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

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, Mis18ß 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 Mis18ß 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 Mis18ß, the latter is indispensable for the optimal level of CENP-A loading required for preserving the centromere identity.

MIS18A upregulation promotes cell viability, migration and tumor immune evasion in lung adenocarcinoma.

Lung adenocarcinoma (LUAD) presents a significant global health challenge owing to its poor prognosis and high mortality rates. Despite its involvement in the initiation and progression of a number of cancer types, the understanding of the precise impact of MIS18 kinetochore protein A (MIS18A) on LUAD remains incomplete. In the present study, the role of MIS18A in LUAD was investigated by analyzing the genomic and clinical data from multiple public datasets. The expression of MIS18A was validated using reverse transcription-quantitative polymerase chain reaction, and in vitro experiments involving small interfering RNA-induced downregulation of MIS18A in lung cancer cells were conducted to further explore its impact. These findings revealed that elevated MIS18A expression in LUAD was associated with advanced clinical features and poor prognosis. Functional analysis also revealed the role of MIS18A in regulating the cell cycle and immune-related pathways. Moreover, MIS18A altered the immune microenvironment in LUAD, influencing its response to immunotherapy and drug sensitivity. The results of the in vitro experiments indicated that suppression of MIS18A expression reduced the proliferative and migratory capacities of LUAD cells. In summary, MIS18A possesses potential as a biomarker and may serve as a possible therapeutic target for LUAD, with significant implications for tumor progression by influencing both cell cycle dynamics and immune infiltration.

Direct recruitment of Mis18 to interphase spindle pole bodies promotes CENP-A chromatin assembly.

CENP-A chromatin specifies mammalian centromere identity, and its chaperone HJURP replenishes CENP-A when recruited by the Mis18 complex (Mis18C) via M18BP1/KNL2 to CENP-C at kinetochores during interphase. However, the Mis18C recruitment mechanism remains unresolved in species lacking M18BP1, such as fission yeast. Fission yeast centromeres cluster at G2 spindle pole bodies (SPBs) when CENP-ACnp1 is replenished and where Mis18C also localizes. We show that SPBs play an unexpected role in concentrating Mis18C near centromeres through the recruitment of Mis18 by direct binding to the major SPB linker of nucleoskeleton and cytoskeleton (LINC) component Sad1. Mis18C recruitment by Sad1 is important for CENP-ACnp1 chromatin establishment and acts in parallel with a CENP-C-mediated Mis18C recruitment pathway to maintain centromeric CENP-ACnp1 but operates independently of Sad1-mediated centromere clustering. SPBs therefore provide a non-chromosomal scaffold for both Mis18C recruitment and centromere clustering during G2. This centromere-independent Mis18-SPB recruitment provides a mechanism that governs de novo CENP-ACnp1 chromatin assembly by the proximity of appropriate sequences to SPBs and highlights how nuclear spatial organization influences centromere identity.

Identification of Novel Gene Variants for Autism Spectrum Disorders in the Lebanese Population Using Whole-Exome Sequencing.

In our previous study, in which array CGH was used on 19 Lebanese ASD subjects and their parents, we identified rare copy number variants (CNVs) in 14 subjects. The five remaining subjects did not show any CNVs related to autism spectrum disorders (ASD). In the present complementary study, we applied whole-exome sequencing (WES), which allows the identification of rare genetic variations such as single nucleotide variations and small insertions/deletions, to the five negative CNV subjects. After stringent filtering of initial data on the five families, three novel genes potentially related to neurodevelopment were identified, including a de novo mutation in the MIS18BP1 gene. In addition, genes already known to be related to ASD contained sequence variations. Our findings outline the potential involvement of the novel de novo mutation in the MIS18BP1 gene in the genetic etiology and pathophysiology of ASD and highlights the genetic complexity of these disorders. Further studies with larger cohorts of subjects are needed to confirm these observations, and functional analyses need to be performed to understand the precise pathophysiology in these cases.

Nuclear translocation of ATG5 induces DNA mismatch repair deficiency (MMR-D)/microsatellite instability (MSI) via interacting with Mis18α in colorectal cancer.

BACKGROUND AND PURPOSE: It is well known that microsatellite instability-high (MSI-H) is associated with 5-fluorouracil (5-FU) resistance in colorectal cancer. MSI-H is the phenotype of DNA mismatch repair deficiency (MMR-D), mainly occurring due to hypermethylation of MLH1 promoter CpG island. However, the mechanisms of MMR-D/MSI-H are unclear. We aim to investigate the pathway of MMR-D/MSI-H involved in 5-FU resistance. EXPERIMENTAL APPROACH: Human colorectal cancer specimens were diagnosed for MSI-H by immunohistochemistry and western blotting. Proteome microarray interactome assay was performed to screen nuclear proteins interacting with ATG5. Nuclear ATG5 and ATG5-Mis18α overexpression were analysed in ATG5high colorectal cancer bearing mice. The methylation assay determined the hypermethylation of hMLH1 promoter CpG island in freshly isolated human colorectal cancer tissue samples and HT29atg5 and SW480atg5 cancer cells. KEY RESULTS: In ATG5high colorectal cancer patients, 5-FU-based therapy resulted in nuclear translocation of ATG5, leading to MSI-H. Colorectal cancer in Atg5 Tg mice demonstrated 5-FU resistance, compared to Atg5+/- and WT mice. Proteome microarray assay identified Mis18α, a protein localized on the centromere and a source for methylation of the underlying chromatin, which responded to the translocated nuclear ATG5 leading to ATG5-Mis18α conjugate overexpression. This resulted in MLH1 deficiency due to hypermethylation of hMLH1 promoter CpG island, while the deletion of nuclear Mis18α failed to induce ATG5-Mis18α complex and MMR-D/MSI-H. CONCLUSIONS AND IMPLICATIONS: Nuclear ATG5 resulted in MMR-D/MSI-H through its interaction with Mis18α in ATG5high colorectal cancer cells. We suggest that ATG5-Mis18α or Mis18α may be a therapeutic target for treating colorectal cancer.

Centromere targeting of Mis18 requires the interaction with DNA and H2A-H2B in fission yeast.

Faithful chromosome segregation during mitosis requires the correct assembly of kinetochore on the centromere. CENP-A is a variant of histone H3, which specializes the centromere region on chromatin and mediates the kinetochore assembly. The Mis18 complex plays a critical role in initiating the centromere loading of the newly-synthesized CENP-A. However, it remains unclear how Mis18 complex (spMis18, spMis16 and spMis19) is located to the centromere to license the recruitment of Cnp1CENP-A in Schizosaccharomyces pombe. We found that spMis18 directly binds to nucleosomal DNA through its extreme C-terminus and interacts with H2A-H2B dimer via the acidic region on the surface of its Yippee-like domain. Live-cell imaging confirmed that mutation of the acidic region and deletion of the extreme C-terminus significantly impairs the localization of spMis18 and Cnp1 to the centromere and delays chromosome segregation during mitosis. Our findings illustrate that the interaction of spMis18 with histone H2A-H2B and DNA plays important roles in the recruitment of spMis18 and Cnp1 to the centromere in fission yeast.

Mis16 Switches Function from a Histone H4 Chaperone to a CENP-ACnp1-Specific Assembly Factor through Eic1 Interaction.

The Mis18 complex, composed of Mis16, Eic1, and Mis18 in fission yeast, selectively deposits the centromere-specific histone H3 variant, CENP-ACnp1, at centromeres. How the intact Mis18 holo-complex oligomerizes and how Mis16, a well-known ubiquitous histone H4 chaperone, plays a centromere-specific role in the Mis18 holo-complex, remain unclear. Here, we report the stoichiometry of the intact Mis18 holo-complex as (Mis16)2:(Eic1)2:(Mis18)4 using analytical ultracentrifugation. We further determine the crystal structure of Schizosaccharomyces pombe Mis16 in complex with the C-terminal portion of Eic1 (Eic1-CT). Notably, Mis16 accommodates Eic1-CT through the binding pocket normally occupied by histone H4, indicating that Eic1 and H4 compete for the same binding site, providing a mechanism for Mis16 to switch its binding partner from histone H4 to Eic1. Thus, our analyses not only determine the stoichiometry of the intact Mis18 holo-complex but also uncover the molecular mechanism by which Mis16 plays a centromere-specific role through Eic1 association.

Mitosis-specific phosphorylation of Mis18α by Aurora B kinase enhances kinetochore recruitment of polo-like kinase 1.

Mis18α, a component of Mis18 complex comprising of Mis18α, Mis18ß, and M18BP1, is known to localize at the centromere from late telophase to early G1 phase and plays a priming role in CENP-A deposition. Although its role in CENP-A deposition is well established, the other function of Mis18α remains unknown. Here, we elucidate a new function of Mis18α that is critical for the proper progression of cell cycle independent of its role in CENP-A deposition. We find that Aurora B kinase phosphorylates Mis18α during mitosis not affecting neither centromere localization of Mis18 complex nor centromere loading of CENP-A. However, the replacement of endogenous Mis18α by phosphorylation-defective mutant causes mitotic defects including micronuclei formation, chromosome misalignment, and chromosomal bridges. Together, our data demonstrate that Aurora B kinase-mediated mitotic phosphorylation of Mis18α is a crucial event for faithful cell cycle progression through the enhanced recruitment of polo-like kinase 1 to the kinetochore.

Kinetochore Components Required for Centromeric Chromatin Assembly Are Impacted by Msc1 in Schizosaccharomyces pombe.

Eukaryotic chromosome segregation requires a protein complex known as the kinetochore that mediates attachment between mitotic spindle microtubules and centromere-specific nucleosomes composed of the widely conserved histone variant CENP-A. Mutations in kinetochore proteins of the fission yeast Schizosaccharomyces pombe lead to chromosome missegregation such that daughter cells emerge from mitosis with unequal DNA content. We find that multiple copies of Msc1-a fission yeast homolog of the KDM5 family of proteins-suppresses the temperature-sensitive growth defect of several kinetochore mutants, including mis16 and mis18, as well as mis6, mis15, and mis17, components of the Constitutive Centromere Associated Network (CCAN). On the other hand, deletion of msc1 exacerbates both the growth defect and chromosome missegregation phenotype of each of these mutants. The C-terminal PHD domains of Msc1, previously shown to associate with a histone deacetylase activity, are necessary for Msc1 function when kinetochore mutants are compromised. We also demonstrate that, in the absence of Msc1, the frequency of localization to the kinetochore of Mis16 and Mis15 is altered from wild-type cells. As we show here for msc1, others have shown that elevating cnp1 levels acts similarly to promote survival of the CCAN mutants. The rescue of mis15 and mis17 by cnp1 is, however, independent of msc1 Thus, Msc1 appears to contribute to the chromatin environment at the centromere: the absence of Msc1 sensitizes cells to perturbations in kinetochore function, while elevating Msc1 overcomes loss of function of critical components of the kinetochore and centromere.

Association of M18BP1/KNL2 with CENP-A Nucleosome Is Essential for Centromere Formation in Non-mammalian Vertebrates.

Centromeres are specified and maintained by sequence-independent epigenetic mechanisms through the incorporation of CENP-A into centromeres. Given that CENP-A incorporation requires the Mis18 complex to be in the centromere region, it is necessary to precisely understand how the Mis18 complex localizes to the centromere region. Here, we showed that centromere localization of the Mis18 complex depends on CENP-A, but not CENP-C or CENP-T, in chicken DT40 cells. Furthermore, we demonstrated that M18BP1/KNL2, a member of the Mis18 complex, contained the CENP-C-like motif in chicken and other vertebrates, which is essential for centromere localization and M18BP1/KNL2 function in DT40 cells. We also showed that in vitro reconstituted CENP-A nucleosome, but not H3 nucleosome, bound to the CENP-C-like motif containing M18BP1/KNL2. Based on these results, we conclude that M18BP1/KNL2 is essential for centromere formation through direct binding to CENP-A nucleosome in non-mammalian vertebrates. This explains how new CENP-A recognizes the centromere position.

Molecular basis for Cdk1-regulated timing of Mis18 complex assembly and CENP-A deposition.

The centromere, a chromosomal locus that acts as a microtubule attachment site, is epigenetically specified by the enrichment of CENP-A nucleosomes. Centromere maintenance during the cell cycle requires HJURP-mediated CENP-A deposition, a process regulated by the Mis18 complex (Mis18α/Mis18ß/Mis18BP1). Spatial and temporal regulation of Mis18 complex assembly is crucial for its centromere association and function. Here, we provide the molecular basis for the assembly and regulation of the Mis18 complex. We show that the N-terminal region of Mis18BP1 spanning amino acid residues 20-130 directly interacts with Mis18α/ß to form the Mis18 complex. Within Mis18α/ß, the Mis18α MeDiY domain can directly interact with Mis18BP1. Mis18α/ß forms a hetero-hexamer with 4 Mis18α and 2 Mis18ß. However, only two copies of Mis18BP1 interact with Mis18α/ß to form a hetero-octameric assembly, highlighting the role of Mis18 oligomerization in limiting the number of Mis18BP1 within the Mis18 complex. Furthermore, we demonstrate the involvement of consensus Cdk1 phosphorylation sites on Mis18 complex assembly and thus provide a rationale for cell cycle-regulated timing of Mis18 assembly and CENP-A deposition.

Centromere localization and function of Mis18 requires Yippee-like domain-mediated oligomerization.

Mis18 is a key regulator responsible for the centromere localization of the CENP-A chaperone Scm3 in Schizosaccharomyces pombe and HJURP in humans, which establishes CENP-A chromatin that defines centromeres. The molecular and structural determinants of Mis18 centromere targeting remain elusive. Here, by combining structural, biochemical, and yeast genetic studies, we show that the oligomerization of S. pombe Mis18, mediated via its conserved N-terminal Yippee-like domain, is crucial for its centromere localization and function. The crystal structure of the N-terminal Yippee-like domain reveals a fold containing a cradle-shaped pocket that is implicated in protein/nucleic acid binding, which we show is required for Mis18 function. While the N-terminal Yippee-like domain forms a homodimer in vitro and in vivo, full-length Mis18, including the C-terminal α-helical domain, forms a homotetramer in vitro We also show that the Yippee-like domains of human Mis18α/Mis18ß interact to form a heterodimer, implying a conserved structural theme for Mis18 regulation.

Transcriptomics analysis and hormonal changes of male and female neonatal rats treated chronically with a low dose of acrylamide in their drinking water.

Acrylamide is known to produce follicular cell tumors of the thyroid in rats. RccHan Wistar rats were exposed in utero to a carcinogenic dose of acrylamide (3 mg/Kg bw/day) from gestation day 6 to delivery and then through their drinking water to postnatal day 35. In order to identify potential mechanisms of carcinogenesis in the thyroid glands, we used a transcriptomics approach. Thyroid glands were collected from male pups at 10 PM and female pups at 10 AM or 10 PM in order to establish whether active exposure to acrylamide influenced gene expression patterns or pathways that could be related to carcinogenesis. While all animals exposed to acrylamide showed changes in expected target pathways related to carcinogenesis such as DNA repair, DNA replication, chromosome segregation, among others; animals that were sacrificed while actively drinking acrylamide-laced water during their active period at night showed increased changes in pathways related to oxidative stress, detoxification pathways, metabolism, and activation of checkpoint pathways, among others. In addition, thyroid hormones, triiodothyronine (T3) and thyroxine (T4), were increased in acrylamide-treated rats sampled at night, but not in quiescent animals when compared to controls. The data clearly indicate that time of day for sample collection is critical to identifying molecular pathways that are altered by the exposures. These results suggest that carcinogenesis in the thyroids of acrylamide treated rats may ensue from several different mechanisms such as hormonal changes and oxidative stress and not only from direct genotoxicity, as has been assumed to date.

Mis16 Independently Recognizes Histone H4 and the CENP-ACnp1-Specific Chaperone Scm3sp.

CENP-A is a centromere-specific histone H3 variant that is required for kinetochore assembly and accurate chromosome segregation. For it to function properly, CENP-A must be specifically localized to centromeres. In fission yeast, Scm3sp and the Mis18 complex, composed of Mis16, Eic1, and Mis18, function as a CENP-A(Cnp1)-specific chaperone and a recruiting factor, respectively, and together ensure accurate delivery of CENP-A(Cnp1) to centromeres. Although how Scm3sp specifically recognizes CENP-A(Cnp1) has been revealed recently, the recruiting mechanism of CENP-A(Cnp1) via the Mis18 complex remains unknown. In this study, we have determined crystal structures of Schizosaccharomyces japonicus Mis16 alone and in complex with the helix 1 of histone H4 (H4α1). Crystal structures followed by mutant analysis and affinity pull-downs have revealed that Mis16 recognizes both H4α1 and Scm3sp independently within the CENP-A(Cnp1)/H4:Scm3sp complex. This observation suggests that Mis16 gains CENP-A(Cnp1) specificity by recognizing both Scm3sp and histone H4. Our studies provide insights into the molecular mechanisms underlying specific recruitment of CENP-A(Cnp1)/H4:Scm3sp into centromeres.

Eic1 links Mis18 with the CCAN/Mis6/Ctf19 complex to promote CENP-A assembly.

CENP-A chromatin forms the foundation for kinetochore assembly. Replication-independent incorporation of CENP-A at centromeres depends on its chaperone HJURP(Scm3), and Mis18 in vertebrates and fission yeast. The recruitment of Mis18 and HJURP(Scm3) to centromeres is cell cycle regulated. Vertebrate Mis18 associates with Mis18BP1(KNL2), which is critical for the recruitment of Mis18 and HJURP(Scm3). We identify two novel fission yeast Mis18-interacting proteins (Eic1 and Eic2), components of the Mis18 complex. Eic1 is essential to maintain Cnp1(CENP-A) at centromeres and is crucial for kinetochore integrity; Eic2 is dispensable. Eic1 also associates with Fta7(CENP-Q/Okp1), Cnl2(Nkp2) and Mal2(CENP-O/Mcm21), components of the constitutive CCAN/Mis6/Ctf19 complex. No Mis18BP1(KNL2) orthologue has been identified in fission yeast, consequently it remains unknown how the key Cnp1(CENP-A) loading factor Mis18 is recruited. Our findings suggest that Eic1 serves a function analogous to that of Mis18BP1(KNL2), thus representing the functional counterpart of Mis18BP1(KNL2) in fission yeast that connects with a module within the CCAN/Mis6/Ctf19 complex to allow the temporally regulated recruitment of the Mis18/Scm3(HJURP) Cnp1(CENP-A) loading factors. The novel interactions identified between CENP-A loading factors and the CCAN/Mis6/Ctf19 complex are likely to also contribute to CENP-A maintenance in other organisms.