of Tudor interactions could provide new avenues for therapeutic intervention.
Project description:Using a library of tagged UNC1215 analogs, we screened a protein domain microarray of methyl-lysine effector molecules to rapidly detect compounds with novel binding profiles. Using this approach, we identified a compound (EML405) that acquired a novel interaction with the Tudor domain-containing protein Spindlin1 (SPIN1). Structural studies revealed that the symmetric nature of EML405 allows it to simultaneously engage two of SPIN1’s Tudor domains, and also facilitated the rational synthesis of more selective SPIN1 inhibitor (EML631). The EML631 compound engages SPIN1 in cells, blocks its ability to “read” H3K4me3 marks, and inhibits its transcriptional coactivator activity. Overall design: RNA-seq of control, SPIN1 siRNA knockdown (24 hour post-transfection) and EML631 treated (10 mM, 3 days) T778 cells
Project description:Spindlin1 is a unique multivalent epigenetic reader that facilitates ribosomal RNA transcription. In this study, we provide molecular and structural basis by which Spindlin1 acts in complex with C11orf84 to preferentially recognize non-canonical bivalent mark of trimethylated lysine 4 and lysine 9 present on the same histone H3 tail (H3K4me3K9me3). We demonstrate that C11orf84 binding stabilizes Spindlin1 and enhances its association with bivalent H3K4me3K9me3 mark. The functional analysis suggests that Spindlin1/C11orf84 complex can displace HP1 proteins from H3K4me3K9me3-enriched rDNA loci, thereby facilitating the conversion of these poised rDNA repeats from the repressed state to the active conformation, and the consequent recruitment of RNA Polymerase I for rRNA transcription. Our study uncovers a previously unappreciated mechanism of bivalent H3K4me3K9me3 recognition by Spindlin1/C11orf84 complex required for activation of rRNA transcription.