Telomere length dynamics and the evolution of cancer genome architecture

Telomeres are progressively eroded during repeated rounds of cell division due to the end replication problem but also undergo additional more substantial stochastic shortening events. In most cases, shortened telomeres induce a cell-cycle arrest or trigger apoptosis, although for those cells that bypass such signals during tumour progression, a critical length threshold is reached at which telomere dysfunction may ensue. Dysfunction of the telomere nucleoprotein complex can expose free chromosome ends to the DNA double-strand break (DSB) repair machinery, leading to telomere fusion with both telomeric and non-telomeric loci. The consequences of telomere fusions in promoting genome instability have long been appreciated through the breakage–fusion–bridge (BFB) cycle mechanism, although recent studies using high-throughput sequencing technologies have uncovered evidence of involvement in a wider spectrum of genomic rearrangements including chromothripsis. A critical step in cancer progression is the transition of a clone to immortality, through the stabilisation of the telomere repeat array. This can be achieved via the reactivation of telomerase, or the induction of the alternative lengthening of telomeres (ALT) pathway. Whilst telomere dysfunction may promote genome instability and tumour progression, by limiting the replicative potential of a cell and enforcing senescence, telomere shortening can act as a tumour suppressor mechanism. However, the burden of senescent cells has also been implicated as a driver of ageing and age-related pathology, and in the promotion of cancer through inflammatory signalling. Considering the critical role of telomere length in governing cancer biology, we review questions related to the prognostic value of studying the dynamics of telomere shortening and fusion, and discuss mechanisms and consequences of telomere-induced genome rearrangements

Genetic association study of UCMA/GRP and OPTN genes (PDB6 locus) with Paget's disease of bone

We performed a genetic association study of rare variants and single nucleotide polymorphisms (SNPs) of UCMA/GRP and OPTN genes, in French-Canadian patients with Paget's disease of bone (PDB) and in healthy controls from the same population. We reproduced the variant found in the UCMA/GRP basal promoter and tested its functionality using in vitro transient transfection assays. Interestingly, this SNP rs17152980 appears to affect the transcription level of UCMA/GRP. In addition, we have identified five rare genetic variants in UCMA/GRP gene, four of them being population-specific, although none were found to be associated with PDB. Six Tag SNPs of UCMA/GRP gene were associated with PDB, particularly the SNP rs17152980 (uncorrected P = 3.8 x 10(-3)), although not significant after Bonferroni's correction. More importantly, we replicated the strong and statistically significant genetic association of two SNPs of the OPTN gene, the rs1561570 (uncorrected P = 5.7 x 10(-7)) and the rs2095388 (uncorrected P = 4.9 x 10(-3)), With PDB. In addition, we identified a very rare variant found to be located close to the basal promoter of the OPTN gene, at -232 bp from its distal transcription start site. Furthermore, depending on the type of allele present (G or A), the binding of several important nuclear factors such as the vitamin D or the retinoic acid receptors is predicted to be altered at this position, suggesting a significant effect in the regulation of transcription of the OPTN gene. In conclusion, we identified a functional SNP located in the basal promoter of the UCMA/GRP gene which provided a weak genetic association with PDB. In addition, we replicated the strong genetic association of two already known SNPs of the OPTN gene, with PDB in a founder effect population. We also identified a very rare variant in the promoter of OPTN, and through bioinformatic analysis, identified putative transcription factor binding sites likely to affect OPTN gene transcription. (C) 2012 Elsevier Inc. All rights reserved.Fonds de la Recherche du Quebec - Sante (FRQS), Canada; Portuguese Science and Technology Foundation, Portugal [SFRH/BPD/48206/2008]; Catalyst Grant (Bone Health) from the Canadian Institutes of Health Research (Canada); CHUQ Foundation (Canada); Groupe de Recherche en Maladies Osseuses (Canada); Canadian Foundation for Innovation (Canada); FRSQ (Canada); Laval University (Canada); CHUQ (CHUL) Research Centre (Canada); Centre of Marine Sciences (CCMAR) (Portugal)info:eu-repo/semantics/publishedVersio

Human telomerase RNA and telomerase activity in immortal cell lines and tumor tissues

Telomerase activity has been detected in many human immortal cells lines and in tumor tissues, whereas it is generally absent from primary cell strains and from many tumor adjacent tissue samples. With the recently cloned human telomerase RNA (hTR), we used Northern analysis to follow the levels of hTR in primary, precrisis, and immortalized cells. It was surprising that the amount of hTR was high in cell strains that lacked telomerase activity, and the levels did not parallel the increases in telomerase activity, which accompanies immortalization. In addition, although the hTR levels were somewhat higher in tumor samples compared to nontumor tissues, the level of hTR in a variety of different human tumors did not predict the level of telomerase activity in the tumor. Thus, whereas hTR was detected in all samples that have telomerase activity, the presence of the RNA was not a good predictor of the presence or amount of telomerase activity

Regulation of Adipose Tissue Stromal Cells Behaviors by Endogenic Oct4 Expression Control

BACKGROUND: To clarify the role of the POU domain transcription factor Oct4 in Adipose Tissue Stromal Cells (ATSCs), we investigated the regulation of Oct4 expression and other embryonic genes in fully differentiated cells, in addition to identifying expression at the gene and protein levels. The ATSCs and several immature cells were routinely expressing Oct4 protein before and after differentiating into specific lineages. METHODOLOGY/PRINCIPAL FINDINGS AND CONCLUSIONS: Here, we demonstrated the role of Oct4 in ATSCs on cell proliferation and differentiation. Exogenous Oct4 improves adult ATSCs cell proliferation and differentiation potencies through epigenetic reprogramming of stemness genes such as Oct4, Nanog, Sox2, and Rex1. Oct4 directly or indirectly induces ATSCs reprogramming along with the activation of JAK/STAT3 and ERK1/2. Exogenic Oct4 introduced a transdifferentiation priority into the neural lineage than mesodermal lineage. Global gene expression analysis results showed that Oct4 regulated target genes which could be characterized as differentially regulated genes such as pluripotency markers NANOG, SOX2, and KLF4 and markers of undifferentiated stem cells FOXD1, CDC2, and EPHB1. The negatively regulated genes included FAS, TNFR, COL6A1, JAM2, FOXQ1, FOXO1, NESTIN, SMAD3, SLIT3, DKK1, WNT5A, BMP1, and GLIS3 which are implicated in differentiation processes as well as a number of novel genes. Finally we have demonstrated the therapeutic utility of Oct4/ATSCs were introduced into the mouse traumatic brain, engrafted cells was more effectively induces regeneration activity with high therapeutic modality than that of control ATSCs. Engrafted Oct4/ATSCs efficiently migrated and transdifferentiated into action potential carrying, functionally neurons in the hippocampus and promoting the amelioration of lesion cavities

A direct physical interaction between Nanog and Sox2 regulates embryonic stem cell self-renewal

Embryonic stem (ES) cell self-renewal efficiency is determined by the Nanog protein level. However, the protein partners of Nanog that function to direct self-renewal are unclear. Here, we identify a Nanog interactome of over 130 proteins including transcription factors, chromatin modifying complexes, phosphorylation and ubiquitination enzymes, basal transcriptional machinery members, and RNA processing factors. Sox2 was identified as a robust interacting partner of Nanog. The purified Nanog–Sox2 complex identified a DNA recognition sequence present in multiple overlapping Nanog/Sox2 ChIP-Seq data sets. The Nanog tryptophan repeat region is necessary and sufficient for interaction with Sox2, with tryptophan residues required. In Sox2, tyrosine to alanine mutations within a triple-repeat motif (S X T/S Y) abrogates the Nanog–Sox2 interaction, alters expression of genes associated with the Nanog-Sox2 cognate sequence, and reduces the ability of Sox2 to rescue ES cell differentiation induced by endogenous Sox2 deletion. Substitution of the tyrosines with phenylalanine rescues both the Sox2–Nanog interaction and efficient self-renewal. These results suggest that aromatic stacking of Nanog tryptophans and Sox2 tyrosines mediates an interaction central to ES cell self-renewal

Use of methanol as cryoprotectant and its effect on sox genes and proteins in chilled zebrafish embryos

Methanol is a widely used cryoprotectant (CPA) in cryopreservation of fish embryos, however little is known about its effect at the molecular level. This study investigated the effect of methanol on sox gene and protein expression in zebrafish embryos (50% epiboly) when they were chilled for 3. h and subsequently warmed and cultured to the hatching stages. Initial experiments were carried out to evaluate the chilling tolerance of 50% epiboly embryos which showed no significant differences in hatching rates for up to 6. h chilling in methanol (0.2-, 0.5- and 1. M). Subsequent experiments in embryos that had been chilled for 3. h in 1. M methanol and warmed and cultured up to the hatching stages found that sox2 and sox3 gene expression were increased significantly in hatched embryos that had been chilled compared to non-chilled controls. Sox19a gene expression also remained above control levels in the chilled embryos at all developmental stages tested. Whilst stable sox2 protein expression was observed between non-chilled controls and embryos chilled for 3. h with or without MeOH, a surge in sox19a protein expression was observed in embryos chilled for 3. h in the presence of 1. M MeOH compared to non-chilled controls and then returned to control levels by the hatching stage. The protective effect of MeOH was increased with increasing concentrations. Effect of methanol at molecular level during chilling was reported here first time which could add new parameter in selection of cryoprotectant while designing cryopreservation protocol

Genetic regulation of pituitary gland development in human and mouse

Normal hypothalamopituitary development is closely related to that of the forebrain and is dependent upon a complex genetic cascade of transcription factors and signaling molecules that may be either intrinsic or extrinsic to the developing Rathke’s pouch. These factors dictate organ commitment, cell differentiation, and cell proliferation within the anterior pituitary. Abnormalities in these processes are associated with congenital hypopituitarism, a spectrum of disorders that includes syndromic disorders such as septo-optic dysplasia, combined pituitary hormone deficiencies, and isolated hormone deficiencies, of which the commonest is GH deficiency. The highly variable clinical phenotypes can now in part be explained due to research performed over the last 20 yr, based mainly on naturally occurring and transgenic animal models. Mutations in genes encoding both signaling molecules and transcription factors have been implicated in the etiology of hypopituitarism, with or without other syndromic features, in mice and humans. To date, mutations in known genes account for a small proportion of cases of hypopituitarism in humans. However, these mutations have led to a greater understanding of the genetic interactions that lead to normal pituitary development. This review attempts to describe the complexity of pituitary development in the rodent, with particular emphasis on those factors that, when mutated, are associated with hypopituitarism in humans

Histone arginine methylation regulates pluripotency in the early mouse embryo

It has been generally accepted that the mammalian embryo starts its development with all cells identical, and only when inside and outside cells form do differences between cells first emerge. However, recent findings show that cells in the mouse embryo can differ in their developmental fate and potency as early as the four-cell stage1,2,3,4. These differences depend on the orientation and order of the cleavage divisions that generated them2,5. Because epigenetic marks are suggested to be involved in sustaining pluripotency6,7, we considered that such developmental properties might be achieved through epigenetic mechanisms. Here we show that modification of histone H3, through the methylation of specific arginine residues, is correlated with cell fate and potency. Levels of H3 methylation at specific arginine residues are maximal in four-cell blastomeres that will contribute to the inner cell mass (ICM) and polar trophectoderm and undertake full development when combined together in chimaeras. Arginine methylation of H3 is minimal in cells whose progeny contributes more to the mural trophectoderm and that show compromised development when combined in chimaeras. This suggests that higher levels of H3 arginine methylation predispose blastomeres to contribute to the pluripotent cells of the ICM. We confirm this prediction by overexpressing the H3-specific arginine methyltransferase CARM1 in individual blastomeres and show that this directs their progeny to the ICM and results in a dramatic upregulation of Nanog and Sox2. Thus, our results identify specific histone modifications as the earliest known epigenetic marker contributing to development of ICM and show that manipulation of epigenetic information influences cell fate determination

Neuronal Migration and Ventral Subtype Identity in the Telencephalon Depend on SOX1

Little is known about the molecular mechanisms and intrinsic factors that are responsible for the emergence of neuronal subtype identity. Several transcription factors that are expressed mainly in precursors of the ventral telencephalon have been shown to control neuronal specification, but it has been unclear whether subtype identity is also specified in these precursors, or if this happens in postmitotic neurons, and whether it involves the same or different factors. SOX1, an HMG box transcription factor, is expressed widely in neural precursors along with the two other SOXB1 subfamily members, SOX2 and SOX3, and all three have been implicated in neurogenesis. SOX1 is also uniquely expressed at a high level in the majority of telencephalic neurons that constitute the ventral striatum (VS). These neurons are missing in Sox1-null mutant mice. In the present study, we have addressed the requirement for SOX1 at a cellular level, revealing both the nature and timing of the defect. By generating a novel Sox1-null allele expressing β-galactosidase, we found that the VS precursors and their early neuronal differentiation are unaffected in the absence of SOX1, but the prospective neurons fail to migrate to their appropriate position. Furthermore, the migration of non-Sox1-expressing VS neurons (such as those expressing Pax6) was also affected in the absence of SOX1, suggesting that Sox1-expressing neurons play a role in structuring the area of the VS. To test whether SOX1 is required in postmitotic cells for the emergence of VS neuronal identity, we generated mice in which Sox1 expression was directed to all ventral telencephalic precursors, but to only a very few VS neurons. These mice again lacked most of the VS, indicating that SOX1 expression in precursors is not sufficient for VS development. Conversely, the few neurons in which Sox1 expression was maintained were able to migrate to the VS. In conclusion, Sox1 expression in precursors is not sufficient for VS neuronal identity and migration, but this is accomplished in postmitotic cells, which require the continued presence of SOX1. Our data also suggest that other SOXB1 members showing expression in specific neuronal populations are likely to play continuous roles from the establishment of precursors to their final differentiation