Integrity of the human centromere DNA repeats is protected by CENP-A, CENP-C, and CENP-T

Centromeres are highly specialized chromatin domains that enable chromosome segregation and orchestrate faithful cell division. Human centromeres are composed of tandem arrays of α-satellite DNA, which spans up to several megabases. Little is known about the mechanisms that maintain integrity of the long arrays of α-satellite DNA repeats. Here, we monitored centromeric repeat stability in human cells using chromosome-orientation fluorescent in situ hybridization (CO-FISH). This assay detected aberrant centromeric CO-FISH patterns consistent with sister chromatid exchange at the frequency of 5% in primary tissue culture cells, whereas higher levels were seen in several cancer cell lines and during replicative senescence. To understand the mechanism(s) that maintains centromere integrity, we examined the contribution of the centromere-specific histone variant CENP-A and members of the constitutive centromere-associated network (CCAN), CENP-C, CENP-T, and CENP-W. Depletion of CENP-A and CCAN proteins led to an increase in centromere aberrations, whereas enhancing chromosome missegregation by alternative methods did not, suggesting that CENP-A and CCAN proteins help maintain centromere integrity independently of their role in chromosome segregation. Furthermore, superresolution imaging of centromeric CO-FISH using structured illumination microscopy implied that CENP-A protects α-satellite repeats from extensive rearrangements. Our study points toward the presence of a centromere-specific mechanism that actively maintains α-satellite repeat integrity during human cell proliferation.

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Full three-dimensional imaging deep through multicellular thick samples with subcellular resolution by structured illumination microscopy and adaptive optics

10.1101/2020.04.15.043026 ◽

2020 ◽

Author(s):

Ruizhe Lin ◽

Edward T. Kipreos ◽

Jie Zhu ◽

Chang Hyun Khang ◽

Peter Kner

Keyword(s):

Adaptive Optics ◽

Three Dimensional ◽

Structured Illumination ◽

Axial Resolution ◽

Structured Illumination Microscopy ◽

Optical Aberrations ◽

C Elegans ◽

Culture Cells ◽

Tissue Culture Cells ◽

Subcellular Resolution

AbstractStructured Illumination Microscopy enables live imaging with resolutions of ~120 nm. Unfortunately, optical aberrations can lead to loss of resolution and artifacts in Structured Illumination Microscopy rendering the technique unusable in samples thicker than a single cell. Here we report on the combination of Adaptive Optics and Structured Illumination Microscopy enabling imaging with 140 nm lateral and 585 nm axial resolution in tissue culture cells, C. elegans, and rice blast fungus. We demonstrate that AO improves resolution and reduces artifacts, making full 3D SIM possible in thicker samples.

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Mammalian NSUN2 introduces 5-methylcytidines into mitochondrial tRNAs

Nucleic Acids Research ◽

10.1093/nar/gkz575 ◽

2019 ◽

Vol 47 (16) ◽

pp. 8734-8745 ◽

Cited By ~ 10

Author(s):

Saori Shinoda ◽

Sho Kitagawa ◽

Shinichi Nakagawa ◽

Fan-Yan Wei ◽

Kazuhito Tomizawa ◽

...

Keyword(s):

Mitochondrial Protein ◽

Spectrometric Analysis ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Respiratory Chain Complexes ◽

Culture Cells ◽

Chain Complexes ◽

Mitochondrial Functions

AbstractPost-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we took advantage of mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48–50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m5C at positions 48–50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine. Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.

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Structure of the human inner kinetochore bound to a centromeric CENP-A nucleosome

10.1101/2022.01.07.475394 ◽

2022 ◽

Author(s):

Stanislau Yatskevich ◽

Kyle W Muir ◽

Dom Bellini ◽

Ziguo Zhang ◽

Jing Yang ◽

...

Keyword(s):

Chromosome Segregation ◽

Satellite Dna ◽

Mitotic Spindle ◽

Genetic Information ◽

Satellite Repeat ◽

Centromeric Chromatin ◽

Histone Fold ◽

Chromosome Congression ◽

Network Complex ◽

Pushing And Pulling

Accurate chromosome segregation, controlled by kinetochore-mediated chromatid attachments to the mitotic spindle, ensures the faithful inheritance of genetic information. Kinetochores assemble onto specialized CENP-A nucleosomes (CENP-ANuc) of centromeric chromatin. In humans, this is mostly organized as thousands of copies of an ~171 bp α-satellite repeat. Here, we describe the cryo-EM structure of the human inner kinetochore CCAN (Constitutive Centromere Associated Network) complex bound to CENP-ANuc reconstituted onto α-satellite DNA. CCAN forms edge-on contacts with CENP-ANuc, while a linker DNA segment of the α-satellite repeat emerges from the fully-wrapped end of the nucleosome to thread through the central CENP-LN channel which tightly grips the DNA. The CENP-TWSX histone-fold module, together with CENP-HIKHead, further augments DNA binding and partially wraps the linker DNA in a manner reminiscent of canonical nucleosomes. Our study suggests that the topological entrapment of the α-satellite repeat linker DNA by CCAN provides a robust mechanism by which the kinetochore withstands the pushing and pulling of centromeres associated with chromosome congression and segregation forces.

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CRISPR/Cas9-Based RGEN-ISL Allows the Simultaneous and Specific Visualization of Proteins, DNA Repeats, and Sites of DNA Replication

Cytogenetic and Genome Research ◽

10.1159/000502600 ◽

2019 ◽

Vol 159 (1) ◽

pp. 48-53 ◽

Cited By ~ 3

Author(s):

Alžběta Němečková ◽

Christina Wäsch ◽

Veit Schubert ◽

Takayoshi Ishii ◽

Eva Hřibová ◽

...

Keyword(s):

Dna Replication ◽

Chromatin Structure ◽

Super Resolution ◽

Dna Repeats ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Replication Sites ◽

And Function ◽

Super Resolution Microscopy

Visualizing the spatiotemporal organization of the genome will improve our understanding of how chromatin structure and function are intertwined. Here, we describe a further development of the CRISPR/Cas9-based RNA-guided endonuclease-in situ labeling (RGEN-ISL) method. RGEN-ISL allowed the differentiation between vertebrate-type (TTAGGG)n and Arabidopsis-type (TTTAGGG)n telomere repeats. Using maize as an example, we established a combination of RGEN-ISL, immunostaining, and EdU labeling to visualize in situ specific repeats, histone marks, and DNA replication sites, respectively. The effects of the non-denaturing RGEN-ISL and standard denaturing FISH on the chromatin structure were compared using super-resolution microscopy. 3D structured illumination microscopy revealed that denaturation and acetic acid fixation impaired and flattened the chromatin. The broad range of adaptability of RGEN-ISL to different combinations of methods has the potential to advance the field of chromosome biology.

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Superresolution expansion microscopy reveals the three-dimensional organization of the Drosophila synaptonemal complex

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1705623114 ◽

2017 ◽

Vol 114 (33) ◽

pp. E6857-E6866 ◽

Cited By ~ 67

Author(s):

Cori K. Cahoon ◽

Zulin Yu ◽

Yongfu Wang ◽

Fengli Guo ◽

Jay R. Unruh ◽

...

Keyword(s):

Synaptonemal Complex ◽

Chromosome Segregation ◽

Biological Sample ◽

3D Model ◽

Three Dimensional ◽

Optical Resolution ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Homologous Chromosomes ◽

Superresolution Microscopy

The synaptonemal complex (SC), a structure highly conserved from yeast to mammals, assembles between homologous chromosomes and is essential for accurate chromosome segregation at the first meiotic division. In Drosophila melanogaster, many SC components and their general positions within the complex have been dissected through a combination of genetic analyses, superresolution microscopy, and electron microscopy. Although these studies provide a 2D understanding of SC structure in Drosophila, the inability to optically resolve the minute distances between proteins in the complex has precluded its 3D characterization. A recently described technology termed expansion microscopy (ExM) uniformly increases the size of a biological sample, thereby circumventing the limits of optical resolution. By adapting the ExM protocol to render it compatible with structured illumination microscopy, we can examine the 3D organization of several known Drosophila SC components. These data provide evidence that two layers of SC are assembled. We further speculate that each SC layer may connect two nonsister chromatids, and present a 3D model of the Drosophila SC based on these findings.

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Mammalian NSUN2 introduces 5-methylcytidines into mitochondrial tRNAs

10.1101/624932 ◽

2019 ◽

Author(s):

Saori Shinoda ◽

Sho Kitagawa ◽

Shinichi Nakagawa ◽

Fan-Yan Wei ◽

Kazuhito Tomizawa ◽

...

Keyword(s):

Mitochondrial Protein ◽

Spectrometric Analysis ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Respiratory Chain Complexes ◽

Culture Cells ◽

Chain Complexes ◽

Mitochondrial Functions

ABSTRACTPost-transcriptional modifications in mitochondrial tRNAs (mt-tRNAs) play critical roles in mitochondrial protein synthesis, which produces respiratory chain complexes. In this study, we used mass spectrometric analysis to map 5-methylcytidine (m5C) at positions 48–50 in eight mouse and six human mt-tRNAs. We also confirmed the absence of m5C in mt-tRNAs isolated from Nsun2 knockout (KO) mice, as well as from NSUN2 KO human culture cells. In addition, we successfully reconstituted m5C at positions 48–50 of mt-tRNA in vitro with NSUN2 protein in the presence of S-adenosylmethionine (SAM). Although NSUN2 is predominantly localized to the nucleus and introduces m5C into cytoplasmic tRNAs and mRNAs, structured illumination microscopy (SIM) clearly revealed NSUN2 foci inside mitochondria. These observations provide novel insights into the role of NSUN2 in the physiology and pathology of mitochondrial functions.

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TOPII and chromosome movement help remove interlocks between entangled chromosomes during meiosis

The Journal of Cell Biology ◽

10.1083/jcb.201803019 ◽

2018 ◽

Vol 217 (12) ◽

pp. 4070-4079 ◽

Cited By ~ 14

Author(s):

Marina Martinez-Garcia ◽

Veit Schubert ◽

Kim Osman ◽

Alice Darbyshire ◽

Eugenio Sanchez-Moran ◽

...

Keyword(s):

Arabidopsis Thaliana ◽

Homologous Recombination ◽

Chromosome Segregation ◽

Meiotic Division ◽

Topoisomerase Ii ◽

Chromosome Movement ◽

Structured Illumination ◽

Structured Illumination Microscopy ◽

Chromosome Synapsis ◽

Active Processes

During the zygotene stage of meiosis, normal progression of chromosome synapsis and homologous recombination frequently lead to the formation of structural interlocks between entangled chromosomes. The persistence of interlocks through to the first meiotic division can jeopardize normal synapsis and occasionally chromosome segregation. However, they are generally removed by pachytene. It has been postulated that interlock removal requires one or more active processes, possibly involving topoisomerase II (TOPII) and/or chromosome movement. However, experimental evidence has been lacking. Analysis of a hypomorphic topII mutant and a meiosis-specific topII RNAi knockdown of Arabidopsis thaliana using immunocytochemistry and structured illumination microscopy (SIM) has now enabled us to demonstrate a role for TOPII in interlock resolution. Furthermore, analysis using a nucleoporin nup136 mutant, which affects chromosome movement, reveals that although TOPII activity is required for the removal of some interlock structures, for others, chromosome movement is also necessary. Thus, our study demonstrates that at least two mechanisms are required to ensure interlock removal.

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