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Abstract Heterochromatin is generally associated with the nuclear periphery, but how the spatial organization of heterochromatin is regulated to ensure epigenetic silencing remains unclear. Here we found that Sad1, an inner nuclear membrane SUN-family protein in fission yeast, interacts with histone H2A-H2B but not H3-H4. We solved the crystal structure of the histone binding motif (HBM) of Sad1 in complex with H2A-H2B, revealing the intimate contacts between Sad1_HBMand H2A-H2B. Structure-based mutagenesis studies revealed that the H2A-H2B-binding activity of Sad1 is required for the dynamic distribution of Sad1 throughout the nuclear envelope (NE). The Sad1-H2A-H2B complex mediates tethering telomeres and the mating-type locus to the NE. This complex is also important for heterochromatin silencing. Mechanistically, H2A-H2B enhances the interaction between Sad1 and HDACs, including Clr3 and Sir2, to maintain epigenetic identity of heterochromatin. Interestingly, our results suggest that Sad1 exhibits the histone-enhanced liquid-liquid phase separation property, which helps recruit heterochromatin factors to the NE. Our results uncover an unexpected role of SUN-family proteins in heterochromatin regulation and suggest a nucleosome-independent role of H2A-H2B in regulating Sad1’s functionality. 

Tandem DNA repeats are often organized into heterochromatin that is crucial for genome organization and stability. Recent studies revealed that individual repeats within tandem DNA repeats can behave very differently. How DNA repeats are assembled into distinct heterochromatin structures remains poorly understood. Here, we developed a genome-wide genetic screen using a reporter gene at different units in a repeat array. This screen led to identification of a conserved protein Rex1BD required for heterochromatin silencing. Our structural analysis revealed that Rex1BD forms a four-helix bundle structure with a distinct charged electrostatic surface. Mechanistically, Rex1BD facilitates the recruitment of Clr6 histone deacetylase (HDAC) by interacting with histones. Interestingly, Rex1BD also interacts with the 14-3-3 protein Rad25, which is responsible for recruiting the RITS (RNA-induced transcriptional silencing) complex to DNA repeats. Our results suggest that coordinated action of Rex1BD and Rad25 mediates formation of distinct heterochromatin structure at DNA repeats via linking RNAi and HDAC pathways. 

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. 

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.