Quantitative Microscopy Reveals Centromeric Chromatin Stability, Size, and Cell Cycle Mechanisms to Maintain Centromere Homeostasis

The deposited item is a book chapter and is part of the series "Centromeres and Kinetochores" published by the publisher Springer Verlag. The deposited book chapter is a post-print version and has been submitted to peer reviewing. There is no public supplementary material available for this publication. This publication hasn't any creative commons license associated.Centromeres are chromatin domains specified by nucleosomes containing the histone H3 variant, CENP-A. This unique centromeric structure is at the heart of a strong self-templating epigenetic mechanism that renders centromeres heritable. We review how specific quantitative microscopy approaches have contributed to the determination of the copy number, architecture, size, and dynamics of centromeric chromatin and its associated centromere complex and kinetochore. These efforts revealed that the key to long-term centromere maintenance is the slow turnover of CENP-A nucleosomes, a critical size of the chromatin domain and its cell cycle-coupled replication. These features come together to maintain homeostasis of a chromatin locus that directs its own epigenetic inheritance and facilitates the assembly of the mitotic kinetochore.There are no funders and sponsors indicated explicitly in the document.info:eu-repo/semantics/publishedVersio

Repeat Composition of CenH3-chromatin and H3K9me2-marked heterochromatin in Sugar Beet (Beta vulgaris)

Kowar T, Zakrzewski F, Macas J, et al. Repeat Composition of CenH3-chromatin and H3K9me2-marked heterochromatin in Sugar Beet (Beta vulgaris). BMC Plant Biology. 2016;16(1): 120.Background Sugar beet (Beta vulgaris) is an important crop of temperate climate zones, which provides nearly 30 % of the world’s annual sugar needs. From the total genome size of 758 Mb, only 567 Mb were incorporated in the recently published genome sequence, due to the fact that regions with high repetitive DNA contents (e.g. satellite DNAs) are only partially included. Therefore, to fill these gaps and to gain information about the repeat composition of centromeres and heterochromatic regions, we performed chromatin immunoprecipitation followed by sequencing (ChIP-Seq) using antibodies against the centromere-specific histone H3 variant of sugar beet (CenH3) and the heterochromatic mark of dimethylated lysine 9 of histone H3 (H3K9me2). Results ChIP-Seq analysis revealed that active centromeres containing CenH3 consist of the satellite pBV and the Ty3-gypsy retrotransposon Beetle7, while heterochromatin marked by H3K9me2 exhibits heterogeneity in repeat composition. H3K9me2 was mainly associated with the satellite family pEV, the Ty1-copia retrotransposon family Cotzilla and the DNA transposon superfamily of the En/Spm type. In members of the section Beta within the genus Beta, immunostaining using the CenH3 antibody was successful, indicating that orthologous CenH3 proteins are present in closely related species within this section. Conclusions The identification of repetitive genome portions by ChIP-Seq experiments complemented the sugar beet reference sequence by providing insights into the repeat composition of poorly characterized CenH3-chromatin and H3K9me2-heterochromatin. Therefore, our work provides the basis for future research and application concerning the sugar beet centromere and repeat rich heterochromatic regions characterized by the presence of H3K9me2

Association of syncoilin and desmin: linking intermediate filament proteins to the dystrophin-associated protein complex.

We recently identified a novel protein called syncoilin, a putative intermediate filament protein that interacts with alpha-dystrobrevin, a member of the dystrophin-associated protein complex. Syncoilin is found at the neuromuscular junction, sarcolemma, and Z-lines and is thought to be important for muscle fiber integrity. Based on the similar protein structure and cellular localization of syncoilin and desmin, we proposed that these proteins interact in vivo. The data presented confirm an interaction between syncoilin and desmin and demonstrate their co-localization in skeletal muscle. Intriguingly, whereas these proteins interact, COS-7 cell expression studies show that desmin and syncoilin do not assemble into heterofilaments. Furthermore, fractionation assay and immunofluorescence study of H2K myoblasts and myotubes suggest that, unlike typical intermediate filament proteins, syncoilin does not participate in filament formation with any protein. However, it is possible that syncoilin is involved in the anchoring of the desmin intermediate filament network at the sarcolemma and the neuromuscular junction. This interaction is likely to be important for maintaining muscle fiber integrity and may also link the dystrophin-associated protein complex to the cytoskeleton. The dysfunction or absence of syncoilin may result in the disruption of the intermediate filament network leading to muscle necrosis. Syncoilin is therefore an ideal candidate gene for muscular dystrophies and desmin-related myopathies

Syncoilin accumulation in two patients with desmin-related myopathy.

We have recently shown that syncoilin interacts with desmin in skeletal muscle and has a role in attaching and organising desmin filaments to the Z-lines. We have analysed patients with desmin accumulation and have found that syncoilin is both upregulated at the sarcolemma and aggregates with desmin indicating the presence of two distinct protein populations. Additional dystrophin-associated protein complex components also accumulate. The striking finding was that alpha-dystrobrevin-1 and neuronal nitric oxide synthase (nNOS) are almost completely lost from the membrane of these patients indicating that the myopathy may result from both the abnormal accumulation of proteins and an increase in ischaemic injury due to the loss of nNOS. We speculate that the loss of alpha-dystrobrevin from the membrane, and subsequent loss of nNOS, is due to the alpha-dystrobrevin-syncoilin-desmin interaction

Syncoilin, a novel member of the intermediate filament superfamily that interacts with alpha-dystrobrevin in skeletal muscle.

Dystrophin coordinates the assembly of a complex of structural and signaling proteins that are required for normal muscle function. A key component of the dystrophin protein complex is alpha-dystrobrevin, a dystrophin-associated protein whose absence results in neuromuscular junction defects and muscular dystrophy. To gain further insights into the role of alpha-dystrobrevin in skeletal muscle, we used the yeast two-hybrid system to identify a novel alpha-dystrobrevin-binding partner called syncoilin. Syncoilin is a new member of the intermediate filament superfamily and is highly expressed in skeletal and cardiac muscle. In normal skeletal muscle, syncoilin is concentrated at the neuromuscular junction, where it colocalizes and coimmunoprecipitates with alpha-dystrobrevin-1. Expression studies in mammalian cells demonstrate that, while alpha-dystrobrevin and syncoilin associate directly, overexpression of syncoilin does not result in the self-assembly of intermediate filaments. Finally, unlike many components of the dystrophin protein complex, we show that syncoilin expression is up-regulated in dystrophin-deficient muscle. These data suggest that alpha-dystrobrevin provides a link between the dystrophin protein complex and the intermediate filament network at the neuromuscular junction, which may be important for the maintenance and maturation of the synapse

Syncoilin upregulation in muscle of patients with neuromuscular disease.

Syncoilin may have a role in linking the desmin-associated intermediate filament network of the muscle fiber with the dystrophin-associated protein complex (DAPC). We have evaluated syncoilin in a range of neuromuscular disorders including Duchenne and Becker muscular dystrophy, central core disease, congenital muscular dystrophies, and neurogenic disorders. Our results show that syncoilin immunolabeling is not only altered in muscle fibers with alterations in the DAPC but also in response to a variety of genetic defects, including those associated with proteins of the extracellular matrix and the intracellular Ca2+-release channel (ryanodine receptor). The pattern of syncoilin immunolabeling in these diseases appeared to reflect a rearrangement of the intermediate filament-associated cytoskeleton that characterizes both muscle fiber development and conditions in which the cytoskeletal organization of the muscle fiber is significantly affected. These observations raise the possibility that mutations in the gene encoding for syncoilin may underlie some forms of muscle disease