Chromatid Ends Research Articles

SU75 - ASSOCIATION OF PTSD WITH TELOMERE LENGTH IN FEMALE RAPE VICTIMS

Background Posttraumatic Stress Disorder (PTSD) is a clinical condition that occurs after experiencing a traumatic external stressful situation, such as exposure to sexual assault or rape. Previous studies suggested that PTSD may be associated with shortened Leukocyte Telomere Length (LTL), in war veterans. Telomeres are repetitive DNA sequences located at the ends of chromatids that become shorter after each cell division. Some findings proposed that stress induced by trauma is a probable cause of accelerated telomere shortening. We examined a cross-sectional association between LTL and PTSD or psychopathological measures in rape victims. Methods The study population consisted of 32 women rape victims with PTSD diagnosis (PTSD group) and 34 healthy controls women without history of sexual trauma and mental disorders (HC group), with ages between 18 and 43 years. All participants were evaluated using the Mini International Neuropsychiatric Interview (MINI) compatible with Diagnostic and Statistical Manual of Mental Disorder (DSM-V), Clinician-Administered PTSD Scale (CAPS), which assesses PTSD symptoms, and Life Events Checklist for DSM-V, which appraises the trauma exposure. DNA was extracted from peripheral blood leukocytes and telomere repeat copy number relative to single gene copy number (relative T/S ratio) was determined by quantitative real-time PCR telomere assay. We used logistic and linear regression models to assess associations between relative T/S ratio and PTSD or its symptoms, respectively. Results The relative T/S ratio was determined in all samples and we found that PTSD diagnosis was associated with longer LTL, after adjusting for age at blood draw (p=0.008; β=3.796; 95% confidence interval: 1.409 – 10.229), indicating elongation LTL in PTSD group (relative T/S ratio mean=0.336; Std Deviation=0.109) compared to HC control (relative T/S ratio mean=-0.316; Std Deviation=0.161). However, LTL was not associated to PTSD symptom severity, measured by CAPS (t=-0.993; p=329). Discussion Our study found evidence of longer telomere length in women with PTSD diagnosis compared to healthy controls. In contrast, only one study showed accelerated LTL shortening in women rape victims with PTSD diagnosis, although, this study must be interpreted with caution, as the sample size was very small (n=9). Nevertheless, the LTL was not significantly associated with CAPS scores, possibly due to our sample size. Only one study found PTSD symptoms were associated to shorter telomeres in women exposed to different traumas, include rape, interpersonal violence, sudden death of a loved one. The telomere dynamics in PTSD is unclear; few studies investigated the relationship between LTL and PTSD, in women rape victims. Different mechanisms may contribute to LTL in PTSD, including neuroprogression it was also associated with elongation of LTL in schizophrenia. Our results show that PTSD was associated to longer LTL in rape victims, perhaps, due to the action of neuroprogression in PTSD.

P300-mediated acetylation of TRF2 is required for maintaining functional telomeres

The human telomeric protein TRF2 is required to protect chromosome ends by facilitating their organization into the protective capping structure. Post-translational modifications of TRF2 such as phosphorylation, ubiquitination, SUMOylation, methylation and poly(ADP-ribosyl)ation have been shown to play important roles in telomere function. Here we show that TRF2 specifically interacts with the histone acetyltransferase p300, and that p300 acetylates the lysine residue at position 293 of TRF2. We also report that p300-mediated acetylation stabilizes the TRF2 protein by inhibiting its ubiquitin-dependent proteolysis and is required for efficient telomere binding of TRF2. Furthermore, overexpression of the acetylation-deficient mutant, K293R, induces DNA-damage response foci at telomeres, thereby leading to induction of impaired cell growth, cellular senescence and altered cell cycle distribution. A small but significant number of metaphase chromosomes show no telomeric signals at chromatid ends, suggesting an aberrant telomere structure. These findings demonstrate that acetylation of TRF2 by p300 plays a crucial role in the maintenance of functional telomeres as well as in the regulation of the telomere-associated DNA-damage response, thus providing a new route for modulating telomere protection function.

Abstract 2121: Nonerythroid alpha spectrin prevents telomere fragility after DNA interstrand crosslink damage

Abstract Telomere dysfunction and increased telomere fragility are important contributing factors to chromosome instability. We have previously demonstrated that the structural protein, nonerythroid -spectrin(IISp), is critical for maintenance of chromosome stability after DNA interstrand crosslink (ICL) damage. However, whether IISp plays an important role in maintaining telomere stability after DNA damage is an extremely important question and the goal of the present study. IISp in normal human lymphoblastoid cells was depleted by siRNA and cells treated with an ICL agent, mitomycin C (MMC). Cells were analyzed using immunofluorescence and in situ hybridization (IF-FISH) and probed with anti–spectrin and a telomeric Cy3-conjugated-PNA probe or antibodies against two telomeric proteins, TRF1 or TRF2. Metaphase spreads of these cells were also examined and stained with the PNA probe and scored for telomeric abnormalities. In cells damaged with MMC but not in undamaged cells, a portion of IISp (50%) was found to co-localize with the PNA probe in the telomeres along with TRF1 and TRF2, indicating that IISp localizes to telomeres after ICL damage. To assess whether there was increased telomere fragility after ICL damage, chromosome ends were examined for loss of telomeric signal. After treatment with MMC, 28% of the chromatid ends showed a loss of telomeric signal compared to 2% in nondamaged cells. However, after depletion of IISp, 74% of the chromatid ends in MMC damaged cells displayed loss of telomeric signal compared to 18% in the nondamaged cells. This indicates that there was increased telomere fragility in MMC treated cells after knockdown of IISp. In addition to an increase in fragile telomeres in these cells, the chromosomes in the IISp siRNA treated cells damaged with MMC showed a significant increase in sister chromatid end fusions compared to the undamaged cells, which is indicative of aberrant telomere structure.These results on telomere fragility after loss of IISp are similar to those obtained using cells from the cancer prone genetic disease, Fanconi anemia complementation group A (FA-A), in which we have shown that there is a deficiency in IISp levels which correlates with a defect in ability to repair DNA ICLs. Examination of FA-A lymphoblastoid cells showed that there is an increase in fragile telomeres after MMC treatment: 76% of the chromatid ends showed loss of telomeric signal after MMC damage compared to 24% in undamaged cells. When levels of IISp were restored to normal in the FA-A cells by knocking down µ-calpain, a protease that cleaves IISp, the percentage of chromatid ends showing loss of telomeric signal (26%) after MMC was similar to that in normal cells. These studies collectively show that IISp plays an important and central role in maintenance of telomere stability after DNA ICL damage and suggests that it is an important factor in telomere function and chromosome stability. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 103rd Annual Meeting of the American Association for Cancer Research; 2012 Mar 31-Apr 4; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2012;72(8 Suppl):Abstract nr 2121. doi:1538-7445.AM2012-2121

Requirement of DDX39 DEAD box RNA helicase for genome integrity and telomere protection

Human chromosome ends associate with shelterin, a six-protein complex that protects telomeric DNA from being recognized as sites of DNA damage. The shelterin subunit TRF2 has been implicated in the protection of chromosome ends by facilitating their organization into the protective capping structure and by associating with several accessory proteins involved in various DNA transactions. Here we describe the characterization of DDX39 DEAD-box RNA helicase as a novel TRF2-interacting protein. DDX39 directly interacts with the telomeric repeat binding factor homology domain of TRF2 via the FXLXP motif (where X is any amino acid). DDX39 is also found in association with catalytically competent telomerase in cell lysates through an interaction with hTERT but has no effect on telomerase activity. Whereas overexpression of DDX39 in telomerase-positive human cancer cells led to progressive telomere elongation, depletion of endogenous DDX39 by small hairpin RNA (shRNA) resulted in telomere shortening. Furthermore, depletion of DDX39 induced DNA-damage response foci at internal genome as well as telomeres as evidenced by telomere dysfunction-induced foci. Some of the metaphase chromosomes showed no telomeric signal at chromatid ends, suggesting an aberrant telomere structure. Our findings suggest that DDX39, in addition to its role in mRNA splicing and nuclear export, is required for global genome integrity as well as telomere protection and represents a new pathway for telomere maintenance by modulating telomere length homeostasis.

AFM images of G 1-phase premature condensed chromosomes: Evidence for 30 nm changed to 50 nm chromatin fibers

To gain evidence for 30 nm changed to 50 nm chromatin fibers, we used atomic force microscopy (AFM) to study the ultrastructural organization of G 1-phase premature condensed chromosomes (PCC). The surface of early G 1-phase PCC is smooth and fibrous structures exist around the chromatids. The height of early G 1-phase PCC is about 410 nm and the width is 1.07 ± 0.11 μm ( n = 30). At late G 1-phase, the surface becomes globular. The height of late G1-phase PCC is about 370 nm and the width is 845.04 ± 82.84 nm ( n = 30). Phase image reveals that early G 1-phase PCC is composed of 50 nm (48.91 ± 6.63 nm, n = 30) chromatin fibers and these 50 nm chromatin fibers tangle together, while late G 1-phase PCC is composed of 30 nm (30.96 ± 4.07 nm, n = 30) chromatin fibers. At high magnification, fibers existing around the chromatids become clear in early G 1-phase PCC. Chromatin fibers revealed by closer view of the end of chromatid are about 50 nm. In late G 1-phase PCC, the surface presents globular structures. The shape of these globular structures is regular and the diameter is 118.96 ± 11.70 nm ( n = 30). Our results clearly show that 30 nm chromatin fibers change to 50 nm chromatin fibers in G 1-phase PCC and suggest that 50 nm chromatin fibers are the basic component of the mitotic chromosomes.

Apollo, an Artemis-Related Nuclease, Interacts with TRF2 and Protects Human Telomeres in S Phase

Human chromosome ends are protected by shelterin, an abundant six-subunit protein complex that binds specifically to the telomeric-repeat sequences, regulates telomere length, and ensures that chromosome ends do not elicit a DNA-damage response (reviewed in). Using mass spectrometry of proteins associated with the shelterin component Rap1, we identified an SMN1/PSO2 nuclease family member that is closely related to Artemis. We refer to this protein as Apollo and report that Apollo has the ability to localize to telomeres through an interaction with the shelterin component TRF2. Although its low abundance at telomeres indicates that Apollo is not a core component of shelterin, Apollo knockdown with RNAi resulted in senescence and the activation of a DNA-damage signal at telomeres as evidenced by telomere-dysfunction-induced foci (TIFs). The TIFs occurred primarily in S phase, suggesting that Apollo contributes to a processing step associated with the replication of chromosome ends. Furthermore, some of the metaphase chromosomes showed two telomeric signals at single-chromatid ends, suggesting an aberrant telomere structure. We propose that the Artemis-like nuclease Apollo is a shelterin accessory factor required for the protection of telomeres during or after their replication.

Importance of TRF1 for Functional Telomere Structure

Telomeres are comprised of telomeric DNA sequences and associated binding molecules. Their structure functions to protect the ends of linear chromosomes and ensure chromosomal stability. One of the mammalian telomere-binding factors, TRF1, localizes telomeres by binding to double-stranded telomeric DNA arrays. Because the overexpression of wild-type and dominant-negative TRF1 induces progressive telomere shortening and elongation in human cells, respectively, a proposed major role of TRF1 is that of a negative regulator of telomere length. Here we report another crucial function of TRF1 in telomeres. In conditional mouse TRF1 null mutant embryonic stem cells, TRF1 deletion induced growth defect and chromosomal instability. Although no clear telomere shortening or elongation was observed in short term cultured TRF1-deficient cells, abnormal telomere signals were observed, and TRF1-interacting telomere-binding factor, TIN2, lost telomeric association. Furthermore, another double-stranded telomeric DNA-binding factor, TRF2, also showed decreased telomeric association. Importantly, end-to-end fusions with detectable telomere signals at fusion points accumulated in TRF1-deficient cells. These results strongly suggest that TRF1 interacts with other telomere-binding molecules and integrates into the functional telomere structure.

Meiosis in holocentric chromosomes: orientation and segregation of an autosome and sex chromosomes in Triatoma infestans (Heteroptera).

The meiotic behaviour of the X chromosome and one autosomal pair of the heteropteran Triatoma infestans was analysed by means of C-banding plus DAPI staining. At first metaphase, the X univalent is oriented with its long axis parallel to the equatorial plate, which suggests a holocentric interaction with the spindle fibres. After this initial orientation, kinetic activity is restricted to one of both chromatid ends. The election of the active chromatid end is random and it is independent of the end selected in the sister chromatid. At second metaphase, the X and Y chromatids associate side by side forming a pseudobivalent. After that, the kinetic activity is again restricted to either of both chromosomal ends in a random fashion. At first metaphase, the fourth autosomal bivalent shows two alternative random orientations depending on the chromosome end showing kinetic activity (DAPI positive or opposite). At second metaphase, half bivalents are oriented with their long axis parallel to the equatorial plate. Three different segregation patterns are observed. The kinetic activity can be localised: (i) in the end with the DAPI signal (46.9%), (ii) in the opposite end (44.6%) or (iii) in one DAPI-positive end in one chromatid and in the opposite end in the other one (8.5%). The existence of the last pattern indicates that the same end can show kinetic activity during both meiotic divisions. Our results provide new information on the comparative meiotic behaviour of autosomes and sex chromosomes in holocentric systems.

Mobilization of a Minos transposon in Drosophila melanogaster chromosomes and chromatid repair by heteroduplex formation.

Transposase-mediated mobilization of the element Minos has been studied in the Drosophila melanogaster genome. Excision and transposition of a nonautonomous Minos transposon in the presence of a Minos transposase gene was detected with a dominant eye color marker carried by the transposon. Frequencies of excision in somatic tissues and in the germ line were higher in flies heterozygous for the transposon than in homozygotes or hemizygotes. Transposition of a X chromosome-linked insertion of Minos into new autosomal sites occurred in 1-12% of males expressing transposase, suggesting that this system is usable for gene tagging and enhancer trapping in Drosophila. Sequence analysis of PCR-amplified donor sites after excision showed precise restoration of the original target sequence in approximately 75% of events in heterozygotes and the presence of footprints or partially deleted elements in the remaining events. Most footprints consisted of the four terminal bases of the transposon, flanked by the TA target duplication. Sequencing of a chromosomal donor site that was directly cloned after excision showed a characteristic two-base mismatch heteroduplex in the center of the 6-bp footprint. Circular extrachromosomal forms of the transposon, presumably representing excised Minos elements, could be detected only in the presence of transposase. A model for chromatid repair after Minos excision is discussed in which staggered cuts are first produced at the ends of the inverted repeats, the broken chromatid ends are joined, and the resulting heteroduplex is subsequently repaired. The model also suggests a simple mechanism for the production of the target site duplication and for regeneration of the transposon ends during reintegration.

Meiosis in holocentric chromosomes: kinetic activity is randomly restricted to the chromatid ends of sex univalents in Graphosoma italicum (Heteroptera).

We have determined the number and location of the nucleolar organizing regions in spermatocytes of Graphosoma italicum (2n = 12A + XYmale/XXfemale) by means of silver impregnation, chromomycin A3/distamycin A staining and fluorescence in situ hybridization. The identification of only one nucleolar organizing region located at one of the X chromosome ends has provided a suitable cytological marker to analyse the segregation of this univalent and that of the XY pseudobivalent during the first and second meiotic divisions respectively. Our results clearly show that at first meiotic metaphase the chromatids of the X chromosome are orientated with their long axes perpendicular to the polar axis. Although the kinetic activity is restricted to only one end in both X chromatids during the first meiotic division, both ends of the same chromatid have the same probability of showing such kinetic activity. In this sense, we also report that the chromatid segregation may be initiated either at the same sister chromatid ends or at opposite ends in each chromatid. Thus, this indicates a sex chromatid independence as regards to the chromatid segregation during the first meiotic division. Throughout the second meiotic division both ends of the X chromatid are involved with the same probability in the end-to-end association to conform the XY pseudobivalent. This also implies a random localization of the kinetic activity at the ends opposite to those involved in the end-to-end association.

The telochore: a telomeric differentiation of the chromosome axis.

We have analysed by means of silver staining the structure of the chromosome axis at the telomeres of meiotic chromosomes in three different grasshopper species. At metaphase I the chromatid axes run the length of the chromatids although they do not reach the chromosome ends. The axes of sister chromatids are associated and show a round differentiation at their distal ends that we have named the 'telochore'. Telochores never contact the chromosome ends: there is always some chromatin beyond them. In late metaphase I bivalents with a distal chiasma, anaphase I and metaphase II half-bivalents and anaphase II chromatids, the axes clearly possess one telochore in each chromosome end. These results seem to indicate that telochores are differentiations of the distal ends of chromatids. We discuss the possible structural significance of telochores according to the current scaffold/radial loop model of chromatin organization of eukaryotic metaphase chromosomes. Additionally, we suggest the possible functional role of the telochore as a nucleoprotein domain forming a protective cap for telomeric DNA.

FLP recombinase induction of the breakage-fusion-bridge cycle and gene conversion in Saccharomyces cerevisiae

A YEp chimaeric plasmid containing URA3 and SMR1 [sulfometuron methyl resistant (SMR) allele of ILV2] as selectable markers, and the 2 μm site-specific recombination FLP recognition target (FRT), was integrated at the ilv2-Δ1 site in chromosome XIII in a cir°] haploid. Southern analysis defined two integrant structures. Structure I had URA3 distal and SMR1 proximal to FRT whereas in structure II both markers were distal to FRT. Selectable markers were stably inherited in [cir°] haploids and [cir°] diploids heterozygous for the integrant and ILV2. Approximately 14% of heterozygous [cir+] diploid cells exhibited homozygotization for the distal (500 kb) ade4 marker in trans. In [cir+] diploids FLP-FRT recombination resulted in the simultaneous loss of both structure II markers, whereas the structure I distal URA3 marker loss always preceded the variable loss of the proximal SMR1 marker. URA− cells continued to segregate for loss of SMR1 until stable URA− SMR or URA−SMS cells were produced. Gene conversion was identified in stable URA−SMR cells that were homozygous SMR1/SMR1 but contained wild type ILV2 restriction endonuclease sites. These observations support a model based on concerted FLP-FRT action resulting from the secondary integration of native 2 μm DNA followed by unequal sister chromatid exchange (USCE) within inverted FRTs. The resultant chromatid bridge resulted in a double-stand break. Fusion of the broken ends of sister chromatids generated a breakage-fusion-bridge cycle (BFBC). Repeated rounds of the BFBC resulted in proximal marker loss and the generation of additional double-strand breaks. Recombinogenic properties of the double-strand break initiated events leading to homozygotization and gene conversion.

The structure of chromatid ends and of the ends of experimentally procuced chromatid fragments as revealed by trypsin treatment of Vicia faba chromosomes

After digestion with trypsin the metaphase chromatids of Vicia faba reveal two length structures (half-chromatids). This confirms the results of other authors. According to our observations the half-chromatids are connected at their ends in such a way that a U-shaped configuration is formed. This finding can be explained in different ways. The ends of chromatid fragments obtained after X-irradiation are either U-shaped (“closed”) like the “natural” chromatid ends or “open”, i.e. the two half-chromatids do not seem to be connected at their ends. As a by-product of our experiments the structure of X-ray-induced achromatic lesions (gaps) was studied in trypsin-treated chromosomes.

A study of newt mitotic chromosomes by negative staining.

A method is described for bursting single, selected mitotic cells on a fluid surface. Cells from cultures of newt heart tissue were burst on dilute solutions containing potassium and sodium with and without added calcium and also on dilute calcium chloride solution. The material was negatively stained with uranyl acetate or sometimes with ammonium molybdate or sodium phosphotungstate. The bodies of chromatids spread on NaCl/KCl solutions showed many parallel fibers about 150 A in diameter. Loops with a complex nodular structure were observed projecting from the sides and ends of chromatids. In calcium-containing solutions there was evidence of fiber coagulation; the chromatid body was more compact and laterally projecting fibers tended to be pulled out straight. Especially in the absence of calcium the chromosomal fibers had a nodular form and appeared to be composed of irregularly folded fibrillar elements. The question as to whether chromosomal fibers, which range in diameter from about 50 to 300 A, consist of single, folded threads or of two or more adjacent subunits is discussed.

An experimental and descriptive study of chromosome elimination in Miastor spec. (Cecidomyidae; Diptera)

1. The descriptive cytology of chromosome elimination has been reinvestigated. The results show that most of the eliminated chromosomes are definitely not homologous with the somatic chromosomes. 2. Evidence from both descriptive and experimental sources shows beyond doubt that the errant feature of the chromosomes eliminated is a failure in the production of normal mid-anaphase tension. Interlocking or sticking of daughter chromatid ends is only rarely if ever responsible for chromosome elimination in the Cecidomyidae. 3. The normal amount of DNA is synthesized just prior to chromosome elimination, and hence no alteration in the normal DNA relationships is involved in the chromosomal behavior here. The distribution of RNA, protein and polysaccharides has also been studied; nothing to indicate their direct involvement in chromosome elimination was detected. 4. Centrifugation studies provide support for the hypothesis that in the cecidomyids chromosome elimination is a largely autonomous act of the chromosomes — it can be prevented but not initiated by cytoplasmic factors. Nucleocytoplasmic interaction therefore will account for the limitation of elimination to only the future somatic nuclei, by permitting normal division of the primordial germ-cell chromosomes.

Centromeric Behaviour of the Univalents in Two Phalaris Hybrids

The hybrid between Phalaris coerulescens (2n = 14) and P. minor (2n = 28) usually forms seven bivalents and seven univalents at the first metaphase of meiosis. The univalents are derived from P. minor. The centromeres of the seven univalents do not divide at the first anaphase, but neocentromeres in distal parts of the chromosomes assume control of their movement. These neocentromeres move towards opposite poles, between which the univalents become stretched. Formation of the cell wall causes breakage of the univalent bridges. Fusions may occur between fragments or between broken ends of sister chromatids in a fragment. Neocentric activity occurs only infrequently at the second anaphase.

Chromosome Studies on <i>Trillium kamtschaticum</i> Pall. and Its Allies. IX

PMCs of T. kamtschaticum were irradiated at their resting stage, and the following results were obtained.1) The irradiation leads to the production of both “chromosome” and “chromatid” breaks.2) The reunion of broken ends occurs between two broken ends as well as between a broken end and a normal end.3) Broken ends do not often reunite. These free broken ends of chromatids reveal themselves in the next division (that is, in the first pollen mitosis) as paired chromatids which are bridged together at their broken ends.4) The frequency breaks per chromosome is proportional to the length of whole chromosomes.5) The frequency of bridges and loops which appear at the first anaphase indicated the ratio of 2 bridges: 1 loop within intra-bivalent aberrations and the ratio of 1:1 in inter-bivalent aberrations. These ratio verify that the mode of disjunction of the paired kinetochores of a bivalent follows the principles of the neo-two plane theory.