Spreading of mammalian DNA-damage response factors studied by ChIP-chip at damaged telomeres

Phosphorylated histone H2AX (gammaH2AX) is generated in nucleosomes flanking sites of DNA double-strand breaks, triggering the recruitment of DNA-damage response proteins such as MDC1 and 53BP1. Here, we study shortened telomeres in senescent human cells. We show that most telomeres trigger gammaH2AX formation, which spreads up to 570 kb into the subtelomeric regions. Furthermore, we reveal that the spreading patterns of 53BP1 and MDC1 are very similar to that of gammaH2AX, consistent with a structural link between these factors. Moreover, different subsets of telomeres signal in different cell lines, with those that signal tending to equate to the shortest telomeres of the corresponding cell line, thus linking telomere attrition with DNA-damage signalling. Notably, we find that, in some cases, gammaH2AX spreading is modulated in a manner suggesting that H2AX distribution or its ability to be phosphorylated is not uniform along the chromosome. Finally, we observe weak gammaH2AX signals at telomeres of proliferating cells, but not in hTERT immortalised cells, suggesting that low telomerase activity leads to telomere uncapping and senescence in proliferating primary cells.     

Subsenescent telomere lengths in fibroblasts immortalized by limiting amounts of telomerase

Human fibroblasts expressing the catalytic component of human telomerase (hTERT) have been followed for 250-400 population doublings. As expected, telomerase activity declined in long term culture of stable transfectants. Surprisingly, however, clones with average telomere lengths several kilobases shorter than those of senescent parental cells continued to proliferate. Although the longest telomeres shortened, the size of the shortest telomeres was maintained. Cells with subsenescent telomere lengths proliferated for an additional 20 doublings after inhibiting telomerase activity with a dominant-negative hTERT mutant. These results indicate that, under conditions of limiting telomerase activity, cis-acting signals may recruit telomerase to act on the shortest telomeres, argue against the hypothesis that the mortality stage 1 mechanism of cellular senescence is regulated by telomere positional effects (in which subtelomeric loci silenced by long telomeres are expressed when telomeres become short), and suggest that catalytically active telomerase is not required to provide a protein-capping role at the end of very short telomeres.     

Lysine residues in N-terminal and C-terminal regions of human histone H2A are targets for biotinylation by biotinidase

In eukaryotic cell nuclei, DNA associates with the core histones H2A, H2B, H3 and H4 to form nucleosomal core particles. DNA binding to histones is regulated by posttranslational modifications of N-terminal tails (e.g., acetylation and methylation of histones). These modifications play important roles in the epigenetic control of chromatin structure. Recently, evidence that biotinidase and holocarboxylase synthetase (HCS) catalyze the covalent binding of biotin to histones has been provided. The primary aim of this study was to identify biotinylation sites in histone H2A and its variant H2AX. Secondary aims were to determine whether acetylation and methylation of histone H2A affect subsequent biotinylation and whether biotinidase and HCS localize to the nucleus in human cells. Biotinylation sites were identified using synthetic peptides as substrates for biotinidase. These studies provided evidence that K9 and K13 in the N-terminus of human histones H2A and H2AX are targets for biotinylation and that K125, K127 and K129 in the C-terminus of histone H2A are targets for biotinylation. Biotinylation of lysine residues was decreased by acetylation of adjacent lysines but was increased by dimethylation of adjacent arginines. The existence of biotinylated histone H2A in vivo was confirmed by using modification-specific antibodies. Antibodies to biotinidase and HCS localized primarily to the nuclear compartment, consistent with a role for these enzymes in regulating chromatin structure. Collectively, these studies have identified five novel biotinylation sites in human histones; histone H2A is unique among histones in that its biotinylation sites include amino acid residues from the C-terminus.     

端粒及端粒酶活性的调控机制

广义的端粒由帽子、双链的串联重复序列的DNA核心部分及亚端粒构成,其结合蛋白是一个复合体,由TRF1、TRF2、TIN2、Pot1、TPP1、RAP1 6个亚单位组成;另外,还结合组蛋白的特定成分H3K9三甲基聚合体和H4K20三甲基聚合体。端粒酶主要由hTerc、hTert、dyskerin构成。端粒的功能主要受端粒酶的活性调控;而端粒酶活性主要受hTert及hTerc的转录水平和转录后的剪切、hTert的翻译等因素的调控。端粒与端粒酶结构和功能的异常与细胞衰老及肿瘤的发生、发展关系密切。     

Suppression of hPOT1 in diploid human cells results in an hTERT-dependent alteration of telomere length dynamics

POT1 is a 3' telomeric single-stranded overhang binding protein that has been implicated in chromosome end protection, the regulation of telomerase function, and defining the 5' chromosome terminus. In human cancer cells that exhibit constitutive hTERT activity, hPOT1 exerts control over telomere length. Primary human fibroblasts express low levels of catalytically active hTERT in an S-phase-restricted manner that fails to counteract telomere attrition with cell division. Here, we show that diploid human fibroblasts in which hPOT1 expression has been suppressed harbor telomeres that are longer than control cells. This difference in telomere length delays the onset of replicative senescence and is dependent on S-phase-restricted hTERT expression. These findings are consistent with the view that hPOT1 promotes a nonextendable telomere state resistant to extension by S-phase-restricted telomerase. Manipulating this function of hPOT1 may thus hasten the cytotoxic effects of telomerase inhibition.     

Transient expression of human telomerase extends the life span of normal human fibroblasts

We utilized the Cre/lox recombination system to transiently express the catalytic subunit of telomerase (hTERT) in normal diploid foreskin fibroblasts (BJ cells). A retroviral construct containing an hTERT cDNA, flanked by loxP-sites was introduced into near senescent BJ cells (population doubling 85). At population doubling (PD) 92, which exceeds the typical life span of these cells, we excised the gene via Cre-mediated recombination. All clones lost telomerase activity and showed telomere shortening over an additional 50 PDs. Interestingly, the average telomere length in these cells became shorter than in untreated BJ cells at senescence. This may be due to hTERT preferentially elongating the shortest telomeres, leading to greater length uniformity. In summary, transient telomerase expression and only a very small average telomere elongation by hTERT resulted in a 50% increase in life span of human fibroblasts. This suggests a potentially safe use of hTERT in tissue engineering.     

Holocarboxylase synthetase is a chromatin protein and interacts directly with histone H3 to mediate biotinylation of K9 and K18

Holocarboxylase synthetase (HCS) mediates the binding of biotin to lysine (K) residues in histones H2A, H3 and H4; HCS knockdown disturbs gene regulation and decreases stress resistance and lifespan in eukaryotes. We tested the hypothesis that HCS interacts physically with histone H3 for subsequent biotinylation. Co-immunoprecipitation experiments were conducted and provided evidence that HCS co-localizes with histone H3 in human cells; physical interactions between HCS and H3 were confirmed using limited proteolysis assays. Yeast two-hybrid (Y2H) studies revealed that the N-terminal and C-terminal domains in HCS participate in H3 binding. Recombinant human HCS was produced and exhibited biological activity, as evidenced by biotinylation of its known substrate, recombinant p67. Recombinant histone H3.2 and synthetic H3-based peptides were also good targets for biotinylation by recombinant HCS (rHCS) in vitro, based on tracing histone-bound biotin with [³H]biotin, streptavidin and anti-biotin antibody. Biotinylation site-specific antibodies were generated and revealed that both K9 and K18 in H3 were biotinylated by HCS. Collectively, these studies provide conclusive evidence that HCS interacts directly with histone H3, causing biotinylation of K9 and K18. We speculate that the targeting of HCS to distinct regions in human chromatin is mediated by DNA sequence, biotin, RNA, epigenetic marks or chromatin proteins.     

Telomerase prevents accelerated senescence in glucose-6-phosphate dehydrogenase (G6PD)-deficient human fibroblasts

Fibroblasts derived from glucose-6-phosphate dehydrogenase (G6PD)-deficient patients display retarded growth and accelerated cellular senescence that is attributable to increased accumulation of oxidative DNA damage and increased sensitivity to oxidant-induced senescence, but not to accelerated telomere attrition. Here, we show that ectopic expression of hTERT stimulates telomerase activity and prevents accelerated senescence in G6PD-deficient cells. Stable clones derived from hTERT-expressing normal and G6PD-deficient fibroblasts have normal karyotypes, and display no sign of senescence beyond 145 and 105 passages, respectively. Activation of telomerase, however, does not prevent telomere attrition in earlier-passage cells, but does stabilize telomere lengths at later passages. In addition, we provide evidence that ectopic expression of hTERT attenuates the increased sensitivity of G6PD-deficient fibroblasts to oxidant-induced senescence. These results suggest that ectopic expression of hTERT, in addition to acting in telomere length maintenance by activating telomerase, also functions in regulating senescence induction.     

The role of holocarboxylase synthetase in genome stability is mediated partly by epigenomic synergies between methylation and biotinylation events

Holocarboxylase synthetase (HLCS) catalyzes the covalent binding of biotin to histones. Biotinylated histones are gene repression marks and are particularly enriched in long terminal repeats, telomeres, and other repeat regions. The effects of HLCS in gene regulation are mediated by its physical interactions with chromatin proteins such as histone H3, DNMT1, MeCP2, and EHMT-1. It appears that histone biotinylation depends on prior methylation of cytosines. De-repression of long terminal repeats in biotin- or HLCS-deficient cell cultures and organisms is associated with genome instability.     

Identification of p38MAPK-dependent genes with changed transcript abundance in H2O2-induced premature senescence of IMR-90 hTERT human fibroblasts

Premature senescence of IMR-90 human diploid fibroblasts expressing telomerase (hTERT) establishes after exposure to an acute sublethal concentration of H2O2. We showed herein that p38(MAPK) was phosphorylated after exposure of IMR-90 hTERT cells to H2O2. Selective inhibition of p38(MAPK) activity attenuated the increase in the proportion of cells positive for senescence associated beta-galactosidase activity. We generated a low density DNA array to study gene expression profiles of 240 senescence-related genes. Using this array, p38(MAPK) inhibitor and p38(MAPK) small interferent RNA, we identified several p38(MAPK)-target genes differentially expressed in H2O2-stressed IMR-90 hTERT fibroblasts.     

K8 and K12 are biotinylated in human histone H4.

Folding of DNA into chromatin is mediated by binding to histones such as H4; association of DNA with histones is regulated by covalent histone modifications, e.g. acetylation, methylation, and biotinylation. We sought to identify amino-acid residues that are biotinylated in histone H4, and to determine whether acetylation and methylation of histones affect biotinylation. Synthetic peptides spanning fragments of human histone H4 were biotinylated enzymatically using biotinidase. Peptide-bound biotin was probed with streptavidin-peroxidase. Peptides based on the N-terminal sequence of histone H4 were effectively recognized by biotinidase as substrates for biotinylation; in contrast, peptides based on the C-terminal sequences were not biotinylated. Substitution of K8 or K12 with alanine or arginine decreased biotinylation, suggesting that these lysines are targets for biotinylation; K8 and K12 are also known targets for acetylation. Chemical acetylation or methylation of a given lysine decreased subsequent enzymatic biotinylation of neighboring lysines, consistent with cross-talk among histone modifications. Substitution of a given lysine (positive charge) with glutamate (negative charge) abolished biotinylation of neighboring lysines, providing evidence that the net charge of histones has a role in biotinylation. An antibody was generated that specifically recognized histone H4 biotinylated at K12. This antibody was used to detect biotinylated histone H4 in nuclear extracts from human cells. These studies suggest that K8 and K12 in histone H4 are targets for biotinylation, that acetylation and biotinylation compete for the same binding sites, and that acetylation and methylation of histones affect biotinylation of neighboring lysines.     

Reversible manipulation of telomerase expression and telomere length. Implications for the ionizing radiation response and replicative senescence of human cells

Most human cells do not express telomerase and irreversibly arrest proliferation after a finite number of divisions (replicative senescence). Several lines of evidence suggest that replicative senescence is caused by short dysfunctional telomeres, which arise when DNA is replicated in the absence of adequate telomerase activity. We describe a method to reversibly bypass replicative senescence and generate mass cultures that have different average telomere lengths. A retrovirus carrying hTERT flanked by excision sites for Cre recombinase rendered normal human fibroblasts telomerase-positive and replicatively immortal. Superinfection with retroviruses carrying wild-type or mutant forms of TIN2, a negative regulator of telomere length, created telomerase-positive, immortal populations with varying average telomere lengths. Subsequent infection with a Cre-expressing retrovirus abolished telomerase activity, creating mortal cells with varying telomere lengths. Using these cell populations, we show that, after hTERT excision, cells senesce with shorter telomeres than parental cells. Moreover, long telomeres, but not telomerase, protected cells from the loss of division potential caused by ionizing radiation. Finally, although telomerase-negative cells with short telomeres senesced after fewer doublings than those with long telomeres, telomere length per se did not correlate with senescence. Our results support a role for telomere structure, rather than length, in replicative senescence.     

Catalytically active human telomerase mutants with allele-specific biological properties

Expression of the catalytic subunit of human telomerase, hTERT, extends human primary fibroblast life span. Such life span extension has generally been reported to be accompanied by net telomere lengthening, which led to the hypothesis that it is the telomere lengthening that causes the life span extension. Here we show that hTERT+C and hTERT-FlagC, mutant telomerase proteins with either 10 additional residues or a FLAG epitope added to the hTERT C-terminus, confer significant but limited life span extension to IMR90 human primary lung fibroblasts. However, as the cells continue to grow for >100 population doublings past their normal senescence point, bulk telomere length continues to erode to lengths much shorter than those seen at the senescence of control telomerase-negative cells. Expression of hTERT+C immortalized IMR90 cells transformed by three different oncogenes. Again, bulk telomeres became much shorter than those of the control cells at crisis. Additional hTERT mutants were constructed and analyzed similarly. Enzymatically active hTERT-N125A+T126A, like other previously reported conserved GQ domain mutants and C-terminally HA-tagged hTERT, failed to extend life span. Another GQ domain mutant, hTERT-E79A, was indistinguishable from wild-type hTERT in its cell growth effects, but there was no net telomere lengthening. These results uncover further hTERT allele-specific phenotypes that uncouple telomerase activity, net telomere lengthening and life span extension.     

Telomerase expression prevents replicative senescence but does not fully reset mRNA expression patterns in Werner syndrome cell strains.

Reduced replicative capacity is a consistent characteristic of cells derived from patients with Werner syndrome. This premature senescence is phenotypically similar to replicative senescence observed in normal cell strains and includes altered cell morphology and gene expression patterns. Telomeres shorten with in vitro passaging of both WRN and normal cell strains; however, the rate of shortening has been reported to be faster in WRN cell strains, and the length of telomeres in senescent WRN cells appears to be longer than that observed in normal strains, leading to the suggestion that senescence in WRN cell strains may not be exclusively associated with telomere effects. We report here that the telomere restriction fragment length in senescent WRN fibroblasts cultures is within the size range observed for normal fibroblasts strains and that the expression of a telomerase transgene in WRN cell strains results in lengthened telomeres and replicative immortalization, thus indicating that telomere effects are the predominant trigger of premature senescence in WRN cells. Microarray analyses showed that mRNA expression patterns induced in senescent WRN cells appeared similar to those in normal strains and that hTERT expression could prevent the induction of most of these genes. However, substantial differences in expression were seen in comparisons of early-passage and telomerase-immortalized derivative lines, indicating that telomerase expression does not prevent the phenotypic drift, or destabilized genotype, resulting from the WRN defect.     

Holocarboxylase synthetase regulates expression of biotin transporters by chromatin remodeling events at the SMVT locus

The sodium-dependent multivitamin transporter (SMVT) is essential for mediating and regulating biotin entry into mammalian cells. In cells, biotin is covalently linked to histones in a reaction catalyzed by holocarboxylase synthetase (HCS); biotinylation of lysine 12-biotinylated histone H4 (K12Bio H4) causes gene silencing. Here, we propose a novel role for HCS in sensing and regulating levels of biotin in eukaryotic cells. We hypothesized that nuclear translocation of HCS increases in response to biotin supplementation; HCS then biotinylates histone H4 at SMVT promoters, silencing biotin transporter genes. Jurkat lymphoma cells were cultured in media containing 0.025, 0.25, or 10 nmol/l biotin. The nuclear translocation of HCS correlated with biotin concentrations in media; the relative enrichment of both HCS and K12Bio H4 at SMVT promoter 1 (but not promoter 2) increased by 91% in cells cultured in medium containing 10 nmol/l biotin compared with 0.25 nmol/l biotin. This increase of K12Bio H4 at the SMVT promoter decreased SMVT expression by up to 86%. Biotin homeostasis by HCS-dependent chromatin remodeling at the SMVT promoter 1 locus was disrupted in HCS knockdown cells, as evidenced by abnormal chromatin structure (K12Bio H4 abundance) and increased SMVT expression. The findings from this study are consistent with the theory that HCS senses biotin, and that biotin regulates its own cellular uptake by participating in HCS-dependent chromatin remodeling events at the SMVT promoter 1 locus in Jurkat cells.