Premature aging syndromes often result from mutations in nuclear proteins involved in the maintenance of genomic integrity. Lamin A is a major component of the nuclear lamina and nuclear skeleton. Truncation in lamin A causes Hutchinson-Gilford progerial syndrome (HGPS), a severe form of early-onset premature aging. Lack of functional Zmpste24, a metalloproteinase responsible for the maturation of prelamin A, also results in progeroid phenotypes in mice and humans. We found that Zmpste24-deficient mouse embryonic fibroblasts (MEFs) show increased DNA damage and chromosome aberrations and are more sensitive to DNA-damaging agents. Bone marrow cells isolated from Zmpste24-/- mice show increased aneuploidy and the mice are more sensitive to DNA-damaging agents. Recruitment of p53 binding protein 1 (53BP1) and Rad51 to sites of DNA lesion is impaired in Zmpste24-/- MEFs and in HGPS fibroblasts, resulting in delayed checkpoint response and defective DNA repair. Wild-type MEFs ectopically expressing unprocessible prelamin A show similar defects in checkpoint response and DNA repair. Our results indicate that unprocessed prelamin A and truncated lamin A act dominant negatively to perturb DNA damage response and repair, resulting in genomic instability which might contribute to laminopathy-based premature aging.

Over the last years, the relevance of the matrix metalloproteinase (MMP) family in cancer research has grown considerably. These enzymes were initially associated with the invasive properties of tumour cells, owing to their ability to degrade all major protein components of the extracellular matrix (ECM) and basement membranes. However, further studies have demonstrated the implication of MMPs in early steps of tumour evolution, including stimulation of cell proliferation and modulation of angiogenesis. The establishment of causal relationships between MMP overproduction in tumour or stromal cells and cancer progression has prompted the development of clinical trials with a series of inhibitors designed to block the proteolytic activity of these enzymes. Unfortunately, the results derived from using broad-spectrum MMP inhibitors (MMPIs) for treating patients with advanced cancer have been disappointing in most cases. There are several putative explanations for the lack of success of these MMPIs including the recent finding that some MMPs may play a paradoxical protective role in tumour progression. These observations together with the identification of novel functions for MMPs in early stages of cancer have made necessary a reformulation of MMP inhibition strategies. A better understanding of the functional complexity of this proteolytic system and global approaches to identify the relevant MMPs which must be targeted in each individual cancer patient, will be necessary to clarify whether MMP inhibition may be part of future therapies against cancer.

Atlantic salmon 5S ribosomal DNA (5S rDNA) was amplified by the polymerase chain reaction, using as primers conserved sequences from the coding region of rainbow trout 5S rRNA. Two amplified products of different molecular weights were obtained, cloned, and sequenced, revealing them to be tandemly arranged. The nucleotide sequences differed between the two clones in the length of the nontranscribed spacer (NTS) and in three nucleotides of the coding sequence. By means of fluorescence in situ hybridization the 5S rDNA was chromosomally located in the heterochromatic arm of the pair bearing the satellite, adjacent to the major ribosomal DNA locus (rDNA).

Therefore, a conserved mechanism for meiotic kinetochore regulation remains elusive.Here we have identified meiosis-specific kinetochore factor MEIKIN in mouse, which functions in meiosis I but neither in meiosis II nor in mitosis. MEIKIN plays a crucial role in both mono-orientation and centromeric cohesion protection, partly by stabilizing the localization of the cohesin protector shugoshin. These functions are mediated largely by the activity of Polo-like kinase PLK1, which is enriched to kinetochores depending on MEIKIN. Our integrative analysis indicates that MEIKIN is the long awaited key regulator of meiotic kinetochore function, which is conserved from yeasts to humans. 2In mitosis, sister chromatid cohesion is established depending on cohesin in S phase and maintained until metaphase when the sister chromatids are captured by spindle microtubules from opposite poles and aligned on the spindle equator. For the onset of anaphase, the anaphase-promoting complex (APC) triggers the degradation of securin, an inhibitory chaperone for separase that cleaves cohesin RAD21 and removes cohesin along the entire chromosome. This removal of cohesin triggers the separation of sister chromatids and their movement to opposite poles, a process called equational division [1][2][3] . During meiosis, however, meiotic cohesin REC8 largely replaces RAD21 along the entire chromosomes; one round of DNA replication is followed by two rounds of nuclear division, which results in four haploid nuclei or gametes (Fig. 1a).In the first division of meiosis (meiosis I), homologous chromosomes connected by chiasmata are captured from the opposite poles, while sisters are captured from the same pole (mono-orientation). At the onset of anaphase I, REC8 cohesin is cleaved by separase along the arm regions, but protected at centromeres until metaphase II 4-6 . Thus, mono-orientation and centromeric cohesion protection are two hallmarks of meiotic kinetochore function, which are widely conserved among eukaryotic organisms 7-9 (Fig. 1a). There is accumulating evidence that cohesion protection is mediated by the centromeric protein shugoshin (SGO) and its partner protein phosphatase 2A (PP2A) [10][11][12][13][14][15] , which antagonizes REC8 phosphorylation, a prerequisite of cleavage 16, 17 . So far, meiosis-specific kinetochore proteins have been identified only in two yeasts (S. cerevisiae Spo13 and Mam1 (monopolin subunit), and S. pombe Moa1) [18][19][20][21][22][23] . Puzzlingly, however, because their structural and functional similarities remain to be identified, conservation of meiotic kinetochore regulation is questionable even between yeasts 8, 9 . Therefore, in this study, we address the long-standing question of whether meiotic kinetochore regulation is conserved from yeasts to mammals, and, if so, how. Mammalian meiotic kinetochore protein MEIKINFission yeast Moa1 interacts directly with the conserved kinetochore protein Cnp3 (CENP-C homolog), and localizes to the kinetochore in meiosis I 24 . To identify an equivalent meiosis...

Meiotic recombination generates crossovers between homologous chromosomes that are essential for genome haploidization. The synaptonemal complex is a ‘zipper'-like protein assembly that synapses homologue pairs together and provides the structural framework for processing recombination sites into crossovers. Humans show individual differences in the number of crossovers generated across the genome. Recently, an anonymous gene variant in C14ORF39/SIX6OS1 was identified that influences the recombination rate in humans. Here we show that C14ORF39/SIX6OS1 encodes a component of the central element of the synaptonemal complex. Yeast two-hybrid analysis reveals that SIX6OS1 interacts with the well-established protein synaptonemal complex central element 1 (SYCE1). Mice lacking SIX6OS1 are defective in chromosome synapsis at meiotic prophase I, which provokes an arrest at the pachytene-like stage and results in infertility. In accordance with its role as a modifier of the human recombination rate, SIX6OS1 is essential for the appropriate processing of intermediate recombination nodules before crossover formation.

Background-Ischemia in the placenta is considered the base of the pathogenesis of preeclampsia, a pregnancy-specific syndrome in which soluble endoglin (sEng) is a prognostic marker and plays a pathogenic role. Here, we investigated the effects of hypoxia and the downstream pathways in the release of sEng. Methods and Results-Under hypoxic conditions, the trophoblast-like cell line JAR showed an increase in sEng parallel to an elevated formation of reactive oxygen species. Because reactive oxygen species are related to the formation of oxysterols, we assessed the effect of 22-(R)-hydroxycholesterol, a natural ligand of the liver X receptor (LXR), and the LXR synthetic agonist T0901317. Treatment of JAR cells or human placental explants with 22-(R)-hydroxycholesterol or T0901317 resulted in a clear increase in sEng that was dependent on LXR. These LXR agonists induced an increased matrix metalloproteinase-14 expression and activity and a significant reduction of its endogenous inhibitor, tissue inhibitor of metalloproteinase-3. In addition, mice treated with LXR agonists underwent an increase in the plasma sEng levels, concomitant with an increase in arterial pressure. Moreover, transgenic mice overexpressing sEng displayed high blood pressure. Finally, administration of an endoglin peptide containing the consensus matrix metalloproteinase-14 cleavage site G-L prevented the oxysterol-dependent increase in arterial pressure and sEng levels in mice. Conclusions-These studies provide a clue to the involvement of the LXR pathway in sEng release and its pathogenic role in vascular disorders such as preeclampsia. (Circulation. 2012;126:2612-2624.)Key Words: cell hypoxia Ⅲ hypertension Ⅲ pre-eclampsia Ⅲ pregnancy Ⅲ peptides P reeclampsia is a pregnancy-specific syndrome characterized by systemic hypertension, proteinuria, and edema in the third trimester of pregnancy. 1,2 It affects both the fetus and the mother and occurs in Ϸ5% of pregnancies. Severe preeclampsia leads to the appearance of the HELLP syndrome (hemolysis, elevated liver enzymes, and low platelets), seizures, or fetal growth restriction and can result in fetal death. Preeclampsia is thought to be the consequence of impaired placentation resulting from inadequate trophoblastic invasion of the maternal spiral arteries. 3 Abnormal placentation is an important predisposing factor for preeclampsia, whereas endothelial dysfunction appears to be central to the pathophysiological changes, possibly indicative of a 2-stage disorder characterized by reduced placental perfusion and a maternal syndrome. Hypoxia, followed by oxidative stress, has been postulated as a critical signal that initiates the pathogenic process in preeclampsia. 4,5 Hypoxia and extracellular inflammatory signals can induce the intracellular accumulation of reactive oxygen species (ROS). 6,7 In turn, the imbalance between ROS production and antioxidant systems induces oxidative stress that negatively affects reproductive processes, including cyclic luteal and endometrial changes, follicular d...

Oligo- and azoospermia are severe forms of male infertility. However, known genetic factors account only for a small fraction of the cases. Recently, whole-exome sequencing in a large consanguineous family with inherited premature ovarian failure (POF) identified a homozygous frameshift mutation in the STAG3 gene leading to a premature stop codon. STAG3 encodes a meiosis-specific subunit of the cohesin complex, a large proteinaceous ring with DNA-entrapping ability that ensures sister chromatid cohesion and enables correct synapsis and segregation of homologous chromosomes during meiosis. The pathogenicity of the STAG3 mutations was functionally validated with a loss-of-function mouse model for STAG3 in oogenesis. However, and since none of the male members of this family was homozygous for the mutant allele, we only could hypothesized its putative involvement in male infertility. In this report, we show that male mice devoid of Stag3 display a severe meiotic phenotype that includes a meiotic arrest at zygonema-like shortening of their chromosome axial elements/lateral elements, partial loss of centromeric cohesion at early prophase and maintenance of the ability to initiate but not complete RAD51- and DMC1-mediated double-strand break repair, demonstrating that STAG3 is a crucial cohesin subunit in mammalian gametogenesis and supporting our proposal that STAG3 is a strong candidate gene for human male infertility.

Meiotic reductional division depends on the synaptonemal complex (SC), a supramolecular protein assembly that mediates homologous chromosomes synapsis and promotes crossover formation. The mammalian SC has eight structural components, including SYCE1, the only central element protein with known causative mutations in human infertility. We combine mouse genetics, cellular, and biochemical studies to reveal that SYCE1 undergoes multivalent interactions with SC component SIX6OS1. The N terminus of SIX6OS1 binds and disrupts SYCE1’s core dimeric structure to form a 1:1 complex, while their downstream sequences provide a distinct second interface. These interfaces are separately disrupted by SYCE1 mutations associated with nonobstructive azoospermia and premature ovarian failure (POF), respectively. Mice harboring SYCE1’s POF mutation and a targeted deletion within SIX6OS1’s N terminus are infertile with failure of chromosome synapsis. We conclude that both SYCE1-SIX6OS1 binding interfaces are essential for SC assembly, thus explaining how SYCE1’s reported clinical mutations give rise to human infertility.