PMC:1440874 / 11599-17737 JSON TXT

Annnotations TAB JSON ListView MergeView

craft-sa-dev

craft-ca-core-ex-dev

2_test

{"project":"2_test","denotations":[{"id":"16628246-206429-85799748","span":{"begin":3523,"end":3525},"obj":"206429"},{"id":"16628246-10742099-85799749","span":{"begin":4681,"end":4683},"obj":"10742099"},{"id":"T34229","span":{"begin":3523,"end":3525},"obj":"206429"},{"id":"T31994","span":{"begin":4681,"end":4683},"obj":"10742099"}],"text":"Perturbed Growth and Methylation Defects in Atrxnull ES Cells\nAtrxnull ES cells could be maintained in culture but were generally slower growing than Atrx+ ES clones, and appeared to undergo higher rates of spontaneous differentiation. We investigated directly the effect of Atrx on ES cell growth by comparing Atrx+ and Atrxnull ES cell clones in competition cultures. Equal numbers of Atrx+ (bearing either an Atrx WT or an Atrx flox allele) and Atrxnull (bearing an Atrx Δ18Δneo allele) ES cells were inoculated into cultures and the mixed cultures were passaged (1:3 split) every 2 d for 8–10 d. The relative abundance of the different alleles in the culture at each time point was analysed by Southern blotting (Figure 3A). The clone containing the Atrx Δ18Δneo allele was rapidly outgrown by both Atrx WT ES cells and cells bearing the Atrx flox allele. In a control competition experiment between different clones bearing functional Atrx alleles (Atrx WT and Atrx flox), both clones continued to be equally represented after 8 d of cocultivation. Thus, although Atrxnull ES cells could be recovered and maintained in culture by a conditional targeting approach, these cocultivation experiments suggested that the absence of Atrx does negatively impact upon normal ES cell growth.\nFigure 3 Growth and Methylation Defects in Atrxnull ES Cells\n(A) Cultures were inoculated with equivalent numbers of ES cells bearing different Atrx alleles as indicated, and were serially passaged. After the indicated days of coculture, DNA extracted from a sample of cells was analysed by Southern blot to detect the Atrx alleles. DNA was digested with SpeI, and the membrane was hybridised with the 20/27 probe shown in Figure 2A. The expected sizes of the different alleles are indicated.\n(B) Schematic diagram of the transcribed portion of the mouse rDNA repeat with the 18S, 5.8S, and 28S genes indicated. The positions of the limit-digesting enzymes BamH (labelled B) and EcoRI (labelled E) and the probes (RIB3 and RIB4) used in the Southern blots shown in (C) are indicated. Below are shown the locations of the methylation-sensitive enzymes (SmaI, PvuI, and MluI) whose methylation status has been analysed in the Southern blots shown in (C).\n(C) DNA from Atrx-positive (Atrx+, bearing either an Atrx WT or Atrx flox allele) or Atrxnull (bearing the Atrx Δ18Δneo allele) ES cells and 7-d embryoid bodies were digested with the enzymes shown and analysed by Southern blotting using the probes indicated. Arrows indicate the fully methylated copies (cut by only the limit-digesting enzyme). Phosphorimager quantitation of the blots are shown below. The y-axis shows the percentage of copies that are undigested by the methylation-sensitive enzyme as a percentage of the total signal from cut and uncut rDNA. Mean values are indicated by horizontal lines, and the significance of the differences between the Atrx-positive and Atrxnull populations are shown for each enzyme. To investigate a possible cell-cycle defect in the absence of Atrx, we analysed the cell cycle distribution of bromodeoxyuridine (BrdU)-pulsed ES cells by flow cytometry (Figure S1A). Surprisingly, both Atrxnull ES cell clones exhibited a cell cycle profile that was indistinguishable from ES cells bearing a functional Atrx allele (Atrx WT or Atrx flox). We also specifically quantitated the mitotic index within each population by flow cytometry after staining ES cells for phosphorylated (Ser10) histone H3, a specific marker of mitosis (Figure S1B) [13]. Consistent with the normal cell-cycle profile observed above, there was no depletion in the size of the mitotic population in the Atrxnull ES clones, despite their slow growth. Finally, we investigated whether the growth defect in the Atrxnull ES cells was due to an up-regulation of apoptosis by staining cells with Annexin V (Figure S2) and found that the proportion of apoptotic cells was not significantly affected by the absence of full-length Atrx. Thus, the growth defect observed in ES cells lacking Atrx is not due to a specific cell cycle block or significant induction of cell death. While the cause of the proliferative delay is not yet clear, since Atrxnull ES cells appear to undergo higher rates of spontaneous differentiation (unpublished data), it seems likely that the observed growth defect reflects the spontaneous transition from fast-cycling, undifferentiated ES cells into more slowly cycling, differentiated cell types in these cultures.\nIt has been shown that disease-causing mutations in the human ATRX gene give rise to changes in the normal pattern of DNA methylation at several repetitive sequences within the human genome [11]. Notably, the transcribed region of the ribosomal DNA (rDNA) repeat was found to be significantly hypomethylated in ATR-X patients relative to normal individuals. Using methylation-sensitive restriction enzymes, we also observed significant hypomethylation at several sites tested within the mouse rDNA repeats in Atrxnull ES cells and 12-d embryoid bodies relative to ES cells and embryoid bodies bearing a functional Atrx allele (Atrx WT or Atrx flox) (Figure 3B and 3C). The observation that rDNA is hypomethylated in the absence of Atrx, even in ES cells, is consistent with the finding that hypomethylation of the human rDNA repeats is detectable from an early developmental stage in ATR-X patients. Other mouse repetitive sequence elements surveyed in ES cell DNA include the heterochromatic major satellite (assayed with MaeII) and minor satellite (assayed with HpaII) repeats, as well as interspersed retroviral repeats of the intracisternal A particle (IAP) type and the Line 1 and Sine B1 families (all assayed with HpaII). These repeats were found to be moderately (Line 1 and Sine B1) or highly (IAP, major satellite, minor satellite) methylated in wild-type ES cells, and this methylation was not detectably perturbed by the absence of Atrx (Figure S3 and unpublished data). Taken together, these data indicate that the subtle interplay between the ATRX protein and DNA methylation observed in human patients is also present in mouse cells."}

craft-ca-core-dev

PMC:1440874 / 11599-17737 JSONTXT

Annnotations TAB JSON ListView MergeView

craft-sa-dev

craft-ca-core-ex-dev

2_test

craft-ca-core-dev

PMC:1440874 / 11599-17737 JSON TXT