RESUMO
Centromeres are epigenetically defined via the presence of the histone H3 variant CENP-A. Contacting CENP-A nucleosomes, the constitutive centromere associated network (CCAN) and the kinetochore assemble, connecting the centromere to spindle microtubules during cell division. The DNA-binding centromeric protein CENP-B is involved in maintaining centromere stability and, together with CENP-A, shapes the centromeric chromatin state. The nanoscale organization of centromeric chromatin is not well understood. Here, we use single-molecule fluorescence and cryoelectron microscopy (cryoEM) to show that CENP-A incorporation establishes a dynamic and open chromatin state. The increased dynamics of CENP-A chromatin create an opening for CENP-B DNA access. In turn, bound CENP-B further opens the chromatin fiber structure and induces nucleosomal DNA unwrapping. Finally, removal of CENP-A increases CENP-B mobility in cells. Together, our studies show that the two centromere-specific proteins collaborate to reshape chromatin structure, enabling the binding of centromeric factors and establishing a centromeric chromatin state.
Assuntos
Cromatina , Proteínas Cromossômicas não Histona , Proteína Centromérica A/metabolismo , Microscopia Crioeletrônica , Proteínas Cromossômicas não Histona/metabolismo , Centrômero/metabolismo , Nucleossomos , DNA/metabolismo , Autoantígenos/metabolismoRESUMO
Protein O-GlcNAcylation is an essential and dynamic regulator of myriad cellular processes, including DNA replication and repair. Proteomic studies have identified the multifunctional nuclear protein HMGB1 as O-GlcNAcylated, providing a potential link between this modification and DNA damage responses. Here, we verify the protein's endogenous modification at S100 and S107 and found that the major modification site is S100, a residue that can potentially influence HMGB1-DNA interactions. Using synthetic protein chemistry, we generated site-specifically O-GlcNAc-modified HMGB1 at S100 and characterized biochemically the effect of the sugar modification on its DNA binding activity. We found that O-GlcNAc alters HMGB1 binding to linear, nucleosomal, supercoiled, cruciform, and interstrand cross-linked damaged DNA, generally resulting in enhanced oligomerization on these DNA structures. Using cell-free extracts, we also found that O-GlcNAc reduces the ability of HMGB1 to facilitate DNA repair, resulting in error-prone processing of damaged DNA. Our results expand our understanding of the molecular consequences of O-GlcNAc and how it affects protein-DNA interfaces. Importantly, our work may also support a link between upregulated O-GlcNAc levels and increased rates of mutations in certain cancer states.
Assuntos
Acetilglucosamina/metabolismo , Dano ao DNA , Proteína HMGB1/metabolismo , Carcinoma Pulmonar de Células não Pequenas , Linhagem Celular Tumoral , Sistema Livre de Células , Reparo do DNA , Proteína HMGB1/genética , Humanos , MutaçãoRESUMO
The centromere is an essential chromatin domain required for kinetochore recruitment and chromosome segregation in eukaryotes. To perform this role, centro-chromatin adopts a unique structure that provides access to kinetochore proteins and maintains stability under tension during mitosis. This is achieved by the presence of nucleosomes containing the H3 variant CENP-A, which also acts as the epigenetic mark defining the centromere. In this review, we discuss the role of CENP-A on the structure and dynamics of centromeric chromatin. We further discuss the impact of the CENP-A binding proteins CENP-C, CENP-N, and CENP-B on modulating centro-chromatin structure. Based on these findings we provide an overview of the higher order structure of the centromere.
Assuntos
Proteína Centromérica A/química , Proteína B de Centrômero/química , Centrômero/ultraestrutura , Cromatina/ultraestrutura , Proteínas Cromossômicas não Histona/química , Adenosina Trifosfatases/química , Adenosina Trifosfatases/genética , Adenosina Trifosfatases/metabolismo , Proteínas de Ciclo Celular/química , Proteínas de Ciclo Celular/genética , Proteínas de Ciclo Celular/metabolismo , Centrômero/química , Centrômero/metabolismo , Proteína Centromérica A/genética , Proteína Centromérica A/metabolismo , Proteína B de Centrômero/genética , Proteína B de Centrômero/metabolismo , Cromatina/química , Cromatina/metabolismo , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Ligação a DNA/química , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Epigênese Genética , Humanos , Mitose , Modelos Moleculares , Complexos Multiproteicos/química , Complexos Multiproteicos/genética , Complexos Multiproteicos/metabolismo , Conformação de Ácido Nucleico , Ligação Proteica , Conformação Proteica em alfa-Hélice , Conformação Proteica em Folha beta , Domínios e Motivos de Interação entre Proteínas , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , CoesinasRESUMO
The kinetochore is an essential structure for the chromosome segregation machinery in eukaryotes; it serves as a bridge between the spindle microtubules and chromosomes. The kinetochore consists of multiple interconnecting components on the centromere; therefore, understanding its formation, molecular function, and regulation has remained an ongoing challenge. Recent studies have provided new insights into centromere identity, kinetochore assembly, and function. In this review, we discuss recent advances in our understanding of the function and regulation of key kinetochore components. We highlight the reciprocal localization dependencies of the different sub-complexes of the kinetochore and describe their regulation during the cell cycle.
Assuntos
Ciclo Celular/genética , Divisão Celular/fisiologia , Centrômero/metabolismo , Segregação de Cromossomos/fisiologia , Cromossomos Humanos/metabolismo , Cinetocoros/metabolismo , Saccharomyces cerevisiae/genética , Fuso Acromático/metabolismo , Autoantígenos/metabolismo , Proteína Centromérica A , Proteína B de Centrômero/metabolismo , Proteínas Cromossômicas não Histona/metabolismo , Humanos , Microtúbulos/metabolismoRESUMO
The kinetochore is a crucial structure for faithful chromosome segregation during mitosis and is formed in the centromeric region of each chromosome. The 16-subunit protein complex known as the constitutive centromere-associated network (CCAN) forms the foundation for kinetochore assembly on the centromeric chromatin. Although the CCAN can be divided into several subcomplexes, it remains unclear how CCAN proteins are organized to form the functional kinetochore. In particular, this organization may vary as the cell cycle progresses. To address this, we analyzed the relationship of centromeric protein (CENP)-C with the CENP-H complex during progression of the cell cycle. We find that the middle portion of chicken CENP-C (CENP-C(166-324)) is sufficient for centromere localization during interphase, potentially through association with the CENP-L-N complex. The C-terminus of CENP-C (CENP-C(601-864)) is essential for centromere localization during mitosis, through binding to CENP-A nucleosomes, independent of the CENP-H complex. On the basis of these results, we propose that CCAN organization changes dynamically during progression of the cell cycle.