Degree Of Chromatin Compaction Research Articles

In-Vitro Effects of Perfluorooctanoic Acid on Human Sperm Function: What Are the Clinical Consequences?

Background: Lifestyle and environmental pollution harm male fertility. Perfluoroalkyl substances (PFAS) are bio-accumulates in the environment as well as in several human tissues, and one of the most common PFAS is perfluorooctanoic acid (PFOA). Therefore, this study aimed to evaluate the in vitro effects of PFOA with hydrophobic and waterproofing properties on motility and bio-functional sperm parameters. Methods: To accomplish this, 50 healthy men with normozoospermia and not exposed to high doses of PFAS were enrolled. Their spermatozoa were incubated for 3 h with increasing concentrations of PFOA (0, 0.01, 0.1, and 1 mM) to evaluate its effects. In particular, we evaluated the effects of PFOA on total and progressive sperm motility and, by flow cytometry, on the following bio-functional sperm parameters: degree of chromatin compactness, viability, early and late apoptosis, mitochondrial membrane potential, the degree of lipoperoxidation, and concentrations of mitochondrial superoxide anion. Results: The results showed that PFOA decreased both total and progressive sperm motility, impaired chromatin compactness, and increased sperm lipid peroxidation and mitochondrial superoxide anion levels. Conclusions: This study showed that PFOA alters several sperm parameters and thus it may play a negative role in male fertility.

Apo-form of recombinant human lactoferrin changes the genome-wide DNA methylation level and the chromatin compaction degree in neuroblastoma cell line IMR-32

BACKGROUND: Neuroblastoma is one of the most common extracranial solid tumors in childhood. At present, epigenetic disorders play a significant role in neoplasms development. Since epigenetic changes in the cell are quite dynamic and reversible, epigenome-modulating exogenous agents can be used in epigenetic targeted therapy for various types of tumors. Therefore, the identification of these agents is still significant. Lactoferrin is one such potential molecule from the transferrin family. Currently, the anti-tumor properties of lactoferrin have been identified, but its effect on the epigenome of cells of various tumors types, particularly on neuroblastomas, is practically unknown.
 AIM: To study the effect of the exogenous recombinant human apolactoferrin on the viability and epigenomic status of IMR-32 neuroblastoma cells.
 MATERIALS AND METHODS: We studied human IMR-32 neuroblastoma cells after 72 hours of exposure to 8 doses of recombinant human apolactoferrin: 0.1, 0.5, 1, 5, 10, 50, 100 and 500 g/ml. The level of genome-wide DNA methylation and the degree of chromatin compaction in IMR-32 cells were quantified using commercial kits 5-mC DNA ELISA Kit, Global DNA Methylation LINE-1 Kit, as well as enzymatic hydrolysis of MspI / HpaII and DNaseI.
 RESULTS: The recombinant apolactoferrin reduces the viability of IMR-32 and, depending on the dose, differentially affects the level of genome-wide DNA methylation (СpG dinucleotides, CCGG sites, LINE-1 repeats) and the degree of chromatin compaction. At the same time, a complex picture of the epigenomic cellular response to the effect of apo-lactoferrin was observed (nonlinear nonmonotonic dose-effect relationship).
 CONCLUSIONS: We assumed that apolactoferrin modulates gene activity through epigenetic mechanisms, in particular, by changing the DNA methylation pattern and affecting the chromatin structure, which may be one of the molecular mechanisms of its anti-tumor effect.

Identification of differentially expressed mRNAs and miRNAs in spermatozoa of bulls of varying fertility.

Bulls used in artificial insemination, with apparently normal semen quality, can vary significantly in their field fertility. This study aimed to characterize the transcriptome of spermatozoa from high (HF) and low (LF) fertility bulls at the mRNA and miRNA level in order to identify potential novel markers of fertility. Holstein-Friesian bulls were assigned to either the HF or LF group (n = 10 per group) based on an adjusted national fertility index from a minimum of 500 inseminations. Total RNA was extracted from a pool of frozen-thawed spermatozoa from three different ejaculates per bull, following which mRNA-seq and miRNA-seq were performed. Six mRNAs and 13 miRNAs were found differentially expressed (P < 0.05, FC > 1.5) between HF and LF bulls. Of particular interest, the gene pathways targeted by the 13 differentially expressed miRNAs were related to embryonic development and gene expression regulation. Previous studies reported that disruptions to protamine 1 mRNA (PRM1) had deleterious consequences for sperm chromatin structure and fertilizing ability. Notably, PRM1 exhibited a higher expression in spermatozoa from LF than HF bulls. In contrast, Western Blot analysis revealed a decrease in PRM1 protein abundance for spermatozoa from LF bulls; this was not associated with increased protamine deficiency (measured by the degree of chromatin compaction) or DNA fragmentation, as assessed by flow cytometry analyses. However, protamine deficiency was positively and moderately correlated with the percentage of spermatozoa with DNA fragmentation, irrespective of fertility group. This study has identified potential biomarkers that could be used for improving semen quality assessments of bull fertility.

Metamorphosis and denucleation of the brain in the miniature wasp Megaphragma viggianii (Hymenoptera: Trichogrammatidae)

Holometabolan brains undergo structural and allometric changes and complex reorganizations during metamorphosis. In minute egg parasitoids, brain formation is shifted to the late larva and young pupa, due to extreme de-embryonization. The brains of Megaphragma wasps undergo denucleation, the details of which remained unknown. We describe the morphological and volumetric changes in the brain of Megaphragma viggianii (Trichogrammatidae) during pupal development with emphasis on the lysis of nuclei and show that the absolute and relative volume of the brain decrease by a factor of 5 from prepupa to adult at the expense of the cell body rind. The first foci of lysis appear during early pupal development, but most nuclei (up to 97%) are lost between pharate adult and adult. The first signs of lysis (destruction of the nuclear envelopes) occur in pupae with red eyes. The number of lysis foci (organelle destruction and increasing number of lysosomes and degree of chromatin compaction) strongly increases in pupae with black eyes. The cell body rind volume strongly decreases during pupal development (in larger insects it increases slightly or remains unchanged). Elucidation of the lysis of nuclei in neurons and of the functioning of an anucleate brain is an important objective for neuroscience.

Balance of osmotic pressures determines the nuclear-to-cytoplasmic volume ratio of the cell

The volume of the cell nucleus varies across cell types and species and is commonly thought to be determined by the size of the genome and degree of chromatin compaction. However, this notion has been challenged over the years by much experimental evidence. Here, we consider the physical condition of mechanical force balance as a determining condition of the nuclear volume and use quantitative, order-of-magnitude analysis to estimate the forces from different sources of nuclear and cytoplasmic pressure. Our estimates suggest that the dominant pressure within the nucleus and cytoplasm of nonstriated muscle cells originates from the osmotic pressure of proteins and RNA molecules that are localized to the nucleus or cytoplasm by out-of-equilibrium, active nucleocytoplasmic transport rather than from chromatin or its associated ions. This motivates us to formulate a physical model for the ratio of the cell and nuclear volumes in which osmotic pressures of localized proteins determine the relative volumes. In accordance with unexplained observations that are a century old, our model predicts that the ratio of the cell and nuclear volumes is a constant, robust to a wide variety of biochemical and biophysical manipulations, and is changed only if gene expression or nucleocytoplasmic transport is modulated.

Balance of osmotic pressures determines the volume of the nucleus

The volume of the cell nucleus varies across cell-types and species, and is commonly thought to be determined by the size of the genome and degree of chromatin compaction. However, this notion has been challenged over the years by multiple experimental evidence. Here, we consider the physical condition of mechanical force balance as a determining condition of the nuclear volume and use quantitative, order-of-magnitude analysis to estimate the forces from different sources of nuclear and cellular pressure.

Relationship between chromatin configuration and in vitro maturation ability in guinea pig oocytes.

BackgroundGerminal vesicle (GV) chromatin configurations of oocytes are proposed to be related to oocyte competence and may reflect the quality of oocyte. Currently, a limited number of published studies investigated the GV chromatin configurations of guinea pig oocytes.ObjectiveIn this study on the in vitro maturation (IVM) of guinea pig oocytes, we examined the changes in their GV chromatin configurations during meiotic progression.MethodsBased on the degree of chromatin compaction, the GV chromatin configurations of guinea pig oocytes could be divided into three categories depending on whether the nucleolus‐like body (NLB) was surrounded or partly surrounded by compacted chromatin, namely the uncondensed (NSN), the intermediate type (SN‐1) and the compacted type (SN‐2).ResultsThe percentage of cells displaying the SN‐2 configuration increased with the growth of guinea pig oocytes, suggesting that this configuration presents the potential for maturation in oocytes. Oocytes derived from larger follicle exhibited increased meiotic potential. Serum starvation affected the GV chromatin configurations of guinea pig oocytes.ConclusionsCollectively, these results suggest that the SN‐2 type might be a more mature form of configuration in guinea pig oocyte, whose proportion was associated with the follicle size and susceptible to the environment (e.g. serum concentration).

Bio-Functional Sperm Parameters: Does Age Matter?

The evaluation of biofunctional sperm parameters can explain some cases of idiopathic male infertility. Among these, sperm DNA fragmentation (fDNA) is the most studied biofunctional sperm parameter. Mitochondrial membrane potential (MMP) correlates positively with sperm motility, the evaluation of sperm apoptosis by flow cytometry allows us to identify a population of spermatozoa not recognizable at the optical microscopy and finally, lipid peroxidation (LP) and mitochondrial superoxide levels measurements are rational oxidative stress indices. Male age seems to affect sperm concentration and sperm fDNA. For these reasons, this study was undertaken to evaluate the correlation, if any, between male age and biofunctional sperm parameters evaluating their possible impact on fDNA. To accomplish this, MMP, degree of chromatin compactness, sperm apoptosis/vitality, fDNA, LP, and mitochondrial superoxide levels were evaluated by flow cytometry in a cohort of 874 men. A significant negative correlation was found between age and the percentage of alive spermatozoa (r = -0.75, p < 0.05). The percentage of spermatozoa with low MMP (L-MMP) correlated positively with the percentage of spermatozoa with abnormal chromatin compactness (r = 0.24, p < 0.05). Spermatozoa with abnormal chromatin compactness and L-MMP correlated negatively with the percentage of alive spermatozoa (r = 0.83, p < 0.05) and positively with spermatozoa with PS externalization (r = 0.13, p < 0.01). The percentage of alive spermatozoa correlated negatively with both the percentage of spermatozoa with PS externalization (r = 0.24, p < 0.01) and of the spermatozoa with fDNA (r = 0.10, p < 0.05). Spermatozoa with PS externalization correlated positively with the percentage of spermatozoa with fDNA (r = 0.09, p < 0.05). Spermatozoa with LP correlated positively with the percentage of spermatozoa with increased mitochondrial superoxide (r = 0.11, p < 0.01) In conclusion, these findings in a large number of men suggest that age, mitochondrial damage, and alteration of chromatin compactness could activate the apoptotic cascade which could result in an increased fDNA rate.

Environment and Male Fertility: Effects of Benzo-α-Pyrene and Resveratrol on Human Sperm Function In Vitro.

Lifestyle, cigarette smoking and environmental pollution have a negative impact on male fertility. Therefore, the aim of this study was to evaluate the in-vitro effects of benzo-α-pyrene (BaP) and aryl hydrocarbon receptor (AHR) agonists on motility and bio-functional sperm parameters. We further assessed whether resveratrol (RES), an AHR antagonist and antioxidant molecule, had any protective effect. To accomplish this, 30 normozoospermic, healthy, non-smoker men not exposed to BaP were enrolled. Spermatozoa of 15 men were incubated with increasing concentrations of BaP to evaluate its effect and to establish its dose response. Then, spermatozoa of the 15 other men were incubated with BaP (15 µM/mL), chosen according to the dose-response and/or RES to evaluate its antagonistic effects. The effects of both substances were evaluated after 3 h of incubation on total and progressive sperm motility and on the following bio-functional sperm parameters evaluated by flow cytometry: Degree of chromatin compactness, viability, phosphatidylserine externalization (PS), late apoptosis, mitochondrial membrane potential (MMP), DNA fragmentation, degree of lipoperoxidation (LP), and concentrations of mitochondrial superoxide anion. Benzo-α-pyrene decreased total and progressive sperm motility, impaired chromatin compactness, and increased sperm lipoperoxidation and mitochondrial superoxide anion levels. All these effects were statistically significant at the lowest concentration tested (15 µM/mL) and they were confirmed at the concentration of 45 µM/mL. In turn, RES was able to counteract the detrimental effects of BaP on sperm motility, abnormal chromatin compactness, lipid peroxidation, and mitochondrial superoxide. This study showed that BaP alters sperm motility and bio-functional sperm parameters and that RES exerts a protective effect on BaP-induced sperm damage.

Differences in cumulus cell gene expression indicate the benefit of a pre-maturation step to improve in-vitro bovine embryo production.

Does the gene expression profile of cumulus cells (CC) accompanying oocytes with different degrees of chromatin compaction within the germinal vesicle (GV) reflect the oocyte's quality and response in culture during in-vitro embryo production (IVP). The transcriptomic profile of the CC is related to oocyte competence, setting the stage for the development of customized pre-maturation strategies to improve IVP. Oocytes complete the acquisition of their competence during antral follicle development. During this period, the chromatin configuration within the GV changes dynamically and is indicative of oocyte's developmental potential. The interactions between somatic and germ cells modulate chromatin morphology and function and are critical for acquisition of oocyte competence. Bovine cumulus-oocyte complexes (COC) were isolated from 0.5 to 6 mm antral follicles. Surrounding CC were separated from the oocyte and classified as GV0, GV1, GV2 and GV3 according to the degree of the oocyte's chromatin compaction. RNA extracted from CC of each group was amplified and hybridized on a bovine embryo-specific 44 K Agilent slide. The CC_GV1, CC_GV2 and CC_GV3 classes were each hybridized against the CC_GV0 class, representing an early oocyte differentiation stage with poor development competence. The data were normalized and fold changes of the differentially expressed genes were determined. Microarray data were validated using quantitative RT-PCR on selected targets. Microarray data were further analyzed through: (i) between-group analysis (BGA), which classifies the samples according to their transcriptomic profiles; (ii) cluster analysis according to the expression profile of each gene; and (iii) Ingenuity Pathway Analysis (IPA) to study gene regulation patterns and predicted functions. Furthermore, CC of each GV group were cultured and apoptotic cells were assessed after 3 h by caspase analysis. Finally, based on the analysis of CC transcriptomic profiles and the relationship between morphological features of the COC and the oocyte chromatin configuration, a customized, stage-dependent oocyte pre-maturation (pre-IVM) system was used to improve oocyte developmental potential before IVM. For this, the blastocyst rate and quality were assessed after in-vitro maturation and fertilization of pre-matured oocytes. Overall, quantitative RT-PCRresults of a subset of five selected genes were consistent with the microarray data. Clustering analysis generated 16 clusters representing the main profiles of transcription modulation. Of the 5571 significantly differentially expressed probes, the majority (25.49%) best fitted with cluster #6 (downregulation between CC_GV0 and CC_GV1 and stable low levels in successive groups). IPA identified the most relevant functions associated with each cluster. Genes included in cluster #1 were mostly related to biological processes such as 'cell cycle' and 'cell death and survival', whereas genes included in cluster #5 were mostly related to 'gene expression'. Interestingly, 'lipid metabolism' was the most significant function identified in clusters #6, #9 and #12. IPA of gene lists obtained from each contrast (i.e., CC_GV0 vs. CC_GV1; CC_GV0 vs. CC_GV2; CC_GV0 vs. CC_GV3) revealed that the main affected function in each contrast was 'cell death and survival'. Importantly, apoptosis was predicted to be inhibited in CC_GV1 and CC_GV2, but activated in CC_GV3. Caspase analysis indicated that a low percentage of CC_GV0 was prone to undergo apoptosis but apoptosis increased significantly in CC from oocytes with condensed chromatin, reaching a peak in CC_GV3 (P <0.05). Finally, the tailored oocyte pre-maturation strategy, based on morphological features of the COC and the oocyte chromatin configuration, demonstrated that pre-IVM improved the developmental capability of oocytes at early stages of differentiation (GV1-enriched COC) but was detrimental for oocytes at more advanced stages of development (GV2 and GV3-enriched COC). The data are available through the GEO series accession number GSE79886. This study was conducted with bovine samples. Whether or not the results are applicable to human oocytes requests further elucidation. Embryo transfer experiments are required to determine whether the improvement in blastocyst rates in the tailored system leads to increased live birth rates. The identification of multiple non-invasive biomarkers predictive of oocyte quality can greatly strengthen the pre-IVM approach aimed to improve IVM outcomes. These results have potentially important implications in treating human infertility and in developing breeding schemes for domestic mammals. This work was supported in part by NSERC Strategic Network EmbryoGENE, Canada and in part by CIG-Marie Curie Actions-Reintegration Grants within the EU 7FP (n. 303640, 'Pro-Ovum'). The authors declare no potential conflict of interest.

In Vitro Effects of Nicotine on Sperm Motility and Bio-Functional Flow Cytometry Sperm Parameters

The aim of this experimental study was to evaluate the effects of nicotine on sperm motility and on non-conventional sperm parameters in vitro. Capacitated spermatozoa isolated from 10 normozoospermic, healthy, non-smoker men were evaluated. Spermatozoa were exposed to increasing concentrations of nicotine (0, 1, 10, and 100 ng/ml) for 3 and 24 hours. Progressive motility and the following non-conventional sperm parameters, evaluated by flow cytometry, were assessed: mitochondrial membrane potential, viability, phosphatidylserine externalization, late apoptosis, degree of chromatin compactness, and DNA fragmentation. Nicotine suppressed, in a concentration-dependent manner, sperm progressive motility starting from the lowest concentration used (1 ng/ml). Similarly, it reduced the percentage of viable spermatozoa and increased the number of spermatozoa in late apoptosis, with altered chromatin compactness, or DNA fragmentation already after 3 hours of incubation. These effects were observed at a concentration similar (100 ng/ml) to that found in the seminal plasma of smokers (70 ng/ml), with the exception of the effects on sperm DNA fragmentation whose significant effect was detected also at a lower concentration (10 ng/ml). Nicotine may be regarded as a noxious component of cigarette smoke on the male reproductive function.

Aberrations of EZH2 in Cancer

Control of gene expression is exerted at a number of different levels, one of which is the accessibility of genes and their controlling elements to the transcriptional machinery. Accessibility is dictated broadly by the degree of chromatin compaction, which is influenced in part by polycomb group proteins. EZH2, together with SUZ12 and EED, forms the polycomb repressive complex 2 (PRC2), which catalyzes trimethylation of histone H3 lysine 27 (H3K27me3). PRC2 may recruit other polycomb complexes, DNA methyltransferases, and histone deacetylases, resulting in additional transcriptional repressive marks and chromatin compaction at key developmental loci. Overexpression of EZH2 is a marker of advanced and metastatic disease in many solid tumors, including prostate and breast cancer. Mutation of EZH2 Y641 is described in lymphoma and results in enhanced activity, whereas inactivating mutations are seen in poor prognosis myeloid neoplasms. No histone demethylating agents are currently available for treatment of patients, but 3-deazaneplanocin (DZNep) reduces EZH2 levels and H3K27 trimethylation, resulting in reduced cell proliferation in breast and prostate cancer cells in vitro. Furthermore, synergistic effects are seen for combined treatment with DNA demethylating agents and histone deacetylation inhibitors, opening up the possibility of refined epigenetic treatments in the future.

Different types of plant chromatin associated with modified histones H3 and H4 and methylated DNA

Eukaryotic chromosomes are organized into two large and distinct domains, euchromatin and heterochromatin, which are cytologically characterized by different degrees of chromatin compaction during interphase/prophase and by post-synthesis modifications of histones and DNA methylation. Typically, heterochromatin remains condensed during the entire cell cycle whereas euchromatin is decondensed at interphase. However, a fraction of the euchromatin can also remain condensed during interphase and appears as early condensing prophase chromatin. 5S and 45S rDNA sites and telomere DNA were used to characterize these regions in metaphase and interphase nuclei. We investigated the chromosomal distribution of modified histones and methylated DNA in the early and late condensing prophase chromatin of two species with clear differentiation between these domains. Both species, Costus spiralis and Eleutherine bulbosa, additionally have a small amount of classical heterochromatin detected by CMA/DAPI staining. The distribution of H4 acetylated at lysine 5 (H4K5ac), H3 phosphorylated at serine 10 (H3S10ph), H3 dimethylated at lysine 4 or 9 (H3K4me2, H3K9me2), and 5-methylcytosine was compared in metaphase, prophase, and interphase cells by immunostaining with specific antibodies. In both species, the late condensing prophase chromatin was highly enriched in H4K5ac and H3K4me2 whereas the early condensing chromatin was very poor in these marks. H3K9me2 was apparently uniformly distributed along the chromosomes whereas the early condensing chromatin was slightly enriched in 5-methylcytosine. Signals of H3S10ph were restricted to the pericentromeric region of all chromosomes. Notably, none of these marks distinguished classical heterochromatin from the early condensing euchromatin. It is suggested that the early condensing chromatin is an intermediate type between classical heterochromatin and euchromatin.

Photoreceptors CRYTOCHROME2 and Phytochrome B Control Chromatin Compaction in Arabidopsis

Development and acclimation processes to the environment are associated with large-scale changes in chromatin compaction in Arabidopsis (Arabidopsis thaliana). Here, we studied the effects of light signals on chromatin organization. A decrease in light intensity induces a large-scale reduction in chromatin compaction. This low light response is reversible and shows strong natural genetic variation. Moreover, the degree of chromatin compaction is affected by light quality signals relevant for natural canopy shade. The photoreceptor CRYPTOCHROME2 appears a general positive regulator of low light-induced chromatin decompaction. Phytochrome B also controls light-induced chromatin organization, but its effect appears to be dependent on the genetic background. We present a model in which chromatin compaction is regulated by the light environment via CRYPTOCHROME2 protein abundance, which is controlled by phytochrome B action.

The ATAC acetyl transferase complex controls mitotic progression by targeting non-histone substrates

All DNA-related processes rely on the degree of chromatin compaction. The highest level of chromatin condensation accompanies transition to mitosis, central for cell cycle progression. Covalent modifications of histones, mainly deacetylation, have been implicated in this transition, which also involves transcriptional repression. Here, we show that the Gcn5-containing histone acetyl transferase complex, Ada Two A containing (ATAC), controls mitotic progression through the regulation of the activity of non-histone targets. RNAi for the ATAC subunits Ada2a/Ada3 results in delayed M/G1 transition and pronounced cell division defects such as centrosome multiplication, defective spindle and midbody formation, generation of binucleated cells and hyperacetylation of histone H4K16 and alpha-tubulin. We show that ATAC localizes to the mitotic spindle and controls cell cycle progression through direct acetylation of Cyclin A/Cdk2. Our data describes a new pathway in which the ATAC complex controls Cyclin A/Cdk2 mitotic function: ATAC/Gcn5-mediated acetylation targets Cyclin A for degradation, which in turn regulates the SIRT2 deacetylase activity. Thus, we have uncovered an essential function for ATAC in regulating Cyclin A activity and consequent mitotic progression.

In Vivo Chromatin Organization of Mouse Rod Photoreceptors Correlates with Histone Modifications

BackgroundThe folding of genetic information into chromatin plays important regulatory roles in many nuclear processes and particularly in gene transcription. Post translational histone modifications are associated with specific chromatin condensation states and with distinct transcriptional activities. The peculiar chromatin organization of rod photoreceptor nuclei, with a large central domain of condensed chromatin surrounded by a thin border of extended chromatin was used as a model to correlate in vivo chromatin structure, histone modifications and transcriptional activity.MethodologyWe investigated the functional relationships between chromatin compaction, distribution of histone modifications and location of RNA polymerase II in intact murine rod photoreceptors using cryo-preparation methods, electron tomography and immunogold labeling. Our results show that the characteristic central heterochromatin of rod nuclei is organized into concentric domains characterized by a progressive loosening of the chromatin architecture from inside towards outside and by specific combinations of silencing histone marks. The peripheral heterochromatin is formed by closely packed 30nm fibers as revealed by a characteristic optical diffraction signal. Unexpectedly, the still highly condensed most external heterochromatin domain contains acetylated histones, which are usually associated with active transcription and decondensed chromatin. Histone acetylation is thus not sufficient in vivo for complete chromatin decondensation. The euchromatin domain contains several degrees of chromatin compaction and the histone tails are hyperacetylated, enriched in H3K4 monomethylation and hypo trimethylated on H3K9, H3K27 and H4K20. The transcriptionally active RNA polymerases II molecules are confined in the euchromatin domain and are preferentially located at the vicinity of the interface with heterochromatin.ConclusionsOur results show that transcription is located in the most decondensed and highly acetylated chromatin regions, but since acetylation is found associated with compact chromatin it is not sufficient to decondense chromatin in vivo. We also show that a combination of histone marks defines distinct concentric heterochromatin domains.

Relationship between chromatin ultrastructure and DNA fragmentation in human spermatozoa

OBJECTIVE: To explore the relationship between the level of DNA fragmentation and the state of chromatin compaction in a population of severely teratozoospermic patients.DESIGN: Retrospective study.MATERIALS AND METHODS: A population of 77 patients with severe teratozoospermia (Kruger ≤4%) was studied by transmission electron microscopy (TEM) and the TUNEL assay for DNA fragmentation. The degree of chromatin compaction was evaluated on 100 sperm heads by TEM and DNA fragmentation was evaluated under epifluorescence microscopy in 500 sperm heads. Abnormal chromatin compaction was characterized by the percentage of hypodense areas of fibrillar or granular structure. When this percentage was ≤49% the incidence was considered low (Group I), and high when this percentage was ≥50 % (Group II). The presence of different conditions that affect fertility, such as varicocele, infections of the seminal pathway, immunologic factors, etc, was also taken into consideration. Statistical comparisons were performed by analysis of variance followed by Student's t-test.RESULTS: Mean value of DNA fragmentation in Group I was 9.2±6.9% and 15.4±13.3% in Group II (p<0.05). Within Group I, the values of DNA fragmentation in those patients that had conditions affecting spermatogenesis or epididymal sperm transit (10.1±7.5%) were not different from those present in patients without other andrological conditions (7.9±6.2%). When the same differences were compared within group II, the values were statistically different (18.0±14.4% vs. 10.0±8.9%, p<0.05).CONCLUSIONS: In conclusion, higher values of chromatin abnormalities are associated with increased levels of DNA fragmentation. This difference becomes more pronounced in the presence of other conditions that affect sperm quality. These results show that there is an association between high levels of chromatin abnormalities and an increased susceptibility to oxidative stress. OBJECTIVE: To explore the relationship between the level of DNA fragmentation and the state of chromatin compaction in a population of severely teratozoospermic patients. DESIGN: Retrospective study. MATERIALS AND METHODS: A population of 77 patients with severe teratozoospermia (Kruger ≤4%) was studied by transmission electron microscopy (TEM) and the TUNEL assay for DNA fragmentation. The degree of chromatin compaction was evaluated on 100 sperm heads by TEM and DNA fragmentation was evaluated under epifluorescence microscopy in 500 sperm heads. Abnormal chromatin compaction was characterized by the percentage of hypodense areas of fibrillar or granular structure. When this percentage was ≤49% the incidence was considered low (Group I), and high when this percentage was ≥50 % (Group II). The presence of different conditions that affect fertility, such as varicocele, infections of the seminal pathway, immunologic factors, etc, was also taken into consideration. Statistical comparisons were performed by analysis of variance followed by Student's t-test. RESULTS: Mean value of DNA fragmentation in Group I was 9.2±6.9% and 15.4±13.3% in Group II (p<0.05). Within Group I, the values of DNA fragmentation in those patients that had conditions affecting spermatogenesis or epididymal sperm transit (10.1±7.5%) were not different from those present in patients without other andrological conditions (7.9±6.2%). When the same differences were compared within group II, the values were statistically different (18.0±14.4% vs. 10.0±8.9%, p<0.05). CONCLUSIONS: In conclusion, higher values of chromatin abnormalities are associated with increased levels of DNA fragmentation. This difference becomes more pronounced in the presence of other conditions that affect sperm quality. These results show that there is an association between high levels of chromatin abnormalities and an increased susceptibility to oxidative stress.

Functions of Site-Specific Histone Acetylation and Deacetylation

Histone acetylation regulates many cellular processes, and specific acetylation marks, either singly or in combination, produce distinct outcomes. For example, the acetylation pattern on newly synthesized histones is important for their assembly into nucleosomes by histone chaperones. Additionally, the degree of chromatin compaction and folding may be regulated by acetylation of histone H4 at lysine 16. Histone acetylation also regulates the formation of heterochromatin; deacetylation of H4 lysine 16 is important for spreading of heterochromatin components, whereas acetylation of this site serves as a barrier to this spreading. Finally, histone acetylation is critical for gene transcription, but recent results suggest that deacetylation of certain sites also plays an important role. There are many histone acetyltransferases (HATs) and deacetylases, with differing preferences for the various histone proteins and for specific sites on individual histones. Determining how these enzymes create distinct acetylation patterns and regulate the functional outcome is an important challenge.

Mechanism of the Long Range Anti-silencing Function of Targeted Histone Acetyltransferases in Yeast

Transcriptionally silent chromatin in Saccharomyces cerevisiae is associated with histone hypoacetylation and is formed through the action of the Sir histone deacetylase complex. A histone acetyltransferase (HAT) targeted near silent chromatin can overcome silencing at a distance by increasing histone acetylation in a sizable region. However, how a tethered HAT acetylates distant nucleosomes has not been resolved. We demonstrate here that targeting the histone H3-specific HAT Gcn5p promotes acetylation of not only histone H3 but also histone H4 in a broad region. We also show that long range anti-silencing and histone acetylation by targeted HATs can be blocked by nucleosome-excluding sequences. These results are consistent with the contention that a tethered HAT promotes stepwise propagation of histone acetylation along the chromatin. Because histone hypoacetylation is key to the formation and maintenance of transcriptionally silent chromatin, it is believed that acetylation promoted by a targeted HAT disrupts silent chromatin thereby overcoming silencing. However, we show that the acetylated and transcriptionally active region created by a tethered HAT retains structural hallmarks of Sir-dependent silent chromatin and remains associated with Sir proteins indicating that tethered HATs overcome silencing without completely dismantling silent chromatin.

Phosphorylation of serine 10 in histone H3, what for?

Eukaryotic cells must possess mechanisms for condensing and decondensing chromatin. Chromatin condensation is particularly evident during mitosis and cell death induced by apoptosis, whereas chromatin decondensation is necessary for replication, repair, recombination and transcription. Histones are among the numerous DNA-binding proteins that control the level of DNA condensation, and post-translational modification of histone tails plays a critical role in the dynamic condensation/decondensation that occurs during the cell cycle. Phosphorylation of Ser10 in the tails of histone H3 has been extensively studied in many organisms. Interestingly, this modification is involved in both transcription and cell division, two events requiring opposite alterations in the degree of chromatin compaction. How does one and the same modification of histone H3 fulfil such roles? For instance, in interphase, phosphorylation of H3 correlates with chromatin relaxation and gene expression, whereas in mitosis it correlates with chromosome condensation. What is the kinase and under what circumstances does Ser10 becomes phosphorylated? Most importantly, what are the consequences of phosphorylation of this residue?