Search for: All records

Abstract Splicing factors have recently been shown to be involved in heterochromatin formation, but their role in controlling heterochromatin structure and function remains poorly understood. In this study, we identified a fission yeast homologue of human splicing factor RBM10, which has been linked to TARP syndrome. Overexpression of Rbm10 in fission yeast leads to strong global intron retention. Rbm10 also interacts with splicing factors in a pattern resembling that of human RBM10, suggesting that the function of Rbm10 as a splicing regulator is conserved. Surprisingly, our deep-sequencing data showed that deletion of Rbm10 caused only minor effect on genome-wide gene expression and splicing. However, the mutant displays severe heterochromatin defects. Further analyses indicated that the heterochromatin defects in the mutant did not result from mis-splicing of heterochromatin factors. Our proteomic data revealed that Rbm10 associates with the histone deacetylase Clr6 complex and chromatin remodelers known to be important for heterochromatin silencing. Deletion of Rbm10 results in significant reduction of Clr6 in heterochromatin. Our work together with previous findings further suggests that different splicing subunits may play distinct roles in heterochromatin regulation. 

Kinetochores, a protein complex assembled on centromeres, mediate chromosome segregation. In most eukaryotes, centromeres are epigenetically specified by the histone H3 variant CENP-A. CENP-T, an inner kinetochore protein, serves as a platform for the assembly of the outer kinetochore Ndc80 complex during mitosis. How CENP-T is regulated through the cell cycle remains unclear. Ccp1 (counteracter of CENP-A loading protein 1) associates with centromeres during interphase but delocalizes from centromeres during mitosis. Here, we demonstrated that Ccp1 directly interacts with CENP-T. CENP-T is important for the association of Ccp1 with centromeres, whereas CENP-T centromeric localization depends on Mis16, a homolog of human RbAp48/46. We identified a Ccp1-interaction motif (CIM) at the N terminus of CENP-T, which is adjacent to the Ndc80 receptor motif. The CIM domain is required for Ccp1 centromeric localization, and the CIM domain–deleted mutant phenocopies ccp1 Δ. The CIM domain can be phosphorylated by CDK1 (cyclin-dependent kinase 1). Phosphorylation of CIM weakens its interaction with Ccp1. Consistent with this, Ccp1 dissociates from centromeres through all stages of the cell cycle in the phosphomimetic mutant of the CIM domain, whereas in the phospho-null mutant of the domain, Ccp1 associates with centromeres during mitosis. We further show that the phospho-null mutant disrupts the positioning of the Ndc80 complex during mitosis, resulting in chromosome missegregation. This work suggests that competitive exclusion between Ccp1 and Ndc80 at the N terminus of CENP-T via phosphorylation ensures precise kinetochore assembly during mitosis and uncovers a previously unrecognized mechanism underlying kinetochore assembly through the cell cycle. 

The centromere is a specialized chromosomal structure essential for chromosome segregation. Centromere dysfunction leads to chromosome segregation errors and genome instability. In most eukaryotes, centromere identity is specified epigenetically by CENP-A, a centromere-specific histone H3 variant. CENP-A replaces histone H3 in centromeres, and nucleates the assembly of the kinetochore complex. Mislocalization of CENP-A to non-centromeric regions causes ectopic assembly of CENP-A chromatin, which has a devastating impact on chromosome segregation and has been linked to a variety of human cancers. How non-centromeric regions are protected from CENP-A misincorporation in normal cells is largely unexplored. Here, we review the most recent advances on the mechanisms underlying the prevention of ectopic centromere formation, and discuss the implications in human disease. 

Abstract Heterochromatin, a transcriptionally silenced chromatin domain, is important for genome stability and gene expression. Histone 3 lysine 9 methylation (H3K9me) and histone hypoacetylation are conserved epigenetic hallmarks of heterochromatin. In fission yeast, RNA interference (RNAi) plays a key role in H3K9 methylation and heterochromatin silencing. However, how RNAi machinery and histone deacetylases (HDACs) are coordinated to ensure proper heterochromatin assembly is still unclear. Previously, we showed that Dpb4, a conserved DNA polymerase epsilon subunit, plays a key role in the recruitment of HDACs to heterochromatin during S phase. Here, we identified a novel RNA-binding protein Dri1 that interacts with Dpb4. GFP-tagged Dri1 forms distinct foci mostly in the nucleus, showing a high degree of colocalization with Swi6/Heterochromatin Protein 1. Deletion of dri1+ leads to defects in silencing, H3K9me, and heterochromatic siRNA generation. We also showed that Dri1 physically associates with heterochromatic transcripts, and is required for the recruitment of the RNA-induced transcriptional silencing (RITS) complex via interacting with the complex. Furthermore, loss of Dri1 decreases the association of the Sir2 HDAC with heterochromatin. We further demonstrated that the C-terminus of Dri1 that includes an intrinsically disordered (IDR) region and three zinc fingers is crucial for its role in silencing. Together, our evidences suggest that Dri1 facilitates heterochromatin assembly via the RNAi pathway and HDAC.