Selenoproteins in mammalian spermatogenesis:role of the nuclear GPx4

The selenoprotein Phospholipid Hydroperoxide Glutathione Peroxidase (PHGPx/GPx4) is an enzyme unique among the various GPxs, because it is able to use protein thiols, beside glutathione, The GPx4 gene encodes for three isoforms having different subcellular localization, being located in the mitochondria (mGPx4), in the cytosol (cGPx4) and in the nucleus (nGPx4), each having distinct functions. The mGPx4 is important to male fertility, as proven by the structural abnormalities of sperm tails from KO mice specifically lacking this isoform, which make these mutants infertile. As for the nuclear isoform, nGPx4-KO mice are fertile but show impaired nuclear condensation of sperm isolated from the caput epididymis, suggesting a role in chromatin stability. To gain more insight into the functions of nGPx4, we first investigated the subnuclear localization of this form in both COS-1 cells overexpressing nGPx4 and mouse male germ cells at different steps of maturation (round spermatids and epididymal spermatozoa). We performed both biochemical and morphological analyses and found that nGPx4 was localized at the level of the nuclear matrix. To test the functional role in chromatin dynamics sperm isolated from the caput and the cauda epididymides from WT and nGPx4-KO mice were subjected to an in vitro chromatin decondensation assay. Our results show that nGPx4-KO mice sperm decondensed earlier than those from WT at all stages of epididymal maturation, providing conclusive evidence that nGPx4 is required for a correct sperm chromatin remodelling. We next addressed the issue whether the sperm nuclear structure instability caused by the lack of nGPx4 might impact on the early events occurring after fertilization. In "in vitro" fertilization experiments, we revealed that, compared to WT, nGPx4-KO mice showed an acceleration of sperm nuclear decondensation, confirming the results previously obtained

Nuclear re-organisation of the Hoxb complex during mouse embryonic development

The spatial and temporal co-linear expression of Hox genes during development is an exquisite example of programmed gene expression. The precise mechanisms underpinning this are not known. Analysis of Hoxb chromatin structure and nuclear organisation, during the differentiation of murine ES cells, has lent support to the idea that there is a progressive 'opening' of chromatin structure propagated through Hox clusters from 3'to 5', which contributes to the sequential activation of gene expression. Here, we show that similar events occur in vivo in at least two stages of development. The first changes in chromatin structure and nuclear organisation were detected during gastrulation in the Hoxb1-expressing posterior primitive streak region: Hoxb chromatin was decondensed and the Hoxb1 locus looped out from its chromosome territory, in contrast to non-expressing Hoxb9, which remained within the chromosome territory. At E9.5, when differential Hox expression along the anteroposterior axis is being established, we found concomitant changes in the organisation of Hoxb. Hoxb organisation differed between regions of the neural tube that had never expressed Hoxb [rhombomeres (r) 1 and 2], strongly expressed Hoxb1 but not b9 (r4), had downregulated Hoxb1 (r5), expressed Hoxb9 but not Hoxb1 (spinal cord), and expressed both genes (tail bud). We conclude that Hoxb chromatin decondensation and nuclear re-organisation is regulated in different parts of the developing embryo, and at different developmental stages. The differential nuclear organisation of Hoxb along the anteroposterior axis of the developing neural tube is coherent with co-linear Hox gene expression. In early development nuclear re-organisation is coupled to Hoxb expression, but does not anticipate it

Encounter time of two loci governed by polymer de-condensation and local chromatin interaction

The time for a DNA sequence to find its homologous depends on a long random search process inside the cell nucleus. Using polymer models, we model and compute here the mean first encounter time (MFET) between two sites located on two different polymer chains and confined by potential wells. We find that reducing the potential (tethering) forces results in a local polymer decondensation near the loci and numerical simulations of the polymer model show that these changes are associated with a reduction of the MFET by several orders of magnitude. We derive here new asymptotic formula for the MFET, confirmed by Brownian simulations. We conclude that the acceleration of the search process after local chromatin decondensation can be used to analyze the local search step during homology search.Comment: 3 figure

Condensation transition in DNA-polyaminoamide dendrimer fibers studied using optical tweezers

When mixed together, DNA and polyaminoamide (PAMAM) dendrimers form fibers that condense into a compact structure. We use optical tweezers to pull condensed fibers and investigate the decondensation transition by measuring force-extension curves (FECs). A characteristic plateau force (around 10 pN) and hysteresis between the pulling and relaxation cycles are observed for different dendrimer sizes, indicating the existence of a first-order transition between two phases (condensed and extended) of the fiber. The fact that we can reproduce the same FECs in the absence of additional dendrimers in the buffer medium indicates that dendrimers remain irreversibly bound to the DNA backbone. Upon salt variation FECs change noticeably confirming that electrostatic forces drive the condensation transition. Finally, we propose a simple model for the decondensing transition that qualitatively reproduces the FECs and which is confirmed by AFM images.Comment: Latex version, 4 pages+3 color figure

Complexation of DNA with positive spheres: phase diagram of charge inversion and reentrant condensation

The phase diagram of a water solution of DNA and oppositely charged spherical macroions is studied. DNA winds around spheres to form beads-on-a-string complexes resembling the chromatin 10 nm fiber. At small enough concentration of spheres these "artificial chromatin" complexes are negative, while at large enough concentrations of spheres the charge of DNA is inverted by the adsorbed spheres. Charges of complexes stabilize their solutions. In the plane of concentrations of DNA and spheres the phases with positive and negative complexes are separated by another phase, which contains the condensate of neutral DNA-spheres complexes. Thus when the concentration of spheres grows, DNA-spheres complexes experience condensation and resolubilization (or reentrant condensation). Phenomenological theory of the phase diagram of reentrant condensation and charge inversion is suggested. Parameters of this theory are calculated by microscopic theory. It is shown that an important part of the effect of a monovalent salt on the phase diagram can be described by the nontrivial renormalization of the effective linear charge density of DNA wound around a sphere, due to the Onsager-Manning condensation. We argue that our phenomenological phase diagram or reentrant condensation is generic to a large class of strongly asymmetric electrolytes. Possible implication of these results for the natural chromatin are discussed.Comment: Many corrections to text. SUbmitted to J. Chem. Phy

ATXR5 and ATXR6 are H3K27 monomethyltransferases required for chromatin structure and gene silencing.

Constitutive heterochromatin in Arabidopsis thaliana is marked by repressive chromatin modifications, including DNA methylation, histone H3 dimethylation at Lys9 (H3K9me2) and monomethylation at Lys27 (H3K27me1). The enzymes catalyzing DNA methylation and H3K9me2 have been identified; alterations in these proteins lead to reactivation of silenced heterochromatic elements. The enzymes responsible for heterochromatic H3K27me1, in contrast, remain unknown. Here we show that the divergent SET-domain proteins ARABIDOPSIS TRITHORAX-RELATED PROTEIN 5 (ATXR5) and ATXR6 have H3K27 monomethyltransferase activity, and atxr5 atxr6 double mutants have reduced H3K27me1 in vivo and show partial heterochromatin decondensation. Mutations in atxr5 and atxr6 also lead to transcriptional activation of repressed heterochromatic elements. Notably, H3K9me2 and DNA methylation are unaffected in double mutants. These results indicate that ATXR5 and ATXR6 form a new class of H3K27 methyltransferases and that H3K27me1 represents a previously uncharacterized pathway required for transcriptional repression in Arabidopsis

Involvement of sperm acetylated histones and the nuclear isoform of Glutathione peroxidase 4 in fertilization

We previously demonstrated that the nuclear form of Glutathione peroxidase 4 (nGPx4) has a peculiar distribution in sperm head, being localized to nuclear matrix and acrosome and that sperm lacking nGPx4 are more prone to decondensation in vitro. In this study we have hypothesized that sperm retained acetylated histones and nGPx4 are implicated in paternal chromatin decondensation and male pronucleus formation at fertilization. Indeed, significant higher amounts of acetylated histone H4 and acetylated histone H3 were observed by both immunofluorescence and western blotting in nGPx4-KO sperm vs WT ones. In vitro fertilization of zona pellucida- deprived oocytes by WT sperm in the presence of trichostatin (TSA) also demonstrated that paternal histone acetylation was inversely related to the timing of sperm nucleus decondensation at fertilization. In contrast, TSA had no effect on nGPx4-KO sperm, indicating they had a maximal level of histone acetylation. Moreover the paternally imprinted gene Igf2/H19 was hypomethylated in KO sperm compared to WT ones. The lack of nGPx4 negatively affected male fertility, causing a marked decrease in total pups and pregnancies with delivery, a significant reduction in pronuclei (PN) embryos in in vitro fertilization assays and an approximately 2 h delay in egg fertilization in vivo. Because the zona pellucida binding and fusion to oolemma of nGPx4-KO and WT sperm were similar, the subfertility of nGPx4 sperm reflected a decreased sperm progression through egg cumulus/zona pellucida, pinpointing a defective acrosome in line with acrosomal nGPx4 localization. We conclude that paternal acetylated histones and acrosomal nGPx4 are directly involved in fertilization