Ki-67 and RepoMan have key roles during mitotic exit. Previously, we showed that Ki-67 organizes the mitotic chromosome periphery and recruits protein phosphatase 1 (PP1) to chromatin at anaphase onset, in a similar manner as RepoMan (Booth et al., 2014). Here we show how Ki-67 and RepoMan form mitotic exit phosphatases by recruiting PP1, how they distinguish between distinct PP1 isoforms and how the assembly of these two holoenzymes are dynamically regulated by Aurora B kinase during mitosis. Unexpectedly, our data also reveal that Ki-67 and RepoMan bind PP1 using an identical, yet novel mechanism, interacting with a PP1 pocket that is engaged only by these two PP1 regulators. These findings not only show how two distinct mitotic exit phosphatases are recruited to their substrates, but also provide immediate opportunities for the design of novel cancer therapeutics that selectively target the Ki-67:PP1 and RepoMan:PP1 holoenzymes.
The three-dimensional organization of tightly condensed chromatin within metaphase chromosomes has been one of the most challenging problems in structural biology since the discovery of the nucleosome. This study shows that chromosome images obtained from typical banded karyotypes and from different multicolour cytogenetic analyses can be used to gain information about the internal structure of chromosomes. Chromatin bands and the connection surfaces in sister chromatid exchanges and in cancer translocations are planar and orthogonal to the chromosome axis. Chromosome stretching produces band splitting and even the thinnest bands are orthogonal and well defined, indicating that short stretches of DNA can occupy completely the chromosome cross-section. These observations impose strong physical constraints on models that attempt to explain chromatin folding in chromosomes. The thin-plate model, which consists of many stacked layers of planar chromatin perpendicular to the chromosome axis, is compatible with the observed orientation of bands, with the existence of thin bands, and with band splitting; it is also compatible with the orthogonal orientation and planar geometry of the connection surfaces in chromosome rearrangements. The results obtained provide a consistent interpretation of the chromosome structural properties that are used in clinical cytogenetics for the diagnosis of hereditary diseases and cancers.
BACKGROUND: Understanding the mechanical properties of chromatin is an essential step towards deciphering the physicalrules of gene regulation. In the past ten years, many single molecule experiments have been carried out, andhigh resolution measurements of the chromatin fiber stiffness are now available. Simulations have been usedin order to link those measurements with structural cues, but so far no clear agreement among different groupshas been reached. RESULTS: We revisit here one of the most precise results obtained with carefully reconstituted fibers. CONCLUSIONS: We show that the mechanical properties of the chromatin fiber can be quantitatively accounted for by thestiffness of the DNA molecule and the 3D structure of the chromatin fiber.
The establishment of a specific nucleoprotein structure, the telomere, is required to ensure the protection of chromosome ends from being recognized as DNA damage sites. Telomere shortening below a critical length triggers a DNA damage response that leads to replicative senescence. In normal human somatic cells, characterized by telomere shortening with each cell division, telomere uncapping is a regulated process associated with cell turnover. Nevertheless, telomere dysfunction has also been associated with genomic instability, cell transformation, and cancer. Despite the essential role telomeres play in chromosome protection and in tumorigenesis, our knowledge of the chromatin structure involved in telomere maintenance is still limited. Here we review the recent findings on chromatin modifications associated with the dynamic changes of telomeres from protected to deprotected state and their role in telomere functions.
While many proteins are involved in the assembly and (re)positioning of nucleosomes, the dynamics of protein-assisted nucleosome formation are not well understood. We study NAP1 (nucleosome assembly protein 1) assisted nucleosome formation at the single-molecule level using magnetic tweezers. This method allows to apply a well-defined stretching force and supercoiling density to a single DNA molecule, and to study in real time the change in linking number, stiffness and length of the DNA during nucleosome formation. We observe a decrease in end-to-end length when NAP1 and core histones (CH) are added to the dsDNA. We characterize the formation of complete nucleosomes by measuring the change in linking number of DNA, which is induced by the NAP1-assisted nucleosome assembly, and which does not occur for non-nucleosomal bound histones H3 and H4. By rotating the magnets, the supercoils formed upon nucleosome assembly are removed and the number of assembled nucleosomes can be counted. We find that the compaction of DNA at low force is about 56 nm per assembled nucleosome. The number of compaction steps and associated change in linking number indicate that NAP1-assisted nucleosome assembly is a two-step process.
Eukaryotic DNA is strongly bent inside fundamental packaging units: the nucleosomes. It is known that their positions are strongly influenced by the mechanical properties of the underlying DNA sequence. Here we discuss the possibility that these mechanical properties and the concomitant nucleosome positions are not just a side product of the given DNA sequence, e.g. that of the genes, but that a mechanical evolution of DNA molecules might have taken place. We first demonstrate the possibility of multiplexing classical and mechanical genetic information using a computational nucleosome model. In a second step we give evidence for genome-wide multiplexing in Saccharomyces cerevisiae and Schizosacharomyces pombe. This suggests that the exact positions of nucleosomes play crucial roles in chromatin function.
Recent studies have revealed the importance of Ki-67 and the chromosome periphery in chromosome structure and segregation, but little is known about this elusive chromosome compartment. Here we used correlative light and serial block-face scanning electron microscopy, which we term 3D-CLEM, to model the entire mitotic chromosome complement at ultra-structural resolution. Prophase chromosomes exhibit a highly irregular surface appearance with a volume smaller than metaphase chromosomes. This may be because of the absence of the periphery, which associates with chromosomes only after nucleolar disassembly later in prophase. Indeed, the nucleolar volume almost entirely accounts for the extra volume found in metaphase chromosomes. Analysis of wild-type and Ki-67-depleted chromosomes reveals that the periphery comprises 30%-47% of the entire chromosome volume and more than 33% of the protein mass of isolated mitotic chromosomes determined by quantitative proteomics. Thus, chromatin makes up a surprisingly small percentage of the total mass of metaphase chromosomes.
Current cell-free DNA assessment of fetal chromosomes does not analyze and report on all chromosomes. Hence, a significant proportion of fetal chromosomal abnormalities are not detectable by current non-invasive methods. Here we report the clinical validation of a novel non-invasive prenatal test designed to detect genome-wide gains and losses of chromosomal material ≥7 Mb and losses associated with specific deletions <7 Mb.
The realization that nuclear distribution of DNA, RNA, and proteins differs between cell types and developmental stages suggests that nuclear organization serves regulatory functions. Understanding the logic of nuclear architecture and how it contributes to differentiation and cell fate commitment remains challenging. Here, we use soft X-ray tomography (SXT) to image chromatin organization, distribution, and biophysical properties during neurogenesis in vivo. Our analyses reveal that chromatin with similar biophysical properties forms an elaborate connected network throughout the entire nucleus. Although this interconnectivity is present in every developmental stage, differentiation proceeds with concomitant increase in chromatin compaction and re-distribution of condensed chromatin toward the nuclear core. HP1β, but not nucleosome spacing or phasing, regulates chromatin rearrangements because it governs both the compaction of chromatin and its interactions with the nuclear envelope. Our experiments introduce SXT as a powerful imaging technology for nuclear architecture.
Migraine is a disabling common brain disorder typically characterized by attacks of severe headache and associated with autonomic and neurological symptoms. Its etiology is far from resolved. This review will focus on evidence that epigenetic mechanisms play an important role in disease etiology. Epigenetics comprise both DNA methylation and post-translational modifications of the tails of histone proteins, affecting chromatin structure and gene expression. Besides playing a role in establishing cellular and developmental stage-specific regulation of gene expression, epigenetic processes are also important for programming lasting cellular responses to environmental signals. Epigenetic mechanisms may explain how non-genetic endogenous and exogenous factors such as female sex hormones, stress hormones and inflammation trigger may modulate attack frequency. Developing drugs that specifically target epigenetic mechanisms may open up exciting new avenues for the prophylactic treatment of migraine.