Centromeric chromatin pliability and memory at a human neocentromere

We show that Trichostatin A (TSA)-induced partial histone hyperacetylation causes a unidirectional shift in the position of a previously defined binding domain for the centromere-specific histone H3 homologue CENP-A at a human neocentromere. The shift of approximately 320 kb is fully reversible when TSA is removed, but is accompanied by an apparent reduction in the density of CENP-A per unit length of genomic DNA at the neocentromere. TSA treatment also instigates a reversible abolition of a previously defined major domain of differentially delayed replication timing that was originally established at the neocentromeric site. None of these changes has any measurable deleterious effects on mitosis or neocentromere function. The data suggest pliability of centromeric chromatin in response to epigenetic triggers, and the non-essential nature of the regions of delayed replication for centromere function. Reversibility of the CENP-A-binding position and the predominant region of delayed replication timing following removal of TSA suggest strong memory at the original site of neocentromeric chromatin formation.     

i-Motif formation and spontaneous deletions in human cells

Concatemers of d(TCCC) that were first detected through their association with deletions at the RACK7 locus, are widespread throughout the human genome. Circular dichroism spectra show that d(GGGA)_n sequences form G-quadruplexes when n > 3, while i-motif structures form at d(TCCC)_n sequences at neutral pH when n ≥ 7 in vitro. In the PC3 cell line, deletions are observed only when the d(TCCC)_n variant is long enough to form significant levels of unresolved i-motif structure at neutral pH. The presence of an unresolved i-motif at a representative d(TCCC)_n element at RACK7 was suggested by experiments showing that that the region containing the d(TCCC)₉ element was susceptible to bisulfite attack in native DNA and that d(TCCC)₉ oligo formed an i-motif structure at neutral pH. This in turn suggested that that the i-motif present at this site in native DNA must be susceptible to bisulfite mediated deamination even though it is a closed structure. Bisulfite deamination of the i-motif structure in the model oligodeoxynucleotide was confirmed using mass spectrometry analysis. We conclude that while G-quadruplex formation may contribute to spontaneous mutation at these sites, deletions actually require the potential for i-motif to form and remain unresolved at neutral pH.     

Genome characterization and CRISPR-Cas9 editing of a human neocentromere

The maintenance of genome integrity is ensured by proper chromosome inheritance during mitotic and meiotic cell divisions. The chromosomal counterpart responsible for chromosome segregation to daughter cells is the centromere, at which the spindle apparatus attaches through the kinetochore. Although all mammalian centromeres are primarily composed of megabase-long repetitive sequences, satellite-free human neocentromeres have been described. Neocentromeres and evolutionary new centromeres have revolutionized traditional knowledge about centromeres. Over the past 20 years, insights have been gained into their organization, but in spite of these advancements, the mechanisms underlying their formation and evolution are still unclear. Today, through modern and increasingly accessible genome editing and long-read sequencing techniques, research in this area is undergoing a sudden acceleration. In this article, we describe the primary sequence of a previously described human chromosome 3 neocentromere and observe its possible evolution and repair results after a chromosome breakage induced through CRISPR-Cas9 technologies. Our data represent an exciting advancement in the field of centromere/neocentromere evolution and chromosome stability.

     

Lipid body formation during maturation of human mast cells

Lipid droplets, also called lipid bodies (LB) in inflammatory cells, are important cytoplasmic organelles. However, little is known about the molecular characteristics and functions of LBs in human mast cells (MC). Here, we have analyzed the genesis and components of LBs during differentiation of human peripheral blood-derived CD34(+) progenitors into connective tissue-type MCs. In our serum-free culture system, the maturing MCs, derived from 18 different donors, invariably developed triacylglycerol (TG)-rich LBs. Not known heretofore, the MCs transcribe the genes for perilipins (PLIN)1-4, but not PLIN5, and PLIN2 and PLIN3 display different degrees of LB association. Upon MC activation and ensuing degranulation, the LBs were not cosecreted with the cytoplasmic secretory granules. Exogenous arachidonic acid (AA) enhanced LB genesis in Triacsin C-sensitive fashion, and it was found to be preferentially incorporated into the TGs of LBs. The large TG-associated pool of AA in LBs likely is a major precursor for eicosanoid production by MCs. In summary, we demonstrate that cultured human MCs derived from CD34(+) progenitors in peripheral blood provide a new tool to study regulatory mechanisms involving LB functions, with particular emphasis on AA metabolism, eicosanoid biosynthesis, and subsequent release of proinflammatory lipid mediators from these cells.     

Transmission of a fully functional human neocentromere through three generations

An unusual Y chromosome with a primary constriction inside the long-arm heterochromatin was found in the amniocytes of a 38-year-old woman. The same Y chromosome was found in her husband and brother-in-law, thus proving that it was already present in the father. FISH with alphoid DNA showed hybridization signals at the usual position of the Y centromere but not at the primary constriction. Centromere proteins (CENP)-A, CENP-C, and CENP-E could not be detected at the site of the canonic centromere but were present at the new constriction, whereas CENP-B was not detected on this Y chromosome. Experiments with 82 Y-specific loci distributed throughout the chromosome confirmed that no gross deletion or rearrangement had taken place, and that the Y chromosome belonged to a haplogroup whose members have a mean alphoid array of 770 kb (range 430-1,600 kb), whereas that of this case was approximately 250 kb. Thus, this Y chromosome appeared to be deleted for part of the alphoid DNA. It seems likely that this deletion was responsible for the silencing of the normal centromere and that the activation of the neocentromere prevented the loss of this chromosome. Alternatively, neocentromere activation could have occurred first and stimulated inactivation of the normal centromere by partial deletion. Whatever the mechanism, the presence of this chromosome in three generations demonstrates that it functions sufficiently well in mitosis for male sex determination and fertility and that neocentromeres can be transmitted normally at meiosis.     

Induction of spontaneous recurrent epileptiform discharges causes long-term changes in intracellular calcium homeostatic mechanisms

Calcium and calcium-dependent systems have been long implicated in the induction of epilepsy. We have previously observed that intracellular calcium ([Ca2+]i) levels remain elevated in cells undergoing epileptogenesis in the hippocampal neuronal culture (HNC) model. In this study, we employed the hippocampal neuronal culture (HNC) model of in vitro 'epilepsy' which produces spontaneous recurrent epileptiform discharges (SREDs) for the life of the neurons in culture to investigate alterations in [Ca2+]i homeostatic mechanisms that may be associated with the 'epileptic' phenotype. [Ca2+]i imaging fluorescence microscopy was performed on control and 'epileptic' neurons with two different fluorescent dyes ranging from high to low affinities for [Ca2+]i. We measured baseline [Ca2+]i levels and the ability to restore resting [Ca2+]i levels after a brief 2-min exposure to the excitatory amino acid glutamate in control neurons and neurons with SREDs. Neurons manifesting SREDs had statistically significantly higher baseline [Ca2+]i levels that persisted for the life of the culture. In addition, the 'epileptic' phenotype was associated with an inability to rapidly restore [Ca2+]i levels to baseline following a glutamate induced [Ca2+]i load. The use of the low affinity dye Fura-FF demonstrated that the difference in restoring baseline [Ca2+]i levels was not due to saturation of the high affinity dye Indo-1, which was utilized for evaluating the [Ca2+]i kinetics at lower [Ca2+]i levels. Peak [Ca2+]i levels in response to glutamate were the same in both 'epileptic' and control neurons. While [Ca2+]i levels recovered in approximately 30 min in control cells, it took more than 90 min to reach baseline levels in cells manifesting SREDs. Alterations of [Ca2+]i homeostatic mechanisms observed with the 'epileptic' phenotype were shown to be independent of the presence of continuous SREDs and persisted for the life of the neurons in culture. Epileptogenesis was shown not to affect the degree or duration of glutamate induced neuronal depolarization in comparing control and 'epileptic' neurons. The results indicate that epileptogenesis in this in vitro model produced long-lasting alterations in [Ca2+]i regulation that may underlie the 'epileptic' phenotype and contribute to the persistent neuroplasticity changes associated with epilepsy.     

A neocentromere in the DAZ region of the human Y chromosome

We describe a novel rearranged human Y chromosome consisting of an inverted duplication of the long arm heterochromatin and a small amount of euchromatin: rea(Y)(qter-q11.2::q11.2-qter). The normal centromere has been deleted and a neocentromere containing CENP-A, -C, -E and Mad2 but not CENP-B has formed close to the breakpoint. A 2.7 Mb yeast artificial chromosome contig spanning the breakpoint was constructed and the breakpoint was localised to a region of <120 kb close to the DAZ gene cluster. Combined immunofluorescence and fluorescence in situ hybridisation showed that the centromeric protein-binding domain of the neocentromere was located near the breakpoint and within the DAZ cluster.     

Attenuation of spontaneous pseudopod formation in human neutrophils by pentoxifylline

The effects of pentoxifylline (PTX) on spontaneous pseudopod formation in neutrophils in response to the tripeptide formyl-Met-Leu-Phe (fMLP), endotoxin, human complement C5a, and leukotriene B₄ (LTB₄) were examined in autologous plasma. Unseparated supernatant leukocyte suspensions from fresh heparinized venous human blood were incubated with PTX (0-5 mM) for 25 min and then stimulated for 5–25 min within a range of concentrations of fMLP, endotoxin, complement C5a, and LTB₄. The cell suspensions were fixed with glutaraldehyde and stained with crystal violet in acetic acid; the percentage of neutrophils with pseudopods was determined under high-resolution light microscope. The results show that PTX significantly decreases formation of pseudopods in the presence of all four stimulators. The mechanism of pseudopod suppression appears to be independent of the adenosine receptor. PTX and its analogues, HWA 138 and HWA 448, decreased pseudopod formation by similar amounts when stimulated with 10⁻⁸M fMLP. These results suggest that PTX may improve microvascular perfusion and attenuate neutrophilmediated injury by reducing the degree of neutrophil pseudopod formation in free suspension and microvascular entrapment.     

Lack of glucose-induced functional maturation during long-term culture of human fetal islet cells

To investigate the long-term effects of glucose on the function of human fetal islets we cultured islet-like cell clusters (ICC) obtained from 12 human fetuses with a mean age of 16.1 weeks in media containing 2.8, 11.1 or 16.7 mM glucose. On the 8th day of culture, the ICC that had been maintained in 16.7 mM glucose contained 60% less insulin than the ICC cultured in 2.8 mM glucose. However, insulin release was similar in both groups, and was not affected by a 24-h incubation in high vs. low glucose. Also (pro) insulin biosynthesis was not significantly affected. During a 24-day culture period, the total release of insulin and glucagon was similar in all glucose concentrations. The ICC released about 75% of their insulin content but only 15% of their glucagon content during the last 48 h of the 24-day culture period, again regardless of glucose concentration in media. Insulin release was insensitive to acute glucose and leucine challenges in perifusion experiments after culture for 1, 5, 8 or 16 days in 11.1 mM glucose, whereas glucagon was always a potent stimulus. In conclusion, the function of cultured young human fetal islet cells is remarkably independent of glucose, even during prolonged exposure. Moreover, the primary role of glucagon in fetal life may be that of a paracrine stimulator of beta-cell function.     

Energy substrates and the completion of spontaneous meiotic maturation

This study was carried out to examine how different combinations of pyruvate and glucose affect spontaneous meiotic maturation of cumulus-cell-enclosed mouse oocytes (CEO) to metaphase II (MII). Most experiments used an open system in which oocytes were cultured in 1 ml medium in plastic tubes. Initial experiments examined the dose response effects of pyruvate or glucose alone in the presence or absence of 2 mM glutamine. When medium lacked both pyruvate and glucose, more than 91% of the oocytes died in glutamine-free medium during 15 h of culture; viability was restored with the addition of glutamine, but only 11% of the CEO reached MII. In the absence of glutamine, 62-68% of oocytes completed maturation in 0.23-2.3 mM pyruvate, while 44-60% MII was observed in 0.55-27.8 mM glucose. The addition of glutamine to these cultures had a general suppressive effect on the completion of maturation. When glucose was added to pyruvate-containing cultures, the combination of 1 mM pyruvate/5.5 mM glucose was most effective in supporting maturation (about 90% MII), with little effect of glutamine. No further increase in maturation was observed when glucose was increased five-fold (to 27.8 mM). The positive effect of glucose was in part attributed to stimulation of glycolysis and increased production of pyruvate, since a reduced culture volume (8 microl), which allows the accumulation of secreted pyruvate, improved maturation in glucose-containing, but not pyruvate-containing, medium, and FSH, which stimulates glycolysis, increased progression to MII in glucose-containing, but not pyruvate-containing, medium. Yet these results also suggest that glucose has a beneficial effect on maturation apart from simple provision of pyruvate. The pyruvate effect was directly on the oocyte, because denuded oocytes responded more effectively than CEO to this energy substrate. The highest percentage of MII oocytes (96-97%) occurred in microdrop cultures containing glucose but lacking glutamine. These results indicate that glutamine supports oocyte viability but is not an adequate energy source for the completion of spontaneous meiotic maturation and may be detrimental. In addition, while pyruvate and glucose alone can each support meiotic progression of CEO to MII, optimal maturation requires the provision of both substrates to the culture medium when a large volume (1 ml) is used. It is concluded that careful attention to specific energy substrate supplementation and culture volume is important to optimise spontaneous meiotic maturation in vitro.     

Attenuation of spontaneous pseudopod formation in human neutrophils by pentoxifylline.

The effects of pentoxifylline (PTX) on spontaneous pseudopod formation in neutrophils in response to the tripeptide formyl-Met-Leu-Phe (fMLP), endotoxin, human complement C5a, and leukotriene B4 (LTB4) were examined in autologous plasma. Unseparated supernatant leukocyte suspensions from fresh heparinized venous human blood were incubated with PTX (0-5 mM) for 25 min and then stimulated for 5-25 min within a range of concentrations of fMLP, endotoxin, complement C5a, and LTB4. The cell suspensions were fixed with glutaraldehyde and stained with crystal violet in acetic acid; the percentage of neutrophils with pseudopods was determined under high-resolution light microscope. The results show that PTX significantly decreases formation of pseudopods in the presence of all four stimulators. The mechanism of pseudopod suppression appears to be independent of the adenosine receptor. PTX and its analogues, HWA 138 and HWA 448, decreased pseudopod formation by similar amounts when stimulated with 10(-8)M fMLP. These results suggest that PTX may improve microvascular perfusion and attenuate neutrophil-mediated injury by reducing the degree of neutrophil pseudopod formation in free suspension and microvascular entrapment.     

A 330 kb CENP-A binding domain and altered replication timing at a human neocentromere

Centromere protein A (CENP-A) is an essential centromere-specific histone H3 homologue. Using combined chromatin immunoprecipitation and DNA array analysis, we have defined a 330 kb CENP-A binding domain of a 10q25.3 neocentromere found on the human marker chromosome mardel(10). This domain is situated adjacent to the 80 kb region identified previously as the neocentromere site through lower-resolution immunofluorescence/FISH analysis of metaphase chromosomes. The 330 kb CENP-A binding domain shows a depletion of histone H3, providing evidence for the replacement of histone H3 by CENP-A within centromere-specific nucleosomes. The DNA within this domain has a high AT-content comparable to that of alpha-satellite, a high prevalence of LINEs and tandem repeats, and fewer SINEs and potential genes than the surrounding region. FISH analysis indicates that the normal 10q25.3 genomic region replicates around mid-S phase. Neocentromere formation is accompanied by a replication time lag around but not within the CENP-A binding region, with this lag being significantly more prominent to one side. The availability of fully sequenced genomic markers makes human neocentromeres a powerful model for dissecting the functional domains of complex higher eukaryotic centromeres.     

Induction of dendritic cell maturation by IL-18

IL-18 is a pluripotent proinflammatory cytokine produced primarily by antigen presenting cells involved in numerous aspects of immune regulation most notably on lymphoid cells. The effect of IL-18 stimulation on cells in the myeloid compartment, however, has been poorly studied. Human monocytes did not respond to IL-18. However, the human myelomonocytic cell line KG-1 and monocyte-derived dendritic cells (generated by GM-CSF+IL-4) showed a marked increase in CD83, HLA-DR, and several costimulatory molecules upon stimulation with IL-18. Furthermore, IL-18 decreased pinocytosis of these cells and increased their ability to stimulate alloreactive T cell proliferation, all characteristics of mature dendritic cells. These results suggest that IL-18 is involved in the maturation of myeloid DCs, but not differentiation of monocytes into DCs. The finding that IL-18 is involved in the maturation of dendritic cells is both novel and unexpected and indicates another important role for IL-18 as a key regulator of immune responses.     

Induction of maturation in small Xenopus laevis oocytes

The competence of Xenopus laevis oocytes in various stages of growth to respond to progesterone treatment was investigated. Full-grown (stage 6) oocytes undergo nuclear membrane dissolution and resume meiosis in response to progesterone exposure, while smaller oocytes (stages 3-5; less than 1100 micron in diameter) do not. The defect which prevents 750- to 1050-micron oocytes from responding to progesterone can be overcome by microinjecting cytoplasm withdrawn from a stage 6 oocyte. Germinal vesicle breakdown in these small oocytes occurs on a timetable similar to that of stage 6 oocytes exposed to progesterone and is accompanied by a twofold increase in protein synthesis as well as the activation of MPF. The results argue that a cytoplasmic factor(s) which probably first appears at late stage 5 is required for progesterone responsiveness. The identity and role of the factor(s) in the development of maturation competence and the regulation of maternal mRNA translation are discussed.     

Induction of starfish oocyte maturation by disulfide-reducing agents

Oocyte maturation was found to be induced by disulfide-reducing agents such as dithiothreitol (DTT) and 2,3-dimercapto-1-propanol (BAL) in the starfish, Asterina pectinifera. The follicular envelopes around the oocytes broke and retracted into small clumps of cells on treatment with these reagents, as in the case of 1-methyladenine. Upon insemination, fertilizable eggs obtained by treatment with DTT formed a tight fertilization membrane and underwent cleavage. Such eggs developed normally to bipinnaria larvae. Cysteine and glutathione-SH had no effect in inducing oocyte maturation. On the other hand, pretreatment with sulfhydryl reagents such as p-chloromercurybenzoate (PCMB), iodoacetamide (IAM) and N-ethylmaleimide (NEM) completely suppressed 1-methyladenine-induced oocyte maturation. This inhibitory effect of sulfhydryl reagents on oocyte maturation was diminished by subsequent treatment with DTT or BAL with or without 1-methyladenine. Pretreatment with o-iodosobenzoate failed to inhibit 1-methyladenine-induced oocyte maturation.     

Induction and inhibition of oocyte maturation by EDCs in zebrafish

Background  

Oocyte maturation in lower vertebrates is triggered by maturation-inducing hormone (MIH), which acts on unidentified receptors on the oocyte surface and induces the activation of maturation-promoting factor (MPF) in the oocyte cytoplasm. We previously described the induction of oocyte maturation in fish by an endocrine-disrupting chemical (EDC), diethylstilbestrol (DES), a nonsteroidal estrogen.     

Induction and formation ofCochliobolus sativus appressoria

Summary GerminatingCochliobolus sativus spores were induced to form appressoria on a variety of artificial surfaces, including replicas of the barley leaf surface. Evidence was obtained for the involvement of chemical and topographic signals during induction of appressorium formation inC. sativus. Germ tube thigmotropism was also observed in vitro. Ultrastructure relevant to appressorium formation was observed, including the germ tube apex, apical swelling of the germ tube apex prior to appressorium formation, the appressorium with associated septation and the penetration peg. Cytochemical probes applied to germlings at the electron microscope level failed to detect -D-mannan, -D-glucan, -D-galactan, D-glcNAc or D-galNAc polymers in the extracellular mucilage associated with the fungal germlings. The ultrastructure of hyphal apices from germlings grown under different nutritional conditions differed with respect to Spitzenkörper morphology, apex shape and in the quantity of associated extracellular mucilage. Experimental findings are discussed relative to current understanding of appressorium induction in more extensively studied systems.Abbreviations PDA potato dextrose agar - DS dilute salts - Con A concanavalin A - RcA₁₂₀ Ricinus communis agglutinin₁₂₀ - WGA wheat germ agglutinin - HpA Helix pomatia agglutinin - DIC differential interference contrast - UV ultraviolet - TEM transmission electron microscopy - NNF National Nanofabrication Facility