Centromeres consist of specialized centrochromatin containing CENP-A nucleosomes intermingled with H3 nucleosomes carrying transcription-associated modifications. We have designed a novel synthetic ...biology 'in situ epistasis' analysis in which H3 dimethylated on lysine 4 (H3K4me2) demethylase LSD2 plus synthetic modules with competing activities are simultaneously targeted to a synthetic alphoid
HAC centromere. This allows us to uncouple transcription from histone modifications at the centromere. Here, we report that H3K4me2 loss decreases centromeric transcription, CENP-A assembly and stability and causes spreading of H3K9me3 across the HAC, ultimately inactivating the centromere. Surprisingly, CENP-28/Eaf6-induced transcription of the alphoid
array associated with H4K12 acetylation does not rescue the phenotype, whereas p65-induced transcription associated with H3K9 acetylation does rescue. Thus mitotic transcription plus histone modifications including H3K9ac constitute the 'epigenetic landscape' allowing CENP-A assembly and centrochromatin maintenance. H3K4me2 is required for the transcription and H3K9ac may form a barrier to prevent heterochromatin spreading and kinetochore inactivation at human centromeres.
Stereochemically inert and positively charged chiral complexes of cobalt(III) prepared from Schiff bases derived from chiral diamines and salicylaldehydes were shown to be efficient catalysts of the ...benchmark asymmetric phase‐transfer Michael addition of nine activated olefins to O’Donnell’s substrate. The reaction products had enantiomeric purities of up to 96%. DFT calculations were invoked to rationalize the stereochemistry of the addition.
Centromere chromatin containing histone H3 variant CENP-A is required for accurate chromosome segregation as a foundation for kinetochore assembly. Human centromere chromatin assembles on a part of ...the long α-satellite (alphoid) DNA array, where it is flanked by pericentric heterochromatin. Heterochromatin spreads into adjacent chromatin and represses gene expression, and it can antagonize centromere function or CENP-A assembly. Here, we demonstrate an interaction between CENP-A assembly factor M18BP1 and acetyltransferase KAT7/HBO1/MYST2. Knocking out KAT7 in HeLa cells reduced centromeric CENP-A assembly. Mitotic chromosome misalignment and micronuclei formation increased in the knockout cells and were enhanced when the histone H3-K9 trimethylase Suv39h1 was overproduced. Tethering KAT7 to an ectopic alphoid DNA integration site removed heterochromatic H3K9me3 modification and was sufficient to stimulate new CENP-A or histone H3.3 assembly. Thus, KAT7-containing acetyltransferases associating with the Mis18 complex provides competence for histone turnover/exchange activity on alphoid DNA and prevents Suv39h1-mediated heterochromatin invasion into centromeres.
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•The histone acetyltransferase KAT7 positively regulates centromeric CENP-A assembly•Human Mis18 complex is a scaffold for assembly of KAT7 and HJURP, a CENP-A chaperone•KAT7 or RSF1 stimulates histone turnover/exchange on alphoid DNA•KAT7 antagonizes H3K9-trimethylase Suv39h1-mediated centromere inactivation
Human centromeres contain histone H3 variant CENP-A and are flanked by pericentric heterochromatin. Spreading of heterochromatin into the centromere can impede centromere function. Ohzeki et al. demonstrate that the KAT7 histone acetyltransferase promotes CENP-A assembly and preserves centromere function by preventing H3K9-trimethylase Suv39h1-mediated heterochromatin invasion via a histone turnover mechanism.
We herein developed a protocol for the asymmetric synthesis of artificial AAs featuring a 3‐spiropyrrolidine oxindole skeletal with continuous tetrasubstituted carbon stereocenters by a 1,3‐dipolar ...cycloaddition reaction of in situ generated azomethine ylides with a chiral dehydroalanine Ni(II) complex. A three‐component reaction of the Ni(II) complex with various isatins and AAs in green solvent (ethanol) provided series of diastereomeric complexes with high dr (>20:1) in 40–86% yields. The formation of different regioisomers in the case of sarcosine and proline was explained using quantum chemical calculation. The acidic decomposition of the obtained Ni(II) complexes led to the target unnatural complex AAs with a 3‐spiropyrrolidine oxindole core. The chiral auxiliary ligand was recovered after decomposition and reused for the synthesis of the starting dehydroalanine complex‐substrate.
Stereochemically inert cationic cobalt(III) complexes were shown to be one‐component catalysts for the synthesis of cyclic carbonates from epoxides and carbon dioxide at 50 °C and 5 MPa carbon ...dioxide pressure. The optimal catalyst possessed an iodide counter anion and could be recycled. A catalytic cycle is proposed in which the ligand of the cobalt complexes acts as a hydrogen‐bond donor, activating the epoxide towards ring opening by the halide anion and activating the carbon dioxide for subsequent reaction with the halo‐alkoxide. No kinetic resolution was observed when terminal epoxides were used as substrates, but chalcone oxide underwent kinetic resolution.
A Co‐catalyst with no co‐catalyst: A chiral cobalt complex catalyses the formation of cyclic carbonates from epoxides and carbon dioxide without the need for a co‐catalyst. Chalcone oxide undergoes kinetic resolution under the reaction conditions. The activity of the one component bifunctional system originates from the hydrogen‐bond donating ability of the coordinated ligand and nucleophilic participation of the counter anion. The complexes are robust, simple to prepare, and easy to recycle.
Since their description in the late 1990s, human artificial chromosomes (HACs) carrying a functional kinetochore were considered as a promising system for gene delivery and expression with a ...potential to overcome many problems caused by the use of viral-based gene transfer systems. Indeed, HACs avoid the limited cloning capacity, lack of copy number control and insertional mutagenesis due to integration into host chromosomes that plague viral vectors. Nevertheless, until recently, HACs have not been widely recognized because of uncertainties of their structure and the absence of a unique gene acceptor site. The situation changed a few years ago after engineering of HACs with a single loxP gene adopter site and a defined structure. In this review, we summarize recent progress made in HAC technology and concentrate on details of two of the most advanced HACs, 21HAC generated by truncation of human chromosome 21 and alphoid
tetO
-HAC generated de novo using a synthetic tetO-alphoid DNA array. Multiple potential applications of the HAC vectors are discussed, specifically the unique features of two of the most advanced HAC cloning systems.
A family of well-defined Λ- and Δ-diastereomeric octahedral cationic chiral-at-cobalt complexes were obtained by a simple two-step reaction of (R,R)-1,2-diaminocyclohexane, ...(R,R)-1,2-diphenylethylenediamine, or (S)-2-(aminomethyl)pyrrolidine and substituted salicylaldehydes with a cobalt(III) salt. It was observed for the first time that the use of an excess of cobalt(III) salt provides both the enantiopure Λ and Δ forms of the corresponding cobalt(III) complexes 1 and 2 in a ratio of diastereomers ranging from 1:1.6 to >20:1 (Λ/Δ) and in 31–95% combined yields. The obtained complexes were robust, air- and bench-stable, soluble in most of organic solvents, and insoluble in water. Through variation of the substituents in the phenyl ring of the salicylaldehyde moiety, it was shown that both steric and electronic effects of substituents have a significant impact on the formation of Λ and Δ isomers. Next, the efficacies of the enantiopure metal-templated complexes 1–3 were investigated in three benchmark asymmetric reactions in order to compare their catalytic activity. The chiral cobalt(III) complexes 1–3 were tested as enantioselective hydrogen-bond-donor catalysts in such important reactions as the Michael addition of the O’Donnell substrate to methyl acrylate, epoxidation of chalcone, and trimethylsilylcyanation of benzaldehyde. It was clearly demonstrated that the chirality at the cobalt center has an impact on the stereochemical outcome of the reactions. In particular, the Λ(R,R)-1 and Δ(R,R)-1 complexes acted as “pseudoenantiomeric” catalysts in the epoxidation and trimethylsilylcyanoation reactions, providing both enantiomers of the products with up to 57% enantiomeric excess.
We have used a human artificial chromosome (HAC) to manipulate the epigenetic state of chromatin within an active kinetochore. The HAC has a dimeric α-satellite repeat containing one natural monomer ...with a CENP-B binding site, and one completely artificial synthetic monomer with the CENP-B box replaced by a tetracycline operator (tetO). This HAC exhibits normal kinetochore protein composition and mitotic stability. Targeting of several tet-repressor (tetR) fusions into the centromere had no effect on kinetochore function. However, altering the chromatin state to a more open configuration with the tTA transcriptional activator or to a more closed state with the tTS transcription silencer caused missegregation and loss of the HAC. tTS binding caused the loss of CENP-A, CENP-B, CENP-C, and H3K4me2 from the centromere accompanied by an accumulation of histone H3K9me3. Our results reveal that a dynamic balance between centromeric chromatin and heterochromatin is essential for vertebrate kinetochore activity.
Here we report the first synthesis of two diastereomeric cationic octahedral Co(III) complexes based on commercially available (R,R)-1,2-diphenylethylenediamine and salicylaldehyde. Both ...diastereoisomers with opposite chiralities at the metal center (Λ and Δ configurations) were prepared. The new Co(III) complexes possessed both acidic hydrogen-bond donating (HBD) NH moieties and nucleophilic counteranions and operate as bifunctional chiral catalysts for the challenging kinetic resolution of terminal and disubstituted epoxides by the reaction with CO2 under mild conditions. The highest selectivity factor (s) of 2.8 for the trans-chalcone epoxide was achieved at low catalyst loading (2 mol %) in chlorobenzene, which is the best achieved result currently for this type of substrate.
Whole chromosomal instability (CIN), manifested as unequal chromosome distribution during cell division, is a distinguishing feature of most cancer types. CIN is generally considered to drive ...tumorigenesis, but a threshold level exists whereby further increases in CIN frequency in fact hinder tumor growth. While this attribute is appealing for therapeutic exploitation, drugs that increase CIN beyond this therapeutic threshold are currently limited. In our previous work, we developed a quantitative assay for measuring CIN based on the use of a nonessential human artificial chromosome (HAC) carrying a constitutively expressed EGFP transgene. Here, we used this assay to rank 62 different anticancer drugs with respect to their effects on chromosome transmission fidelity. Drugs with various mechanisms of action, such as antimicrotubule activity, histone deacetylase inhibition, mitotic checkpoint inhibition, and targeting of DNA replication and damage responses, were included in the analysis. Ranking of the drugs based on their ability to induce HAC loss revealed that paclitaxel, gemcitabine, dactylolide, LMP400, talazoparib, olaparib, peloruside A, GW843682, VX-680, and cisplatin were the top 10 drugs demonstrating HAC loss at a high frequency. Therefore, identification of currently used compounds that greatly increase chromosome mis-segregation rates should expedite the development of new therapeutic strategies to target and leverage the CIN phenotype in cancer cells.
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