ABSTRACT
Centromeres are the foundation for mitotic kinetochore assembly and thus are essential for chromosome segregation. Centromeres are epigenetically defined by nucleosomes containing the histone H3 variant CENP-A. CENP-A nucleosome assembly is uncoupled from replication and occurs in G1, but how cells control this timing is incompletely understood. The formation of CENP-A nucleosomes in vertebrates requires CENP-C and the Mis18 complex which recruit the CENP-A chaperone HJURP to centromeres. Using a cell-free system for centromere assembly in X. laevis egg extracts, we discover two activities that inhibit CENP-A assembly in metaphase. HJURP phosphorylation prevents the interaction between HJURP and CENP-C in metaphase, blocking the delivery of soluble CENP-A to centromeres. Non-phosphorylatable mutants of HJURP constitutively bind CENP-C in metaphase but are not sufficient for new CENP-A assembly. We find that the M18BP1.S subunit of the Mis18 complex also binds to CENP-C to competitively inhibit HJURP's access to centromeres. Removal of these two inhibitory activities causes CENP-A assembly in metaphase.
Subject(s)
Carrier Proteins , Centromere Protein A , Centromere , DNA-Binding Proteins , Nucleosomes , Xenopus Proteins , Animals , Autoantigens/metabolism , Carrier Proteins/metabolism , Centromere/metabolism , Centromere Protein A/metabolism , Metaphase , Nucleosomes/metabolism , Phosphorylation , Xenopus laevis/genetics , Xenopus Proteins/metabolism , DNA-Binding Proteins/metabolismABSTRACT
Background: The American Academy of Dermatology and the Food and Drug Administration recommend consultation with a dermatologist prior to undergoing laser tattoo removal. However, non-health care professionals offer tattoo removal. Understanding the information available on the internet for patients regarding tattoo removal is important given that individuals are increasingly consulting digital sources to make decisions regarding skin care. Prior research has identified that YouTube contains misinformation on dermatologic health. Objective: Here, we present a cross-sectional study that determined the sources of information in YouTube videos that discuss tattoo removal and described the content presented to viewers. Methods: Using the query "tattoo removal," we reviewed English-language YouTube videos that explicitly discussed tattoo removal. The following data were recorded: profession of the presenter, tattoo removal method discussed, whether an explicit recommendation to see a dermatologist or physician was present in the video, and number of views. Results: We analyzed 162 YouTube videos. We found that the majority were presented by non-health care professionals (n=125, 77%), with only 4 (3.7%) records of this subset recommending viewers to seek consultation from a dermatologist to ensure safe and adequate tattoo removal. Conclusions: Based on our findings, we recommend that dermatologists and other health care professionals provide high-quality, evidence-based information to viewers on tattoo removal and encourage dermatology societies to share via their social media platforms information about the importance of consulting a dermatologist for tattoo removal.
ABSTRACT
During cell division, chromosomes must be equally segregated to daughter cells. Centromeres, the primary interaction site between chromosomes and microtubules, mediate faithful chromosome segregation during mitosis. Functional studies of centromere proteins in cells have proven difficult, as mutation or deletion of most centromeric proteins often results in cell lethality. In this protocol, sperm chromatin or reconstituted chromatin arrays, together with Xenopus laevis egg extracts, are used to overcome these limitations and study centromere and kinetochore assembly in vitro. X. laevis egg extract is a powerful tool, as it can be readily cycled in vitro by addition of calcium and easily modified biochemically. Coupled with the addition of customizable reconstituted chromatin arrays or sperm chromatin, X. laevis egg extract provides distinct advantages over cell-based approaches in which similar experiments would not be feasible. Following incubation in egg extract, reconstituted centromeric chromatin arrays and sperm chromatin specifically assemble core centromere and kinetochore components that can be analyzed via immunofluorescence.
