Human male meiotic sex chromosome inactivation

In mammalian male gametogenesis the sex chromosomes are distinctive in both gene activity and epigenetic strategy. At first meiotic prophase the heteromorphic X and Y chromosomes are placed in a separate chromatin domain called the XY body. In this process, X,Y chromatin becomes highly phosphorylated at S139 of H2AX leading to the repression of gonosomal genes, a process known as meiotic sex chromosome inactivation (MSCI), which has been studied best in mice. Post-meiotically this repression is largely maintained. Disturbance of MSCI in mice leads to harmful X,Y gene expression, eventuating in spermatocyte death and sperm heterogeneity. Sperm heterogeneity is a characteristic of the human male. For this reason we were interested in the efficiency of MSCI in human primary spermatocytes. We investigated MSCI in pachytene spermatocytes of seven probands: four infertile men and three fertile controls, using direct and indirect in situ methods. A considerable degree of variation in the degree of MSCI was detected, both between and within probands. Moreover, in post-meiotic stages this variation was observed as well, indicating survival of spermatocytes with incompletely inactivated sex chromosomes. Furthermore, we investigated the presence of H3K9me3 posttranslational modifications on the X and Y chromatin. Contrary to constitutive centromeric heterochromatin, this heterochromatin marker did not specifically accumulate on the XY body, with the exception of the heterochromatic part of the Y chromosome. This may reflect the lower degree of MSCI in man compared to mouse. These results point at relaxation of MSCI, which can be explained by genetic changes in sex chromosome composition during evolution and candidates as a mechanism behind human sperm heterogeneity.     

Evidence that meiotic sex chromosome inactivation is essential for male fertility

The mammalian X and Y chromosomes share little homology and are largely unsynapsed during normal meiosis. This asynapsis triggers inactivation of X- and Y-linked genes, or meiotic sex chromosome inactivation (MSCI). Whether MSCI is essential for male meiosis is unclear. Pachytene arrest and apoptosis is observed in mouse mutants in which MSCI fails, e.g., Brca1(-/-), H2afx(-/-), Sycp1(-/-), and Msh5(-/-). However, these also harbor defects in synapsis and/or recombination and as such may activate a putative pachytene checkpoint. Here we present evidence that MSCI failure is sufficient to cause pachytene arrest. XYY males exhibit Y-Y synapsis and Y chromosomal escape from MSCI without accompanying synapsis/recombination defects. We find that XYY males, like synapsis/recombination mutants, display pachytene arrest and that this can be circumvented by preventing Y-Y synapsis and associated Y gene expression. Pachytene expression of individual Y genes inserted as transgenes on autosomes shows that expression of the Zfy 1/2 paralogs in XY males is sufficient to phenocopy the pachytene arrest phenotype; insertion of Zfy 1/2 on the X chromosome where they are subject to MSCI prevents this response. Our findings show that MSCI is essential for male meiosis and, as such, provide insight into the differential severity of meiotic mutations' effects on male and female meiosis.     

The Caenorhabditis elegans dosage compensation machinery is recruited to X chromosome DNA attached to an autosome

The dosage compensation machinery of Caenorhabditis elegans is targeted specifically to the X chromosomes of hermaphrodites (XX) to reduce gene expression by half. Many of the trans-acting factors that direct the dosage compensation machinery to X have been identified, but none of the proposed cis-acting X chromosome-recognition elements needed to recruit dosage compensation components have been found. To study X chromosome recognition, we explored whether portions of an X chromosome attached to an autosome are competent to bind the C. elegans dosage compensation complex (DCC). To do so, we devised a three-dimensional in situ approach that allowed us to compare the volume, position, and number of chromosomal and subchromosomal bodies bound by the dosage compensation machinery in wild-type XX nuclei and XX nuclei carrying an X duplication. The dosage compensation complex was found to associate with a duplication of the right 30% of X, but the complex did not spread onto adjacent autosomal sequences. This result indicates that all the information required to specify X chromosome identity resides on the duplication and that the dosage compensation machinery can localize to a site distinct from the full-length hermaphrodite X chromosome. In contrast, smaller duplications of other regions of X appeared to not support localization of the DCC. In a separate effort to identify cis-acting X recognition elements, we used a computational approach to analyze genomic DNA sequences for the presence of short motifs that were abundant and overrepresented on X relative to autosomes. Fourteen families of X-enriched motifs were discovered and mapped onto the X chromosome.     

Caenorhabditis elegans prom-1 is required for meiotic prophase progression and homologous chromosome pairing

A novel gene, prom-1, was isolated in a screen for Caenorhabditis elegans mutants with increased apoptosis in the germline. prom-1 encodes an F-box protein with limited homology to the putative human tumor suppressor FBXO47. Mutations in the prom-1 locus cause a strong reduction in bivalent formation, which results in increased embryonic lethality and a Him phenotype. Furthermore, retarded and asynchronous nuclear reorganization as well as reduced homologous synapsis occur during meiotic prophase. Accumulation of recombination protein RAD-51 in meiotic nuclei suggests disturbed repair of double-stranded DNA breaks. Nuclei in prom-1 mutant gonads timely complete mitotic proliferation and premeiotic replication, but they undergo prolonged delay upon meiotic entry. We, therefore, propose that prom-1 regulates the timely progression through meiotic prophase I and that in its absence the recognition of homologous chromosomes is strongly impaired.     

Extensive meiotic asynapsis in mice antagonises meiotic silencing of unsynapsed chromatin and consequently disrupts meiotic sex chromosome inactivation

Chromosome synapsis during zygotene is a prerequisite for the timely homologous recombinational repair of meiotic DNA double-strand breaks (DSBs). Unrepaired DSBs are thought to trigger apoptosis during midpachytene of male meiosis if synapsis fails. An early pachytene response to asynapsis is meiotic silencing of unsynapsed chromatin (MSUC), which, in normal males, silences the X and Y chromosomes (meiotic sex chromosome inactivation [MSCI]). In this study, we show that MSUC occurs in Spo11-null mouse spermatocytes with extensive asynapsis but lacking meiotic DSBs. In contrast, three mutants (Dnmt3l, Msh5, and Dmc1) with high levels of asynapsis and numerous persistent unrepaired DSBs have a severely impaired MSUC response. We suggest that MSUC-related proteins, including the MSUC initiator BRCA1, are sequestered at unrepaired DSBs. All four mutants fail to silence the X and Y chromosomes (MSCI failure), which is sufficient to explain the midpachytene apoptosis. Apoptosis does not occur in mice with a single additional asynapsed chromosome with unrepaired meiotic DSBs and no disturbance of MSCI.     

Characterization of regions of the Caenorhabditis elegans X chromosome containing vitellogenin genes

Caenorhabditis elegans contains a family of vitellogenin genes consisting of five closely related genes (vit-1 to vit-5) coding for 186,000 Mr yolk proteins, and one distantly related gene (vit-6) encoding a 200,000 Mr precursor to two smaller yolk proteins. We demonstrate here that, although vit-1 to vit-5 are not clustered (with the exception of vit-3 and vit-4), they are all on the X chromosome. In contrast, vit-6 is autosomal. The genes are strictly regulated during development: they are activated in the intestine of the hermaphrodite worm, following the last larval molt. In order to determine whether the vit genes are contained within chromosomal domains of similarly regulated genes, we have used the chromosomal "walking" technique to isolate 55,000 to 60,000 base-pairs of DNA surrounding each of the X-linked genes and determined the developmental specificity of nearby genes. In the total of 235,400 base-pairs of cloned DNA, seven genes, in addition to the five vit genes, were found. The average gene spacing is approximately 20,000 base-pairs per gene but is highly variable, ranging from less than 2000 to more than 38,000 base-pairs. The seven newly identified genes, called uvt-1 to uvt-7, specify RNAs varying in size from 500 to 2700 bases. With the exception of uvt-4, all of the genes are developmentally regulated; but the patterns of regulation are quite variable, and all are different from the vitellogenin genes. The vit genes, therefore, are not contained within co-regulated chromosomal domains. We also searched for the presence of repetitive DNA, but only four such sequences were found.     

Multiple forms of histone H2B from the nematode Caenorhabditis elegans.

The complete amino acid sequence of histone H2B from the nematode Caenorhabditis elegans was determined. The protein as obtained by us is a mixture of multiple forms. Approx. 90% of the molecules consist of a polypeptide chain of 122 amino acids with alanine as N-terminal residue and proline at the second position. In the remaining 10% alanine is lacking and the chain starts with proline. In addition to the heterogeneity of chain length, polymorphism occurs at the positions 7 (Ala/Lys), 14 (Ala/Lys) and 72 (Ala/Ser) of the major chain and at position 6 (Ala/Lys) of the shorter chain. In the N-terminal third of the molecule there is a high degree of sequence homology to the corresponding region in H2B from Drosophila (insect), Patella (mollusc) and Asterias (starfish). In contrast, this part of the molecule differs considerably from mammalian histone H2B.     

Re-analysis of the larval testis data on meiotic sex chromosome inactivation revealed evidence for tissue-specific gene expression related to the drosophila X chromosome

Background

Molecular oxygen (O₂) is one of the key metabolites of all obligate and facultative aerobic pro- and eukaryotes. It plays a fundamental role in energy homeostasis whereas oxygen deprivation, in turn, broadly affects various physiological and pathophysiological processes. Therefore, real-time monitoring of cellular oxygen levels is basically a prerequisite for the analysis of hypoxia-induced processes in living cells and tissues.

Results

We developed a genetically encoded F?rster resonance energy transfer (FRET)-based biosensor allowing the observation of changing molecular oxygen concentrations inside living cells. This biosensor named FluBO (fluorescent protein-based biosensor for oxygen) consists of the yellow fluorescent protein (YFP) that is sensitive towards oxygen depletion and the hypoxia-tolerant flavin-binding fluorescent protein (FbFP). Since O₂ is essential for the formation of the YFP chromophore, efficient FRET from the FbFP donor domain to the YFP acceptor domain only occurs in the presence but not in the absence of oxygen. The oxygen biosensor was used for continuous real-time monitoring of temporal changes of O₂ levels in the cytoplasm of Escherichia coli cells during batch cultivation.

Conclusions

FluBO represents a unique FRET-based oxygen biosensor which allows the non-invasive ratiometric readout of cellular oxygen. Thus, FluBO can serve as a novel and powerful probe for investigating the occurrence of hypoxia and its effects on a variety of (patho)physiological processes in living cells.     

HIM-8 binds to the X chromosome pairing center and mediates chromosome-specific meiotic synapsis

The him-8 gene is essential for proper meiotic segregation of the X chromosomes in C. elegans. Here we show that loss of him-8 function causes profound X chromosome-specific defects in homolog pairing and synapsis. him-8 encodes a C2H2 zinc-finger protein that is expressed during meiosis and concentrates at a site on the X chromosome known as the meiotic pairing center (PC). A role for HIM-8 in PC function is supported by genetic interactions between PC lesions and him-8 mutations. HIM-8 bound chromosome sites associate with the nuclear envelope (NE) throughout meiotic prophase. Surprisingly, a point mutation in him-8 that retains both chromosome binding and NE localization fails to stabilize pairing or promote synapsis. These observations indicate that stabilization of homolog pairing is an active process in which the tethering of chromosome sites to the NE may be necessary but is not sufficient.     

The primary structure of histone H3 from the nematode Caenorhabditis elegans

The complete amino acid sequence of histone H3 (135 residues) from the nematode Caenorhabditis elegans has been established. Microheterogeneity occurs at positions 96 and 100 of the chain. The sequences of the nematode H3 isoforms are very similar to the major chain of calf thymus H3 with which they show 4 substitutions in total. The major variant has cysteine in position 96. This is the first report of cysteine in this position in H3 from non-mammalian tissue. An exceptional methylation site has been detected at position 79. Various other sites of secondary modification are of a conservative nature.     

SDX on the X chromosome is required for male sex determination

Dear Editor, Sex determination is one of the most fundamental develop-ment processes,as gender is the first and most important identity of human.In most mammals...     

The primary structure of histone H4 from the nematode Caenorhabditis elegans

1. The complete amino acid sequence of histone H4 from the nematode Caenorhabditis elegans has been established. 2. The polypeptide chain consists of 102 amino acids and has a completely alpha-N-blocked serine at residue 1. 3. The sequence differs from vertebrate H4 in position 73 by substitution of cysteine for threonine. 4. Lysine in position 20 is monomethylated.