A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.     

A unique epigenetic signature is associated with active DNA replication loci in human embryonic stem cells

The cellular epigenetic landscape changes as pluripotent stem cells differentiate to somatic cells or when differentiated cells transform to a cancerous state. These epigenetic changes are commonly correlated with differences in gene expression. Whether active DNA replication is also associated with distinct chromatin environments in these developmentally and phenotypically diverse cell types has not been known. Here, we used BrdU-seq to map active DNA replication loci in human embryonic stem cells (hESCs), normal primary fibroblasts and a cancer cell line, and correlated these maps to the epigenome. In all cell lines, the majority of BrdU peaks were enriched in euchromatin and at DNA repetitive elements, especially at microsatellite repeats, and coincided with previously determined replication origins. The most prominent BrdU peaks were shared between all cells but a sizable fraction of the peaks were specific to each cell type and associated with cell type-specific genes. Surprisingly, the BrdU peaks that were common to all cell lines were associated with H3K18ac, H3K56ac, and H4K20me1 histone marks only in hESCs but not in normal fibroblasts or cancer cells. Depletion of the histone acetyltransferases for H3K18 and H3K56 dramatically decreased the number and intensity of BrdU peaks in hESCs. Our data reveal a unique epigenetic signature that distinguishes active replication loci in hESCs from normal somatic or malignant cells.     

Cathepsin D deficiency is associated with a human neurodegenerative disorder

Cathepsin D is a ubiquitously expressed lysosomal protease that is involved in proteolytic degradation, cell invasion, and apoptosis. In mice and sheep, cathepsin D deficiency is known to cause a fatal neurodegenerative disease. Here, we report a novel disorder in a child with early blindness and progressive psychomotor disability. Two missense mutations in the CTSD gene, F229I and W383C, were identified and were found to cause markedly reduced proteolytic activity and a diminished amount of cathepsin D in patient fibroblasts. Expression of cathepsin D mutants in cathepsin D(-/-) mouse fibroblasts revealed disturbed posttranslational processing and intracellular targeting for W383C and diminished maximal enzyme velocity for F229I. The structural effects of cathepsin D mutants were estimated by computer modeling, which suggested larger structural alterations for W383C than for F229I. Our studies broaden the group of human neurodegenerative disorders and add new insight into the cellular functions of human cathepsin D.     

Derivation of human embryonic stem cells with NEMO deficiency

Deficiency of the nuclear factor-kappa-B essential modulator (NEMO) is a rare X-linked disorder that presents in boys as hypohydrotic ectodermal dysplasia with immunodeficiency due to defective nuclear factor-κB activation. Here we report on the generation of 2 human embryonic stem cell lines from discarded in vitro fertilization (IVF) embryos ascertained via preimplantation genetic diagnosis. We have derived two human embryonic stem cell lines that carry a T458G hypomorphic mutation in exon 4 of the NEMO (or IKBKG) gene. One of the lines is diploid male; the other is diploid female but has clonally inactivated the X-chromosome that harbors the wild-type IKBKG gene. We show that both lines are pluripotent, have the capacity to differentiate into hematopoietic progenitors, and have defective inhibitor of nuclear factor kappa-B kinase activity. These NEMO deficiency hES cell lines provide an unlimited source for differentiated cell types and may serve as a unique tool to study NEMO deficiency and potentially lead to the development of new therapies for this disease.     

Telomere length is associated with obesity parameters but with a gender difference

Cardiovascular disease (CVD) and obesity have been coupled to short telomere length in peripheral blood. The biological background to this observation is not obvious from the literature. In this study we have analyzed a large set of known risk factors for CVD in relation to telomere length in blood cells on a merged cohort of 989 individuals recruited in the Malmö Diet and Cancer Cohort (MDCC) and the Northern Sweden MONICA project. We found a significant or borderline association between obesity parameters and telomere length in women after age and center adjustments (BMI: r = ?0.106, P = 0.021, weight: r = ?0.087, P = 0.060, waist circumference: r = ?0.099, P = 0.032, hip circumference: r = ?0.128, P = 0.005). In men, a positive borderline correlation to high‐density lipoprotein (HDL) (r = 0.111, P = 0.053) and a negative correlation to 2‐h post‐oral glucose‐tolerance test (OGTT) was observed (r = ?0.202, P = 0.045). In neither group any association was found between telomere length and cholesterol, serum triglycerides, serum low‐density lipoprotein, plasma insulin, blood pressure, pulse pressure, or smoking habits. Our data indicate that telomere length is associated with an “obesity‐phenotype” but only in women.     

Telomere shortening is associated with malformation in p53-deficient mice after irradiation during specific stages of development

The natural ends of linear chromosomes, the telomeres, recruit specific proteins in the formation of protective caps that preserve the integrity of the genome. Unprotected chromosomes induce DNA damage checkpoint cascades and ultimately lead to senescence both in mouse and man in a p53 dependent manner and initial telomere length setting therefore determines the proliferative capacity of each cell. Yet, only little information is available on telomere biology during embryonic development. We have previously shown that the p53 gene plays a crucial role in the development of malformations (exencephaly, gastroschisis, polydactyly, cleft palate and dwarfism) in control and irradiated mouse embryos. Here, we investigated telomere biology and the outcome of radiation exposure in wild type (p53+/+) and p53-mutant (p53+/-- and--/--) C57BL mouse foetuses irradiated at three different developmental stages. We show that telomeres are significantly shorter in malformed foetuses as compared to normal counterparts. In addition, our results indicate that the observed telomere attrition is primarily associated with p53-deficiency but is also modulated by irradiation, more specifically during the gastrulation and organogenesis stages. In conclusion, we formulate a hypothesis in which telomere shortening is linked to the absence of p53 in mouse foetuses and that when, in the presence of shorter telomeres, these foetuses are irradiated, the chance for the occurrence of developmental defects increases substantially.     

Structural instability of a transgene locus in tobacco is associated with aneuploidy

This paper describes molecular and cytogenetic evidence for the instability of a transgene locus that is present on the triplicated chromosome in an aneuploid tobacco line. This instability was manifested in several ways in trisomics including a major chromosome rearrangement that was detectable cytogenetically, smaller scale DNA rearrangements that occurred both germinally and somatically, and methylation/epigenetic silencing. In a deletion derivative of the locus, DNA breakpoints were found in AT-rich regions. One of these regions binds to nuclear scaffolds in vitro , suggesting a possible role for aberrant topoisomerase II cleavage in destabilization of the locus. The implications of increased chromosome instability in aneuploids for plant karyotype evolution and human carcino-genesis are discussed.     

Telomere and centromere DNA are associated with the cores of meiotic prophase chromosomes

Mouse (Mus musculus) whole-mount, surface-spread, meiotic prophase chromosomes have an axial which extend chromatin loops. This arrangement permits a novel approach to the analysis of chromosome structure. Using in situ hybridization, the types of DNA sequences preferentially associated with the SC and the types located primarily in the chromatin loops can be determined. With biotinylated probes, detected by avidin conjugated to FITC, we present evidence for differential chromatin-SC interaction. The telomere sequence (TTAGGG)_n is associated exclusively with the two ends of each autosomal SC rather than with the chromatin loops. The minor satellite DNA sequences are predominantly localized to the centromeric region of the SC, as defined by CREST serum anti-centromere antibodies. In contrast, the major satellite DNA probe hybridizes to the chromatin loops of the centromeric heterochromatin, and a probe containing a LINE sequence hybridizes to chromatin loops in general with no obvious preference for the SC. These observations demonstrate that, depending on the type of DNA sequence, the chromatin has different properties in regard to its association with the SC.D.P. Bazett-Jones     

5-Hydroxymethylcytosine is associated with enhancers and gene bodies in human embryonic stem cells

Background  

5-Hydroxymethylcytosine (5hmC) was recently found to be abundantly present in certain cell types, including embryonic stem cells. There is growing evidence that TET proteins, which convert 5-methylcytosine (5mC) to 5hmC, play important biological roles. To further understand the function of 5hmC, an analysis of the genome-wide localization of this mark is required.     

Telomere shortening is associated with corticosterone stress response in adult barn swallows

When vertebrates face stressful events, the hypothalamic–pituitary–adrenal (HPA) axis is activated, generating a rapid increase in circulating glucocorticoid (GC) stress hormones followed by a return to baseline levels. However, repeated activation of HPA axis may lead to increase in oxidative stress. One target of oxidative stress is telomeres, nucleoprotein complexes at the end of chromosomes that shorten at each cell division. The susceptibility of telomeres to oxidizing molecules has led to the hypothesis that increased GC levels boost telomere shortening, but studies on this link are scanty. We studied if, in barn swallows Hirundo rustica, changes in adult erythrocyte telomere length between 2 consecutive breeding seasons are related to corticosterone (CORT) (the main avian GC) stress response induced by a standard capture-restraint protocol. Within-individual telomere length did not significantly change between consecutive breeding seasons. Second-year individuals showed the highest increase in circulating CORT concentrations following restraint. Moreover, we found a decline in female stress response along the breeding season. In addition, telomere shortening covaried with the stress response: a delayed activation of the negative feedback loop terminating the stress response was associated with greater telomere attrition. Hence, among-individual variation in stress response may affect telomere dynamics.     

Mannosidase 2, alpha 1 deficiency is associated with ricin resistance in embryonic stem (ES) cells

Host gene products required for mediating the action of toxins are potential targets for reversing or controlling their pathogenic impact following exposure. To identify such targets libraries of insertional gene-trap mutations generated with a PiggyBac transposon in Blm-deficient embryonic stem cells were exposed to the plant toxin, ricin. Resistant clones were isolated and genetically characterised and one was found to be a homozygous mutant of the mannosidase 2, alpha 1 (Man2α1) locus with a matching defect in the homologous allele. The causality of the molecular lesion was confirmed by removal of the transposon following expression of PB-transposase. Comparative glycomic and lectin binding analysis of the Man2α1 (−/−) ricin resistant cells revealed an increase in the levels of hybrid glycan structures and a reduction in terminal β-galactose moieties, potential target receptors for ricin. Furthermore, naïve ES cells treated with inhibitors of the N-linked glycosylation pathway at the mannosidase 2, alpha 1 step exhibited either full or partial resistance to ricin. Therefore, we conclusively identified mannosidase 2, alpha 1 deficiency to be associated with ricin resistance.     

The origin and impact of embryonic aneuploidy

Despite the clinical importance of aneuploidy, surprisingly little is known concerning its impact during the earliest stages of human development. This study aimed to shed light on the genesis, progression, and survival of different types of chromosome anomaly from the fertilized oocyte through the final stage of preimplantation development (blastocyst). 2,204 oocytes and embryos were examined using comprehensive cytogenetic methodology. A diverse array of chromosome abnormalities was detected, including many forms never recorded later in development. Advancing female age was associated with dramatic increase in aneuploidy rate and complex chromosomal abnormalities. Anaphase lag and congression failure were found to be important malsegregation causing mechanisms in oogenesis and during the first few mitotic divisions. All abnormalities appeared to be tolerated until activation of the embryonic genome, after which some forms started to decline in frequency. However, many aneuploidies continued to have little impact, with affected embryos successfully reaching the blastocyst stage. Results from the direct analyses of female meiotic divisions and early embryonic stages suggest that chromosome errors present during preimplantation development have origins that are more varied than those seen in later pregnancy, raising the intriguing possibility that the source of aneuploidy might modulate impact on embryo viability. The results of this study also narrow the window of time for selection against aneuploid embryos, indicating that most survive until the blastocyst stage and, since they are not detected in clinical pregnancies, must be lost around the time of implantation or shortly thereafter.     

Maternal smoking is associated with mitochondrial DNA depletion and respiratory chain complex III deficiency in placenta

Maternal smoking during pregnancy is often associated with a decrease in placental function, which might lead to intrauterine growth retardation. Because tobacco is known to alter the mitochondrial respiratory function in cardiomyocytes and lung tissue, we hypothesized that placental mitochondrial function could be altered by maternal smoking. Placental mitochondria from 9 smoking and 19 nonsmoking mothers were isolated by differential centrifugation. Mitochondrial oxygen consumption was measured by polarography, and the enzymatic activity of each complex of the electron transport chain was assessed by spectrophotometry. In addition, the relative content in mitochondrial DNA (mtDNA) was determined by real-time quantitative PCR in placentas from seven smoking and seven nonsmoking mothers. We observed a 29% reduction in the enzymatic activity of complex III in the placental mitochondria from smokers compared with nonsmokers (P = 0.03). The relative content of mtDNA (with respect to the beta-globin gene) was reduced by 37% in the placental tissue from smokers compared with nonsmokers (P < 0.02). Both the enzymatic activity of complex III and mtDNA content were inversely related with the daily consumption of cigarettes, and mtDNA content was correlated with cord blood insulin-like growth factor-binding protein-3 (r = 0.74, P < 0.01), a marker of fetal growth. These results show that maternal smoking is associated with placental mitochondrial dysfunction, which might contribute to restricted fetal growth by limiting energy availability in cells.     

DNA primase of human mitochondria is associated with structural RNA that is essential for enzymatic activity

DNA primase isolated from human mitochondria sediments in glycerol density gradients at 30S and 70S. These unusually high sedimentation coefficients are a result of association of the primase activity with RNA. Treatment of primase with nuclease not only affects its sedimentation behavior, but also inactivates the primase activity. The major RNA species that cofractionates with primase activity is shown by direct sequence analysis to be cytosolic 5.8S ribosomal RNA (rRNA). Specific degradation of endogenous 5.8S rRNA using ribonuclease H and oligonucleotides complementary to 5.8S rRNA results in reduction of primase activity. Other small RNAs may play a structural role in the formation of an active DNA primase complex.     

Telomere maintenance in telomerase-deficient mouse embryonic stem cells: characterization of an amplified telomeric DNA

Telomere dynamics, chromosomal instability, and cellular viability were studied in serial passages of mouse embryonic stem (ES) cells in which the telomerase RNA (mTER) gene was deleted. These cells lack detectable telomerase activity, and their growth rate was reduced after more than 300 divisions and almost zero after 450 cell divisions. After this growth crisis, survivor cells with a rapid growth rate did emerge. Such survivors were found to maintain functional telomeres in a telomerase-independent fashion. Although telomerase-independent telomere maintenance has been reported for some immortalized mammalian cells, its molecular mechanism has not been elucidated. Characterization of the telomeric structures in one of the survivor mTER(-/-) cell lines showed amplification of the same tandem arrays of telomeric and nontelomeric sequences at most of the chromosome ends. This evidence implicates cis/trans amplification as one mechanism for the telomerase-independent maintenance of telomeres in mammalian cells.     

Radiation-induced genomic instability is associated with DNA methylation changes in cultured human keratinocytes

The mechanism by which radiation-induced genomic instability is initiated, propagated and effected is currently under intense scrutiny. We have investigated the potential role of altered genomic methylation patterns in the cellular response to irradiation and have found evidence for widespread dysregulation of CpG methylation persisting up to 20 population doublings post-irradiation. Similar effects are seen with cells treated with medium from irradiated cells (the 'bystander effect') rather than subjected to direct irradiation. Using an arbitrarily primed methylation sensitive PCR screening method we have demonstrated that irradiation causes reproducible alterations in the methylation profile of a human keratinocyte cell line, HPV-G, and have further characterised one of these sequences as being a member of a retrotransposon element derived sequence family on chromosome 7; MLT1A. Multiple changes were also detected in the screen, which indicate that although the response of cells is predominantly hypermethylation, specific hypomethylation occurs as well. Sequence specific changes are also reported in the methylation of the pericentromeric SAT2 satellite sequence. This is the first demonstration that irradiation results in the induction of heritable methylation changes in mammalian cells, and provides a link between the various non-radiological instigators of genomic instability, the perpetuation of the unstable state and several of its manifestations.     

Beclin 1 deficiency is associated with increased hypoxia-induced angiogenesis

Beclin 1, a tumor suppressor protein, acts as an initiator of autophagy in mammals. Heterozygous disruption of Beclin 1 accelerates tumor growth, but the underlying mechanisms remain unclear. We examined the role of Beclin 1 in tumor proliferation and angiogenesis, using a primary mouse melanoma tumor model. Beclin 1 (Becn1^+/-) hemizygous mice displayed an aggressive tumor growth phenotype with increased angiogenesis under hypoxia, associated with enhanced levels of circulating erythropoietin but not vascular endothelial growth factor, relative to wild-type mice. Using in vivo and ex vivo assays, we demonstrated increased angiogenic activity in Becn1^+/- mice relative to wild-type mice. Endothelial cells from Becn1^+/- mice displayed increased proliferation, migration and tube formation in response to hypoxia relative to wild-type cells. Moreover, Becn1^+/- cells subjected to hypoxia displayed increased hypoxia-inducible factor-2α (HIF-2α) expression relative to HIF-1α. Genetic interference of HIF-2α but not HIF-1α, dramatically reduced hypoxia-inducible proliferation, migration and tube formation in Becn1^+/- endothelial cells. We demonstrated that mice deficient in the autophagic protein Beclin 1 display a pro-angiogenic phenotype associated with the upregulation of HIF-2α and increased erythropoietin production. These results suggest a relationship between Beclin 1 and the regulation of angiogenesis, with implications in tumor growth and development.     

MeCP2 deficiency is associated with impaired microtubule stability

Rett syndrome (RTT) is a neurodevelopmental disorder caused by MECP2 mutations. Previous studies performed on Mecp2-deficient brain showed striking changes in neuronal maturation. We recently showed that MeCP2 deficiency affects microtubule (MT) dynamics in RTT astrocytes. Here, we analyze MT stability in primary fibroblast cultures from patients with RTT syndrome and identify a significant decrease in stability compared to controls. Furthermore, we found that MT stability was reduced both in cells expressing the mutant or the wild-type allele in RTT fibroblasts, suggesting that mutated cells could damage wild-type ones through a non-cell-autonomous pathway. These results suggest that MeCP2 has a stabilizing role on MT dynamics and that its deficiency could lead to impaired MT stability that may explain in part the dendritic abnormalities observed in RTT brains.