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Structural Basis for Mis18 Complex Assembly: Implications for Centromere Maintenance

ABSTRACT: The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18β and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18β and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18β the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

ORGANISM(S): Homo sapiens (human)

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PROVIDER: S-BSST1408 | biostudies-other |

REPOSITORIES: biostudies-other

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Structural basis for Mis18 complex assembly and its implications for centromere maintenance.

Project description:The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18β and Mis18BP1), which recruits the CENP-A-specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18β and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18β, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

| S-EPMC11315898 | biostudies-literature

Structural Basis for Mis18 Complex Assembly and its Implications for Centromere Maintenance

Project description:The centromere, defined by the enrichment of CENP-A (a Histone H3 variant) containing nucleosomes, is a specialised chromosomal locus that acts as a microtubule attachment site. To preserve centromere identity, CENP-A levels must be maintained through active CENP-A loading during the cell cycle. A central player mediating this process is the Mis18 complex (Mis18α, Mis18β and Mis18BP1), which recruits the CENP-A specific chaperone HJURP to centromeres for CENP-A deposition. Here, using a multi-pronged approach, we characterise the structure of the Mis18 complex and show that multiple hetero- and homo-oligomeric interfaces facilitate the hetero-octameric Mis18 complex assembly composed of 4 Mis18α, 2 Mis18β and 2 Mis18BP1. Evaluation of structure-guided/separation-of-function mutants reveals structural determinants essential for cell cycle controlled Mis18 complex assembly and centromere maintenance. Our results provide new mechanistic insights on centromere maintenance, highlighting that while Mis18α can associate with centromeres and deposit CENP-A independently of Mis18β, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

| S-SCDT-10_1038-S44319-024-00183-W | biostudies-other

Structural Basis for CAL1-Mediated Centromere Maintenance

Project description:Intermolecular interactions stabilising the CAL1–CENP-A/H4 complex were analysed using chemical Cross-Linking Mass Spectrometry (CLMS). Purified recombinant CAL11-160–CENP-A101-225–H4 complex was crosslinked using EDC and BS3. The crosslinked peptides were analysed by mass spectrometry to identify intra- and intermolecular contacts. Notably, the data revealed several intramolecular crosslinks between the N- and C-terminal regions of CAL11-160, suggesting a direct interaction between these regions.

2020-03-11 | PXD017238 | Pride

Structural basis for centromere maintenance by Drosophila CENP-A chaperone CAL1.

Project description:Centromeres are microtubule attachment sites on chromosomes defined by the enrichment of histone variant CENP-A-containing nucleosomes. To preserve centromere identity, CENP-A must be escorted to centromeres by a CENP-A-specific chaperone for deposition. Despite this essential requirement, many eukaryotes differ in the composition of players involved in centromere maintenance, highlighting the plasticity of this process. In humans, CENP-A recognition and centromere targeting are achieved by HJURP and the Mis18 complex, respectively. Using X-ray crystallography, we here show how Drosophila CAL1, an evolutionarily distinct CENP-A histone chaperone, binds both CENP-A and the centromere receptor CENP-C without the requirement for the Mis18 complex. While an N-terminal CAL1 fragment wraps around CENP-A/H4 through multiple physical contacts, a C-terminal CAL1 fragment directly binds a CENP-C cupin domain dimer. Although divergent at the primary structure level, CAL1 thus binds CENP-A/H4 using evolutionarily conserved and adaptive structural principles. The CAL1 binding site on CENP-C is strategically positioned near the cupin dimerisation interface, restricting binding to just one CAL1 molecule per CENP-C dimer. Overall, by demonstrating how CAL1 binds CENP-A/H4 and CENP-C, we provide key insights into the minimalistic principles underlying centromere maintenance.

| S-EPMC7110144 | biostudies-literature

Structural basis of the DEK-nucleosome complex and its functional implications

Project description:The nuclear factor Dek is notably enriched within chromatin; nevertheless, the precise binding mechanism of Dek to the nucleosome remains elusive. In this study, we employ cryo-electron microscopy (cryo-EM) to elucidate the high-resolution structure of the Dek-Nucleosome Core Particle (NCP) complex. We identify specific domains responsible for DEK interaction with the histone octamer and DNA within the nucleosome. The veracity of these binding domains is confirmed through a series of meticulous biochemical experiments. Subsequently, cellular experiments reveal that Dek lacking nucleosome-binding capacity exhibits a deficiency in chromatin interaction. Remarkably, this impairment induces a shift towards the primitive endoderm fate in mouse embryonic stem cells, underscoring the pivotal role of Dek in determining cell fate through its nucleosomal interactions.

2023-12-31 | GSE247796 | GEO

Structural basis of the DEK-nucleosome complex and its functional implications (CUT&Tag)

2024-01-15 | GSE253062 | GEO

Structural basis for assembly of the CBF3 kinetochore complex.

Project description:Eukaryotic chromosomes contain a specialised region known as the centromere, which forms the platform for kinetochore assembly and microtubule attachment. The centromere is distinguished by the presence of nucleosomes containing the histone H3 variant, CENP-A. In budding yeast, centromere establishment begins with the recognition of a specific DNA sequence by the CBF3 complex. This in turn facilitates CENP-ACse4 nucleosome deposition and kinetochore assembly. Here, we describe a 3.6 Å single-particle cryo-EM reconstruction of the core CBF3 complex, incorporating the sequence-specific DNA-binding protein Cep3 together with regulatory subunits Ctf13 and Skp1. This provides the first structural data on Ctf13, defining it as an F-box protein of the leucine-rich-repeat family, and demonstrates how a novel F-box-mediated interaction between Ctf13 and Skp1 is responsible for initial assembly of the CBF3 complex.

| S-EPMC5771398 | biostudies-literature

A structural basis for kinetochore recruitment of the Ndc80 complex via two distinct centromere receptors.

Project description:The Ndc80 complex is the key microtubule-binding element of the kinetochore. In contrast to the well-characterized interaction of Ndc80-Nuf2 heads with microtubules, little is known about how the Spc24-25 heterodimer connects to centromeric chromatin. Here, we present molecular details of Spc24-25 in complex with the histone-fold protein Cnn1/CENP-T illustrating how this connection ultimately links microtubules to chromosomes. The conserved Ndc80 receptor motif of Cnn1 is bound as an α helix in a hydrophobic cleft at the interface between Spc24 and Spc25. Point mutations that disrupt the Ndc80-Cnn1 interaction also abrogate binding to the Mtw1 complex and are lethal in yeast. We identify a Cnn1-related motif in the Dsn1 subunit of the Mtw1 complex, necessary for Ndc80 binding and essential for yeast growth. Replacing this region with the Cnn1 peptide restores viability demonstrating functionality of the Ndc80-binding module in different molecular contexts. Finally, phosphorylation of the Cnn1 N-terminus coordinates the binding of the two competing Ndc80 interaction partners. Together, our data provide structural insights into the modular binding mechanism of the Ndc80 complex to its centromere recruiters.

| S-EPMC3567489 | biostudies-literature

Structural basis of preinitiation complex assembly on human pol II promoters.

Project description:Transcription initiation requires the assembly of a preinitiation complex (PIC), which is nucleated through binding of the TATA-box binding protein (TBP) to the promoter. Biochemical studies have shown, however, that TBP recognizes the TATA-box in both orientations and, therefore, cannot account for the directionality of PIC assembly. Transcription factor IIB (TFIIB) is essential for transcription initiation from RNA polymerase II promoters. Recent functional studies have identified a specific 7 bp TFIIB recognition element (BRE) immediately upstream of the TATA-box. We present here the 2.65 A resolution crystal structure of a human TFIIBc-TBPc complex bound to an idealized and extended adenovirus major late promoter. This structure now reveals that human TFIIBc binds to the promoter asymmetrically through base-specific contacts in the major groove upstream and in the minor groove downstream of the TATA-box. Binding of TFIIBc is, therefore, synergistic with TBPc requiring the distortion of the TATA-box. Thus, the newly described TFIIBc-DNA interface is likely to be a key determinant for the unidirectional assembly of a functional PIC.

| S-EPMC1171774 | biostudies-other

The structural basis for intramembrane assembly of an activating immunoreceptor complex.

Project description:Many receptors that activate cells of the immune system are multisubunit membrane protein complexes in which ligand recognition and signaling functions are contributed by separate protein modules. Receptors and signaling subunits assemble through contacts among basic and acidic residues in their transmembrane domains to form the functional complexes. Here we report the nuclear magnetic resonance (NMR) structure of the membrane-embedded, heterotrimeric assembly formed by association of the DAP12 signaling module with the natural killer (NK) cell-activating receptor NKG2C. The main intramembrane contact site is formed by a complex electrostatic network involving five hydrophilic transmembrane residues. Functional mutagenesis demonstrated that similar polar intramembrane motifs are also important for assembly of the NK cell-activating NKG2D-DAP10 complex and the T cell antigen receptor (TCR)-invariant signaling protein CD3 complex. This structural motif therefore lies at the core of the molecular organization of many activating immunoreceptors.

| S-EPMC3215083 | biostudies-literature

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