Effects of chromatin decondensation on alternative NHEJ

In cells of higher eukaryotes, repair of DNA double strand breaks (DSBs) utilizes different forms of potentially error-prone non-homologous end joining (NHEJ): canonical DNA-PK-dependent (C-NHEJ) and alternative backup pathways (A-NHEJ). In contrast to C-NHEJ, A-NHEJ shows pronounced efficiency fluctuations throughout the cell cycle and is severely compromised as cells cease proliferating and enter the plateau phase (Windhofer et al., 2007 [23]). The molecular mechanisms underpinning this response remain unknown but changes in chromatin structure are prime candidate-A-NHEJ-modulators. Since parameters beyond chromatin acetylation appear to determine A-NHEJ efficiency (Manova et al., 2012 0210 and 0380), we study here the role of chromatin decondensation mediated either by treatment with 5′-aza-2′-deoxycytidine (AzadC) or growth in hypotonic conditions, on A-NHEJ. We report that both treatments have no detectable effect on C-NHEJ but provoke, specifically for A-NHEJ, cell-growth-dependent effects. These results uncover for the first time a link between A-NHEJ and chromatin organization and provide means for understanding the regulatory mechanisms underpinning the growth-state dependency of A-NHEJ. A-NHEJ is implicated in the formation of chromosomal translocations and in chromosome fusions that underlie genomic instability and carcinogenesis. The observations reported here may therefore contribute to the development of drug-based A-NHEJ suppression-strategies aiming at optimizing cancer treatment outcomes and possibly also at suppressing carcinogenesis.     

Effect of in vivo oocyte aging on sperm chromatin decondensation in the golden hamster

Aged spontaneously activated hamster oocytes recovered from adult females 18 and 24 hours after ovulation were at the pronuclear stage. These oocytes and fresh controls were inseminated in vitro with capacitated hamster spermatozoa and observed with the phase-contrast microscope. The percentage of fertilization in fresh control oocytes was 98%, as compared to 36% and 18% when the oocytes were recovered 18 and 24 hours after ovulation, respectively. The mean number of sperm decondensations per egg in control oocytes was 10, and in the aged ones it was 0.69 and 0.12 when the oocytes were recovered 18 and 24 hours after ovulation, respectively. When similarly treated oocytes were studied with scanning and transmission electron microscopy, it was found that the degree of gamete membrane fusion was greater than that observed with the phase-contrast microscope, but that most of the spermatozoa failed to decondense the chromatin. We suggest that parthenogenetic oocytes at the pronuclear stage are in a similar stage of the cell cycle as in fertilized eggs, in which the cytoplasm does not have the ability to decondense the sperm chromatin.     

The effect of chromatin decondensation on DNA damage and repair

The effects of chromatin compaction on X-radiation-induced cell killing and the induction and repair of DNA damage were studied in Chinese hamster ovary cells deprived of isoleucine for 24 h (Ile- cells) and compared to untreated controls. The results show that chromatin is decondensed in Ile- cells; i.e., in Ile- cells the nuclear area occupied by heterochromatin decreased 30-fold over control cells, both the rate and limit of micrococcal nuclease digestion were greater for Ile- cells, and 14.2% more propidium iodide was intercalated into the Ile- cell chromatin. The X-ray-induced cytotoxicity did not change in Ile- cells versus the control cells (D0 = 0.99 Gy) nor did the X-ray-induced DNA damage. However, the repair of DNA damage produced by 10 Gy proceeded with different kinetics in Ile- cells when compared to the controls. The initial rate of DNA damage repair was slower in Ile- cells by a factor of 2 compared to controls (the time required to rejoin 50% of the lesions was 6 versus 3 min, respectively). However, after 2 h of repair no DNA damage was detected in either group. Therefore, we conclude that this decondensation of chromatin, per se, does not directly modify the induction or ultimate repair of DNA damage by X radiation or cell clonogenicity and thus does not appear to be a primary factor in cell survival.     

Structural state of active and inactive genes during chromatin decondensation

Chromatin structure of globin and ovalbumin genes in chicken erythrocyte nuclei has been investigated by means of the "nuclease criterion" (described earlier). In intact nuclei (i.e. in the presence of 3 mM MgCl2) DNase I cleaves chromatin of both genes generating fragments multiple of a double-nucleosome repeat (2N-periodicity). However, in the case of the globin gene, apart from the 2N-periodicity, fragments were observed that are multiple of 100 b.p. and are characteristic for partially unfolded chromatin. This distinction in nuclease cleavage patterns correlates with a higher sensitivity of the globin gene as compared with the inactive ovalbumin gene. At 0.5-0.7 mM MgCl2 the transition from dinucleosomal fragmentation with DNase I and DNase II to fragmentation via a 100 b.p. interval occurs and the difference in digestibility of both genes is dramatically increased. If chromatin has been decondensed by incubation of nuclei in 10 mM Tris-buffer DNase Il generates an usual nucleosomal repeat, and in this ionic conditions one may not observe any difference in nuclease sensitivity of the analyzed genes. The data allow to suggest that the high nuclease sensitivity of potentially active genes can be conditioned by more relaxed arrangement of nucleosomes in higher order chromatin structure.     

DDB2 promotes chromatin decondensation at UV-induced DNA damage

Nucleotide excision repair (NER) is the principal pathway that removes helix-distorting deoxyribonucleic acid (DNA) damage from the mammalian genome. Recognition of DNA lesions by xeroderma pigmentosum group C (XPC) protein in chromatin is stimulated by the damaged DNA-binding protein 2 (DDB2), which is part of a CUL4A-RING ubiquitin ligase (CRL4) complex. In this paper, we report a new function of DDB2 in modulating chromatin structure at DNA lesions. We show that DDB2 elicits unfolding of large-scale chromatin structure independently of the CRL4 ubiquitin ligase complex. Our data reveal a marked adenosine triphosphate (ATP)-dependent reduction in the density of core histones in chromatin containing UV-induced DNA lesions, which strictly required functional DDB2 and involved the activity of poly(adenosine diphosphate [ADP]-ribose) polymerase 1. Finally, we show that lesion recognition by XPC, but not DDB2, was strongly reduced in ATP-depleted cells and was regulated by the steady-state levels of poly(ADP-ribose) chains.     

Increase in intranuclear birefringence during chromatin activation reaction

The lymphocytes of rat spleen are birefringent after staining with neutral red. The negative birefringence of the DNA increases remarkably after in vitro incubation with phytohemagglutinin (PHA). The effect of PHA on lymphocytes is demonstrable even in the early stage of stimulation. This early stage is named ‘chromatin activation reaction’. Demonstration of the chromatin activation reaction by means of polarization optical analysis can be applied to the investigation of the lymphocyte transformation reaction.     

Electrostatic interactions at the C-terminal domain of nucleoplasmin modulate its chromatin decondensation activity

The chromatin decondensation activity, thermal stability, and secondary structure of recombinant nucleoplasmin, of two deletion mutants, and of the protein isolated from Xenopus oocytes have been characterized. As previously reported, the chromatin decondensation activity of recombinant, unphosphorylated nucleoplasmin is almost negligible. Our data show that deletion of 50 residues at the C-terminal domain of the protein, containing the positively charged nuclear localization sequence, activates its chromatin decondensation ability and decreases its stability. Interestingly, both the decondensation activity and thermal stability of this deletion mutant resemble those of the phosphorylated protein isolated from Xenopus oocytes. Deletion of 80 residues at the C-terminal domain, containing the above-mentioned positively charged region and a poly(Glu) tract, inactivates the protein and increases its thermal stability. These findings, along with the effect of salt on the thermal stability of these proteins, suggest that electrostatic interactions between the positive nuclear localization sequence and the poly(Glu) tract, at the C-terminal domain, modulate protein activity and stability.     

Involvement of mouse nucleoplasmin 2 in the decondensation of sperm chromatin after fertilization

The tightly condensed chromatin of spermatozoa is rapidly decondensed after the spermatozoa enter oocytes. Although no factor involved in sperm chromatin decondensation (SCD) has been identified in mammals, it has been suggested that a factor related to SCD activity is present in the germinal vesicle (GV) of oocytes. Here, we found that the nucleolus-like body (NLB), which is a component of the GV, is involved in SCD in murine oocytes. When NLBs were microsurgically removed from GV-stage oocytes, SCD was significantly retarded in the paternal genome after fertilization following meiotic maturation. We found that the retardation of SCD in the NLB-removed oocytes was restored by the microinjection of mRNA encoding nucleoplasmin 2 (NPM2), a component of NLBs. Furthermore, SCD was retarded in the fertilized oocytes from Npm2-knockout females, and recombinant NPM2 alone could induce the SCD in vitro. These data provide evidence that NPM2 is involved in sperm chromatin remodeling in mammals.     

Study of chromatin decondensation factors in human spermatozoids by flow cytometry

To date, the mechanisms responsible for radical change of chromatin structure in male germ cells during fertilization are unclear. Evidence suggesting the existence of proteolytic nuclear enzymes in mature human spermatozoids are presented in this work. The possible role of these previously unknown proteases in decondensation of chromatin of spermatozoids in a fertilized ovum is discussed. Application of the flow cytometry technique has shown that treatment of human spermatozoid nuclei with SH-reagents leads not only to destruction of disulfide bonds between protamine molecules that is necessary for their effective utilization but also induces specific endogenous proteolytic activity that consequently results in rather fast decondensation of chromatin followed by proteolytic cleavage of nuclear proteins. A chromatin decondensation process can be almost totally blocked by serine protease inhibitors and components of seminal fluid. An original cytochemical approach of binding of fluorescently labeled protease inhibitor to the target of investigation has been used in order to visualize the localization of proteases in male germ cell nuclei. The results of our study suggest that one of the factors of chromatin reorganization involved in the formation of male pronucleus is endogenous nuclear protease of spermatozoids, which is activated by glutathione or other SH-components of ovum cytoplasm.     

Proposed mechanism for sperm chromatin condensation/decondensation in the male rat

Condensation of sperm chromatin occurs after spermatozoa have left the caput epididymis and are in transit to the cauda epididymis, during which time large numbers of disulfide bonds are formed. The formation of these disulfide bonds requires the repeated oxidation of the cofactor, NAD(P)H. To date, the means by which this oxidation is achieved has yet to be elucidated. Spermatozoa lose the bulk of their cytoplasm prior to leaving the testis; and, as a result, any shuttle systems for removing and transferring reducing equivalents into the mitochondria are unlikely to be operational. In an apparent preparation for the loss of cytoplasm, however, the following events occur during spermatogenesis. First, androgen-binding protein (ABP) is produced by the Sertoli cells of the testis; second, high affinity binding sites for ABP are inserted into the membrane surrounding the nucleus; and third, a nuclear location is acquired for the enzyme, 3α-hydroxysteroid dehydrogenase (3α-HSD).     

Gold nanoparticles disturb nuclear chromatin decondensation in mouse sperm in vitro

The effect of gold nanoparticles on mouse epididymal sperm has been studied using the model system of nuclear chromatin decondensation in vitro. It is shown that the treatment of gametes, preliminary membrane-freed by sodium dodecyl sulfate, in the mediums containing gold nanoparticles (with diameter ∼2.5 nm) in concentrations 1.0 × 10¹⁵ or 0.5 × 10¹⁵ particles/ml and following incubation in dithiothreitol solution (DTT) resulted in failure of chromatin decondensation process and nucleus structure. We conclude that gold nanoparticles possess spermatotoxicity. The mechanism of cytotoxic effect of gold nanoparticles may be related with their interaction with molecules of double-helix DNA. The model system studied in this research is applicable for further investigations of cytotoxic effects of nanoparticles of different origin and made of different metals.     

Functional studies of MP62 during male chromatin decondensation in sea urchins

In amphibians, sperm histone transition post‐fertilization during male pronucleus formation is commanded by histone chaperone Nucleoplasmin (NPM). Here, we report the first studies to analyze the participation of a Nucleoplasmin‐like protein on male chromatin remodeling in sea urchins. In this report, we present the molecular characterization of a nucleoplasmin‐like protein that is present in non fertilized eggs and early zygotes in sea urchin specie Tetrapygus niger. This protein, named MP62 can interact with sperm histones in vitro. By male chromatin decondensation assays and immunodepletion experiments in vitro, we have demonstrated that this protein is responsible for sperm nucleosome disorganization. Furthermore, as amphibian nucleoplasmin MP62 is phosphorylated in vivo immediately post‐fertilization and this phosphorylation is dependent on CDK‐cyclin activities found after fertilization. As we shown, olomoucine and roscovitine inhibits male nucleosome decondensation, sperm histone replacement in vitro and MP62 phosphorylation in vivo. This is the first report of a nucleoplasmin‐like activity in sea urchins participating during male pronucleus formation post‐fecundation. J. Cell. Biochem. 114: 1779–1788, 2013. © 2013 Wiley Periodicals, Inc.     

Dynamics of chromatin decondensation reveals the structural integrity of a mechanically prestressed nucleus

Genome organization within the cell nucleus is a result of chromatin condensation achieved by histone tail-tail interactions and other nuclear proteins that counter the outward entropic pressure of the polymeric DNA. We probed the entropic swelling of chromatin driven by enzymatic disruption of these interactions in isolated mammalian cell nuclei. The large-scale decondensation of chromatin and the eventual rupture of the nuclear membrane and lamin network due to this entropic pressure were observed by fluorescence imaging. This swelling was accompanied by nuclear softening, an effect that we quantified by measuring the fluctuations of an optically trapped bead adhered onto the nucleus. We also measured the pressure at which the nuclear scaffold ruptured using an atomic force microscope cantilever. A simple theory based on a balance of forces in a swelling porous gel quantitatively explains the diffusive dynamics of swelling. Our experiments on decondensation of chromatin in nuclei suggest that its compaction is a critical parameter in controlling nuclear stability.     

An intranuclear frame for chromatin compartmentalization and higher-order folding

Recent ultrastructural, immunoelectron, and confocal microscopy observations done in our laboratory [Barboro et al. [2002] Exp. Cell. Res. 279:202-218] have confirmed that lamins and the nuclear mitotic apparatus protein (NuMA) are localized inside the interphase nucleus in a polymerized form. This provided evidence of the existence of a RNA stabilized lamin/NuMA frame, consisting of a web of thin ( approximately 3 and approximately 5 nm) lamin filaments to which NuMA is anchored mainly in the form of discrete islands, which might correspond to the minilattices described by Harborth et al. [1999] (EMBO. J. 18:1689-1700). In this article we propose that this scaffold is involved in the compartmentalization of both chromatin and functional domains and further determines the higher-order nuclear organization. This hypothesis is strongly supported by the scrutiny of different structural transitions which occur inside the nucleus, such as chromatin displacement and rearrangements, the collapse of the internal nuclear matrix after RNA digestion and the disruption of chromosome territories induced by RNase A and high salt treatment. All of these destructive events directly depend on the loss of the stabilizing effect exerted on the different levels of structural organization by the interaction of RNA with lamins and/or NuMA. Therefore, the integrity of nuclear RNA must be safeguarded as far as possible to isolate the matrix in the native form. This material will allow for the first time the unambiguous ultrastructural localization inside the INM of the components of the functional domains, so opening new avenues of investigation on the mechanisms of gene expression in eukaryotes.     

Barrier-to-autointegration factor: major roles in chromatin decondensation and nuclear assembly

Barrier-to-autointegration factor (BAF) is a DNA-bridging protein, highly conserved in metazoans. BAF binds directly to LEM (LAP2, emerin, MAN1) domain nuclear membrane proteins, including LAP2 and emerin. We used site-directed mutagenesis and biochemical analysis to map functionally important residues in human BAF, including those required for direct binding to DNA or emerin. We also tested wild-type BAF and 25 point mutants for their effects on nuclear assembly in Xenopus egg extracts, which contain approximately 12 microM endogenous BAF dimers. Exogenous BAF caused two distinct effects: at low added concentrations, wild-type BAF enhanced chromatin decondensation and nuclear growth; at higher added concentrations, wild-type BAF completely blocked chromatin decondensation and nuclear growth. Mutants fell into four classes, including one that defines a novel functional surface on the BAF dimer. Our results suggest that BAF, unregulated, potently compresses chromatin structure, and that BAF interactions with both DNA and LEM proteins are critical for membrane recruitment and chromatin decondensation during nuclear assembly.     

During male pronuclei formation chromatin remodeling is uncoupled from nucleus decondensation

Male pronucleus formation involves sperm nucleus decondensation and sperm chromatin remodeling. In sea urchins, male pronucleus decondensation was shown to be modulated by protein kinase C and a cdc2-like kinase sensitive to olomoucine in vitro assays. It was further demonstrated that olomoucine blocks SpH2B and SpH1 phosphorylation. These phosphorylations were postulated to participate in the initial steps of male chromatin remodeling during male pronucleus formation. At final steps of male chromatin remodeling, all sperm histones (SpH) disappear from male chromatin and are subsequently degraded by a cysteine protease. As a result of this remodeling, the SpH are replaced by maternal histone variants (CS). To define if sperm nucleus decondensation is coupled with sperm chromatin remodeling, we have followed the loss of SpH in zygotes treated with olomoucine. SpH degradation was followed with anti-SpH antibodies that had no cross-reactivity with CS histone variants. We found that olomoucine blocks SpH1 and SpH2B phosphorylation and inhibits male pronucleus decondensation in vivo. Interestingly, the normal schedule of SpH degradation remains unaltered in the presence of olomoucine. Taken together these results, it was concluded that male nucleus decondensation is uncoupled from the degradation of SpH associated to male chromatin remodeling. From these results, it also emerges that the phosphorylation of SpH2B and SpH1 is not required for the degradation of the SpH that is concurrent to male chromatin remodeling.     

Cation-dependent solubilization of rat thymocyte chromatin is closely related to decondensation of the nuclei

The cation-dependent solubilization of rat thymocyte chromatin has been compared with decondensation of the nuclei as a function of sodium phosphate-mediated changes in the concentration of Mg2+ and Na+. After digestion of the nuclei with DNase I or Micrococcus nuclease for a time just sufficient to permit extraction of a maximal amount of chromatin (minimum digestion), solubilization of most of the chromatin was found to occur with the same cation dependency as decondensation of untreated nuclei, while further digestion changed the ionic requirements for solubilization. The cation-dependency of the chromatin solubility and of the nuclear decondensation also exhibited the same variations with temperature. The chromatin in the nuclei became up to 4-times more sensitive to DNase I by decondensation, which also induced a shift in the DNase I cleavage mode from a 200 bp to a 100 bp repeat pattern. In contrast, the sensitivity to Micrococcus nuclease appeared to be nearly unchanged. These results suggest that solubilization of chromatin prepared by a mild endonuclease treatment occurs as a direct consequence of structural changes in the chromatin which take place during decondensation of the nuclei.     

Transient decondensation of chromatin in liver nuclei of rats treated with tannic acid

Communicated by Ramaswamy H. Sarma     

A new approach of successful denaturation of human sperm chromatin without undergoing decondensation treatment

Due to the highly folded chromatin in human sperm, a proper nuclear swelling was highly required to localize certain DNA inside the sperm nuclei. Therefore, previous method for denaturation of sperm chromatin had to adopt chemical agents of decondensation treatment using Heparin/DTT or LIS, directly applied into the sperm cell before further examinations by FISH. Nevertheless, authors still had questions arising on the efficiency of decondensation process which is directly related to the quality of fluorescence signals, which, in turn, underlies the reliability of the results in frequencies and compositions as that still not a proper solution to overcome the major limitation in sperm studies. In this study, we approached a newly improved denaturation process of sperm chromatin without undergoing decondensation treatments that intact human sperms were used as the first time to localize examined DNA, and also two rounds of sequential FISH was carried out in the same sperm cell for the first time to investigate an idea of nullisomy of given chromosomes. From the results, all the variable centromeric compositions of sperm chromosomes 7, 8, and sex chromosomes revealed with significantly given frequencies of monosomy, disomy and nullisomy. Moreover, nullisomy was identified as a true absence of given chromosome rather than technical error of hybridization failure under decondensation. From the findings by our modified denaturation of human sperm chromatin without undergoing decondensation treatment, we strongly believe that more advanced and deep studies in human sperm of nuclear architecture and frequencies can be progressed with significantly reliable results.