Shake It Off: The Elimination of Erroneous Kinetochore-Microtubule Attachments and Chromosome Oscillation

Cell proliferation and sexual reproduction require the faithful segregation of chromosomes. Chromosome segregation is driven by the interaction of chromosomes with the spindle, and the attachment of chromosomes to the proper spindle poles is essential. Initial attachments are frequently erroneous due to the random nature of the attachment process; however, erroneous attachments are selectively eliminated. Proper attachment generates greater tension at the kinetochore than erroneous attachments, and it is thought that attachment selection is dependent on this tension. However, studies of meiotic chromosome segregation suggest that attachment elimination cannot be solely attributed to tension, and the precise mechanism of selective elimination of erroneous attachments remains unclear. During attachment elimination, chromosomes oscillate between the spindle poles. A recent study on meiotic chromosome segregation in fission yeast has suggested that attachment elimination is coupled to chromosome oscillation. In this review, the possible contribution of chromosome oscillation in the elimination of erroneous attachment is discussed in light of the recent finding.     

Active Transport Can Greatly Enhance Cdc20:Mad2 Formation

To guarantee genomic integrity and viability, the cell must ensure proper distribution of the replicated chromosomes among the two daughter cells in mitosis. The mitotic spindle assembly checkpoint (SAC) is a central regulatory mechanism to achieve this goal. A dysfunction of this checkpoint may lead to aneuploidy and likely contributes to the development of cancer. Kinetochores of unattached or misaligned chromosomes are thought to generate a diffusible “wait-anaphase” signal, which is the basis for downstream events to inhibit the anaphase promoting complex/cyclosome (APC/C). The rate of Cdc20:C-Mad2 complex formation at the kinetochore is a key regulatory factor in the context of APC/C inhibition. Computer simulations of a quantitative SAC model show that the formation of Cdc20:C-Mad2 is too slow for checkpoint maintenance when cytosolic O-Mad2 has to encounter kinetochores by diffusion alone. Here, we show that an active transport of O-Mad2 towards the spindle mid-zone increases the efficiency of Mad2-activation. Our in-silico data indicate that this mechanism can greatly enhance the formation of Cdc20:Mad2 and furthermore gives an explanation on how the “wait-anaphase” signal can dissolve abruptly within a short time. Our results help to understand parts of the SAC mechanism that remain unclear.     

DNA Damage Response Regulation by Histone Ubiquitination

Cells are constantly exposed to numerous genotoxic stresses that induce DNA damage. DNA double-strand breaks (DSBs) are among the most serious damages and should be systematically repaired to preserve genomic integrity. The efficiency of repair is closely associated with chromatin structure, which is regulated by posttranslational modifications of histones, including ubiquitination. Recent evidence shows crosstalk between histone ubiquitination and DNA damage responses, suggesting an integrated model for the systematic regulation of DNA repair. There are two major pathways for DSB repair, viz., nonhomologous end joining and homologous recombination, and the choice of the pathway is partially controlled by posttranslational modifications of histones, including ubiquitination. Histone ubiquitination changes chromatin structure in the vicinity of DSBs and serves as a platform to select and recruit repair proteins; the removal of these modifications by deubiquitinating enzymes suppresses the recruitment of repair proteins and promotes the convergence of repair reactions. This article provides a comprehensive overview of the DNA damage response regulated by histone ubiquitination in response to DSBs.     

Histone Modifications and Non-Coding RNAs: Mutual Epigenetic Regulation and Role in Pathogenesis

In the last few years, more and more scientists have suggested and confirmed that epigenetic regulators are tightly connected and form a comprehensive network of regulatory pathways and feedback loops. This is particularly interesting for a better understanding of processes that occur in the development and progression of various diseases. Appearing on the preclinical stages of diseases, epigenetic aberrations may be prominent biomarkers. Being dynamic and reversible, epigenetic modifications could become targets for a novel option for therapy. Therefore, in this review, we are focusing on histone modifications and ncRNAs, their mutual regulation, role in cellular processes and potential clinical application.     

Physiological Ovarian Aging Is Associated with Altered Expression of Post-Translational Modifications in Mice

Post-translational modifications (PTMs) have been confirmed to be involved in multiple female reproductive events, but their role in physiological ovarian aging is far from elucidated. In this study, mice aged 3, 12 or 17 months (3M, 12M, 17M) were selected as physiological ovarian aging models. The expression of female reproductive function-related genes, the global profiles of PTMs, and the level of histone modifications and related regulatory enzymes were examined during physiological ovarian aging in the mice by quantitative real-time PCR and western blot, respectively. The results showed that the global protein expression of Kbhb (lysineβ-hydroxybutyryllysine), Khib (lysine 2-hydroxyisobutyryllysine), Kglu (lysineglutaryllysine), Kmal (lysinemalonyllysine), Ksucc (lysinesuccinyllysine), Kcr (lysinecrotonyllysine), Kbu (lysinebutyryllysine), Kpr (lysinepropionyllysine), SUMO1 (SUMO1 modification), ub (ubiquitination), P-Typ (phosphorylation), and 3-nitro-Tyr (nitro-tyrosine) increased significantly as mice aged. Moreover, the modification level of Kme2 (lysinedi-methyllysine) and Kac (lysineacetyllysine) was the highest in the 3M mice and the lowest in 12M mice. In addition, only trimethylation of histone lysine was up-regulated progressively and significantly with increasing age (p < 0.001), H4 ubiquitination was obviously higher in the 12M and 17M mice than 3M (p < 0.001), whereas the modification of Kpr (lysinepropionylation) and O-GlcNA in 17M was significantly decreased compared with the level in 3M mice (p < 0.05, p < 0.01). Furthermore, the expression levels of the TIP60, P300, PRDM9, KMT5B, and KMT5C genes encoding PTM regulators were up-regulated in 17M compared to 3M female mice (p < 0.05). These findings indicate that altered related regulatory enzymes and PTMs are associated with physiological ovarian aging in mice, which is expected to provide useful insights for the delay of ovarian aging and the diagnosis and treatment of female infertility.     

Substrate Scope for Human Histone Lysine Acetyltransferase KAT8

Biomedically important histone lysine acetyltransferase KAT8 catalyses the acetyl coenzyme A-dependent acetylation of lysine on histone and other proteins. Here, we explore the ability of human KAT8 to catalyse the acetylation of histone H4 peptides possessing lysine and its analogues at position 16 (H4K16). Our synthetic and enzymatic studies on chemically and structurally diverse lysine mimics demonstrate that KAT8 also has a capacity to acetylate selected lysine analogues that possess subtle changes on the side chain and main chain. Overall, this work highlights that KAT8 has a broader substrate scope beyond natural lysine, and contributes to the design of new chemical probes targeting KAT8 and other members of the histone lysine acetyltransferase (KAT) family.     

Histone Acetylation Dynamics during In Vivo and In Vitro Oocyte Aging in Common Carp Cyprinus carpio

Aging is the most critical factor that influences the quality of post-ovulatory oocytes. Age-related molecular pathways remain poorly understood in fish oocytes. In this study, we examined the effect of oocyte aging on specific histone acetylation in common carp Cyprinus carpio. The capacity to progress to the larval stage in oocytes that were aged for 28 h in vivo and in vitro was evaluated. Global histone modifications and specific histone acetylation (H3K9ac, H3K14ac, H4K5ac, H4K8ac, H4K12ac, and H4K16ac) were investigated during oocyte aging. Furthermore, the activity of histone acetyltransferase (HAT) was assessed in fresh and aged oocytes. Global histone modifications did not exhibit significant alterations during 8 h of oocyte aging. Among the selected modifications, H4K12ac increased significantly at 28 h post-stripping (HPS). Although not significantly different, HAT activity exhibited an upward trend during oocyte aging. Results of our current study indicate that aging of common carp oocytes for 12 h results in complete loss of egg viability rates without any consequence in global and specific histone modifications. However, aging oocytes for 28 h led to increased H4K12ac. Thus, histone acetylation modification as a crucial epigenetic mediator may be associated with age-related defects, particularly in oocytes of a more advanced age.     

Unraveling the Role of Histone Variant CENP-A and Chaperone HJURP Expression in Thymic Epithelial Neoplasms

Background: Recent advances demonstrate the role of chromatin regulators, including histone variants and histone chaperones, in cancer initiation and progression. Methods: Histone H3K4me3, histone variant centromere protein (CENP-A) and histone chaperones Holliday junction recognition protein (HJURP) as well as DAXX expression were examined immunohistochemically in 95 thymic epithelial tumor (TET) specimens. Our results were compared with the expression profile of DAXX, HJURP and CENP-A in gene expression profiling interactive analysis (GEPIA2). Results: The lymphocyte-poor B3- and C-type TETs were more frequently DAXX negative (p = 0.043). B3 and C-Type TETs showed higher cytoplasmic and nuclear CENP-A (p = 0.007 and p = 0.002) and higher cytoplasmic HJURP H-score (p < 0.001). Higher nuclear CENP-A and cytoplasmic HJURP expression was associated with advanced Masaoka–Koga stage (p = 0.048 and p < 0.001). A positive correlation between HJURP and CENP-A was also observed. The presence of cytoplasmic CENP-A expression was correlated with a favorable overall survival (p = 0.03). CENP-A overexpression in survival analysis of TCGA TETs showed similar results. H3K4me3 expression was not associated with any clinicopathological parameters. Conclusions: Our results suggest a significant interaction between CENP-A and HJURP in TETs. Moreover, we confirmed the presence of a cytoplasmic CENP-A immunolocalization, suggesting also a possible favorable prognostic value of this specific immunostaining pattern.     

OTUD6A Is an Aurora Kinase A-Specific Deubiquitinase

Aurora kinases are serine/threonine kinases required for cell proliferation and are overexpressed in many human cancers. Targeting Aurora kinases has been a therapeutic strategy in cancer treatment. Here, we attempted to identify a deubiquitinase (DUB) that regulates Aurora kinase A (Aurora-A) protein stability and/or kinase activity as a potential cancer therapeutic target. Through pull-down assays with the human DUB library, we identified OTUD6A as an Aurora-A-specific DUB. OTUD6A interacts with Aurora-A through OTU and kinase domains, respectively, and deubiquitinates Aurora-A. Notably, OTUD6A promotes the protein half-life of Aurora-A and activates Aurora-A by increasing phosphorylation at threonine 288 of Aurora-A. From qPCR screening, we identified and validated that the cancer gene CKS2 encoding Cyclin-dependent kinases regulatory subunit 2 is the most upregulated cell cycle regulator when OTUD6A is overexpressed. The results suggest that OTUD6A may serve as a therapeutic target in human cancers.     

Live‐Cell Studies of p300/CBP Histone Acetyltransferase Activity and Inhibition

Histone acetyltransferase enzymes (HATs) are important therapeutic targets, but there are few cell‐based assays available for evaluating the pharmacodynamics of HAT inhibitors. Here we present the application of a FRET‐based reporter, Histac, in live‐cell studies of p300/CBP HAT inhibition, by both genetic and pharmacologic disruption. shRNA knockdown of p300/CBP led to increased Histac FRET, thus suggesting a role for p300/CBP in the acetylation of the histone H4 tail. Additionally, we describe a new p300/CBP HAT inhibitor, C107, and show that it can also increase cellular Histac FRET. Taken together, these studies provide a live‐cell strategy for identifying and evaluating p300/CBP inhibitors.     

Sperm Energy Restriction and Recovery (SER) Alters Epigenetic Marks during the First Cell Cycle of Development in Mice

The sperm energy restriction and recovery (SER) treatment developed in our laboratory was shown to improve fertilization and blastocyst development following in vitro fertilization (IVF) in mice. Here, we investigated the effects of SER on early embryogenesis. Developmental events observed during the first cell cycle indicated that progression through the pronuclear stages of SER-generated embryos is advanced in comparison with control-generated embryos. These findings prompted further analysis of potential effects of SER on pronuclear chromatin dynamics, focusing on the key H3K4me3 and H3K27ac histone modifications. Nearly all the SER-generated embryos displayed H3K4me3 in the male pronuclei at 12 h post-insemination (HPI), while a subset of the control-generated embryos did not. Additionally, SER-generated embryos displayed a more homogenous intensity of H3K27ac at 8 and 12 HPI compared to control embryos. These changes in histone modifications during the first cell cycle were accompanied by differences in gene expression at the two-cell stage; both of these changes in early embryos could potentially play a role in the improved developmental outcomes of these embryos later in development. Our results indicate that sperm incubation conditions have an impact on early embryo development and can be useful for the improvement of assisted reproductive technology outcomes.     

Control of Chromatin Organization and Chromosome Behavior during the Cell Cycle through Phase Separation

Phase-separated condensates participate in various biological activities. Liquid–liquid phase separation (LLPS) can be driven by collective interactions between multivalent and intrinsically disordered proteins. The manner in which chromatin—with various morphologies and activities—is organized in a complex and small nucleus still remains to be fully determined. Recent findings support the claim that phase separation is involved in the regulation of chromatin organization and chromosome behavior. Moreover, phase separation also influences key events during mitosis and meiosis. This review elaborately dissects how phase separation regulates chromatin and chromosome organization and controls mitotic and meiotic chromosome behavior.     

Recent Progress on the Localization of PLK1 to the Kinetochore and Its Role in Mitosis

The accurate distribution of the replicated genome during cell division is essential for cell survival and healthy organismal development. Errors in this process have catastrophic consequences, such as birth defects and aneuploidy, a hallmark of cancer cells. PLK1 is one of the master kinases in mitosis and has multiple functions, including mitotic entry, chromosome segregation, spindle assembly checkpoint, and cytokinesis. To dissect the role of PLK1 in mitosis, it is important to understand how PLK1 localizes in the specific region in cells. PLK1 localizes at the kinetochore and is essential in spindle assembly checkpoint and chromosome segregation. However, how PLK1 localizes at the kinetochore remains elusive. Here, we review the recent literature on the kinetochore recruitment mechanisms of PLK1 and its roles in spindle assembly checkpoint and attachment between kinetochores and spindle microtubules. Together, this review provides an overview of how the local distribution of PLK1 could regulate major pathways in mitosis.     

Comparative Analysis of Histone H3K4me3 Distribution in Mouse Liver in Different Diets Reveals the Epigenetic Efficacy of Cyanidin-3-O-glucoside Dietary Intake

Background: Different diets result in significantly different phenotypes through metabolic and genomic reprogramming. Epigenetic marks, identified in humans and mouse models through caloric restriction, a high-fat diet or the intake of specific bioactives, suggest that genomic reprogramming drives this metabolic reprogramming and mediates the effect of nutrition on health. Histone modifications encode the epigenetic signal, which adapts genome functions to environmental conditions, including diets, by tuning the structure and properties of chromatin. To date, the effect of different diets on the genome-wide distribution of critical histone marks has not been determined. Methods: Using chromatin immunoprecipitation sequencing, we investigated the distribution of the trimethylation of lysine 4 of histone H3 in the liver of mice fed for one year with five different diets, including: chow containing yellow corn powder as an extra source of plant bioactives or specifically enriched with cyanidin-3-O-Glucoside, high-fat-enriched obesogenic diets, and caloric-restricted pro-longevity diets. Conclusions: Comparison of the resulting histone mark profiles revealed that functional food containing cyanidin determines a broad effect.     

The Dynamics and Plasticity of Epigenetics in Diabetic Kidney Disease: Therapeutic Applications Vis-à-Vis

Chronic kidney disease (CKD) refers to the phenomenon of progressive decline in the glomerular filtration rate accompanied by adverse consequences, including fluid retention, electrolyte imbalance, and an increased cardiovascular risk compared to those with normal renal function. The triggers for the irreversible renal function deterioration are multifactorial, and diabetes mellitus serves as a major contributor to the development of CKD, namely diabetic kidney disease (DKD). Recently, epigenetic dysregulation emerged as a pivotal player steering the progression of DKD, partly resulting from hyperglycemia-associated metabolic disturbances, rising oxidative stress, and/or uncontrolled inflammation. In this review, we describe the major epigenetic molecular mechanisms, followed by summarizing current understandings of the epigenetic alterations pertaining to DKD. We highlight the epigenetic regulatory processes involved in several crucial renal cell types: Mesangial cells, podocytes, tubular epithelia, and glomerular endothelial cells. Finally, we highlight epigenetic biomarkers and related therapeutic candidates that hold promising potential for the early detection of DKD and the amelioration of its progression.