3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin

Booth, Daniel G; Beckett, Alison J.; Molina, Òscar; Samejima, Itaru; Masumoto, Hiroshi; Kouprina, Natalay; Larionov, Vladimir; Prior, Ian A.

doi:10.1016/j.molcel.2016.10.009

Cited by 107 publications

(135 citation statements)

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“…Use of 3D correlative light electron microscopy (3d-CLEM) allowed the determination of the HAC volume at prometaphase. Assuming a normal density of chromatin packing, this yielded an estimated size of 5.5 MB for the HAC, essentially identical to the 5.1 MB calculated from the molecular cloning analysis (Kouprina et al 2012;Booth et al 2016). This agreement confirmed that the packing density of the alphoid tetO HAC is comparable to the other endogenous chromosomes.…”

Section: The Synthetic Alphoid Teto Hac For Epigenetic Engineering Ofsupporting

confidence: 65%

“…Whereas none of the de novo HACs constructed before were physically mapped in molecular detail, largely due to the fact that they contain huge blocks of repeated DNA, the containing, in addition to its kinetochore, chromosome scaffold (revealed by staining for condensin subunit SMC2) and a chromosome periphery compartment (revealed by staining for Ki-67) (Booth et al 2016). Use of 3D correlative light electron microscopy (3d-CLEM) allowed the determination of the HAC volume at prometaphase.…”

Section: The Synthetic Alphoid Teto Hac For Epigenetic Engineering Ofmentioning

confidence: 99%

“…This agreement confirmed that the packing density of the alphoid tetO HAC is comparable to the other endogenous chromosomes. Thus, the mechanisms used to condense mitotic chromosomes are apparently independent of the chromosome shape and size (Booth et al 2016). In HAC formation experiments, the most common fate of the input DNA constructs is integration into the chromosome arm of a host chromosome (Figure 2b) Masumoto et al 1998;Nakano et al 2008).…”

Section: The Synthetic Alphoid Teto Hac For Epigenetic Engineering Ofmentioning

confidence: 99%

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Using human artificial chromosomes to study centromere assembly and function

et al. 2017

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Abstract:Centromeres are the site of assembly of the kinetochore, which directs chromosome segregation during cell division. Active centromeres are characterized by the presence of nucleosomes containing CENP-A and a specific chromatin environment that resembles that of active genes. Recent work using Human Artificial Chromosomes (HAC) sheds light on the fine balance of different histone post-translational modifications and transcription that exists at centromeres for kinetochore assembly and maintenance. Here, we review the use of HAC technology to understand centromere assembly and function. We put particular emphasis on studies using the alphoidtetO HAC, whose centromere can be specifically modified for epigenetic engineering studies. Author Comments:We suggest Professor Erich Nigg as an Editor (not found in the "Request Editor" list), who invited us to write this review.Response to Reviewers: Comments for the Author:Reviewer #1: Since there has been significant progress over the last few years with regards to HAC technology, particularly with alphoidtetO HACs, a review on this topic would be welcomed in the field, especially by these authors, who are world experts on Powered by Editorial Manager® and ProduXion Manager® from Aries Systems Corporationthis topic. The review is mostly satisfying, but I would suggest two areas where the authors would be well served to make additions/changes to avoid disappointing readers hoping for more focus/organization/clarity. First, since the goal of this review is to "review the use of HAC technology to understand centromere assembly and function", it would be especially helpful if the authors summarized the evolution of HAC technology and the findings that came from targeting different tetR fusion proteins to the alphoidtetO HACs (probably most effectively in a table or schematic).We thank the reviewer for the thoughtful review of our MS. Following the reviewer´s advice, a table has been added explaining the findings that came from the alphoidtetO HAC studies in a timeline manner (Table 1).Second, more discussion on the pitfalls of current HACs would be desirable (currently only a couple sentences is mentioned in the future directions). From how it is written they sound so great that a reader would probably wonder why they are not used more widely in the research lab and/or clinic. There are problems with them, and it would be more clear and interesting, in my opinion, if more of the problems were laid out/challenges addressed.A description of limitations of the HAC technology have been added in the text, first limitations for HAC clinical use (p. 8-9) and additional limitations that should be overcome in the final remarks (p. 23). Some other changes that I'd suggest:1. It's confusing to refer to the same DNA as "alpha satellite", "α-satellite", and "alpha-satellite" throughout the review (most notably in the first two lines of the second paragraph). Why not just refer to it as "α-satellite" DNA?We changed the term to α-satellite DNA throughout the text to maintain consist...

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Section: The Synthetic Alphoid Teto Hac For Epigenetic Engineering Ofsupporting

confidence: 65%

Section: The Synthetic Alphoid Teto Hac For Epigenetic Engineering Ofmentioning

confidence: 99%

Section: The Synthetic Alphoid Teto Hac For Epigenetic Engineering Ofmentioning

confidence: 99%

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Using human artificial chromosomes to study centromere assembly and function

et al. 2017

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“…This is particularly true for its mitotic roles. Specifically, Ki-67 is required for formation of the mitotic perichromosomal layer (11, 12), a proteinaceous sheath that coats mitotic chromosomes (13,14). In this layer, Ki-67's large size and highly positively charged amino acid composition keep individual mitotic chromosomes dispersed rather than aggregated upon nuclear envelope disassembly, thereby ensuring normal kinetics of anaphase progression (15).…”

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confidence: 99%

Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells

Sun

Bizhanova

Matheson

et al. 2017

Molecular and Cellular Biology

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The Ki-67 protein is widely used as a tumor proliferation marker. However, whether Ki-67 affects cell cycle progression has been controversial. Here we demonstrate that depletion of Ki-67 in human hTERT-RPE1, WI-38, IMR90, and hTERT-BJ cell lines and primary fibroblast cells slowed entry into S phase and coordinately downregulated genes related to DNA replication. Some gene expression changes were partially relieved in Ki-67-depleted hTERT-RPE1 cells by codepletion of the Rb checkpoint protein, but more thorough suppression of the transcriptional and cell cycle defects was observed upon depletion of the cell cycle inhibitor p21. Notably, induction of p21 upon depletion of Ki-67 was a consistent hallmark of cell types in which transcription and cell cycle distribution were sensitive to Ki-67; these responses were absent in cells that did not induce p21. Furthermore, upon Ki-67 depletion, a subset of inactive X (Xi) chromosomes in female hTERT-RPE1 cells displayed several features of compromised heterochromatin maintenance, including decreased H3K27me3 and H4K20me1 labeling. These chromatin alterations were limited to Xi chromosomes localized away from the nuclear lamina and were not observed in checkpoint-deficient 293T cells. Altogether, our results indicate that Ki-67 integrates normal S-phase progression and Xi heterochromatin maintenance in p21 checkpoint-proficient human cells.KEYWORDS cell cycle, heterochromatin K i-67 was first identified via an antibody raised against Hodgkin's lymphoma cell nuclei (1). Because Ki-67 is generally expressed strongly in proliferating cells and poorly in quiescent cells (2), anti-Ki-67 antibodies are frequently used to detect proliferative cells in clinical studies (3, 4). In interphase cells, Ki-67 primarily localizes to the nucleolus (5-7), whereas during mitosis, it coats the chromosomes (8-10). In the past few years, several studies have greatly increased our understanding of Ki-67 function. This is particularly true for its mitotic roles. Specifically, Ki-67 is required for formation of the mitotic perichromosomal layer (11, 12), a proteinaceous sheath that coats mitotic chromosomes (13,14). In this layer, Ki-67's large size and highly positively charged amino acid composition keep individual mitotic chromosomes dispersed rather than aggregated upon nuclear envelope disassembly, thereby ensuring normal kinetics of anaphase progression (15). At anaphase onset, Ki-67 binds protein phosphatase 1␥ (PP1␥) to form a holoenzyme (16) important for targeting substrates that must be dephosphorylated during mitotic exit (10). In contrast to its structural role on the

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“…It is performed by laser welding to join two adjacent pieces of genetic material. Unlike the tissues in laser tissue welding [24,25], chromosomes are very small in size and are extremely thin (about 0.5-0.7 μm for human chromosomes) [26]. To weld the two tiny pieces of chromosome together without thermal and mechanical damage upon them is a great challenge in biotechnology and optical engineering.…”

Section: Introductionmentioning

confidence: 99%

Technique of laser chromosome welding for chromosome repair and artificial chromosome creation

Huang¹,

Li²,

Liu³

et al. 2018

Biomed. Opt. Express

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Abstract:Here we report a technique of laser chromosome welding that uses a violet pulse laser micro-beam for welding. The technique can integrate any size of a desired chromosome fragment into recipient chromosomes by combining with other techniques of laser chromosome manipulation such as chromosome cutting, moving, and stretching. We demonstrated that our method could perform chromosomal modifications with high precision, speed and ease of use in the absence of restriction enzymes, DNA ligases and DNA polymerases. Unlike the conventional methods such as de novo artificial chromosome synthesis, our method has no limitation on the size of the inserted chromosome fragment. The inserted DNA size can be precisely defined and the processed chromosome can retain its intrinsic structure and integrity. Therefore, our technique provides a high quality alternative approach to directed genetic recombination, and can be used for chromosomal repair, removal of defects and artificial chromosome creation. The technique may also have applicability on the manipulation and extension of large pieces of synthetic DNA.

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3D-CLEM Reveals that a Major Portion of Mitotic Chromosomes Is Not Chromatin

Cited by 107 publications

References 58 publications

Using human artificial chromosomes to study centromere assembly and function

Using human artificial chromosomes to study centromere assembly and function

Ki-67 Contributes to Normal Cell Cycle Progression and Inactive X Heterochromatin in p21 Checkpoint-Proficient Human Cells

Technique of laser chromosome welding for chromosome repair and artificial chromosome creation

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