Many Saccharomyces telomeres bear one or more copies of the repetitive Y element followed by ϳ350 bp of telomerase-generated C 1-3 A/TG 1-3 repeats. Although most cells lacking a gene required for the telomerase pathway die after 50 to 100 cell divisions, survivors arise spontaneously in such cultures. These survivors have one of two distinct patterns of telomeric DNA (V. Lundblad and E. H. Blackburn, Cell 73:347-360, 1993). The more common of the two patterns, seen in type I survivors, is tandem amplification of Y followed by very short tracts of C 1-3 A/TG 1-3 DNA. By determining the structure of singly tagged telomeres, chromosomes in type II survivors were shown to end in very long and heterogeneous-length tracts of C 1-3 A/TG 1-3 DNA, with some telomeres having 12 kb or more of C 1-3 A/TG 1-3 repeats. Maintenance of these long telomeres required the continuous presence of Rad52p. Whereas type I survivors often converted to the type II structure of telomeric DNA, the type II pattern was maintained for at least 250 cell divisions. However, during outgrowth, the structure of type II telomeres was dynamic, displaying gradual shortening as well as other structural changes that could be explained by continuous gene conversion events with other telomeres. Although most type II survivors had a growth rate similar to that of telomerase-proficient cells, their telomeres slowly returned to wild-type lengths when telomerase was reintroduced. The very long and heterogeneous-length telomeres characteristic of type II survivors in Saccharomyces are reminiscent of the telomeres in immortal human cell lines and tumors that maintain telomeric DNA in the absence of telomerase.

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Type II survivors arise in Saccharomyces cells lacking telomerase by a recombinational pathway that results in very long and heterogeneous length telomeres. Here we show that type II telomeres appeared abruptly in a population of cells with very short telomeres. Once established, these long telomeres progressively shortened. Short telomeres were substrates for rare, one-step lengthening events. The generation of type II survivors was absolutely Rad50p dependent. In a telomerase-proficient cell, the telomere-binding Rif proteins inhibited telomerase lengthening of telomeres. In a telomerase-deficient strain, Rif proteins, especially Rif2p, inhibited type II recombination. These data argue that only short telomeres are substrates for type II recombination and suggest that the donor for this recombination is not a chromosomal telomere.

BackgroundHypoxia induces the epithelial-mesenchymal transition, EMT, to promote cancer metastasis. In addition to transcriptional regulation mediated by hypoxia-inducible factors, HIFs, other epigenetic mechanisms of gene regulation, such as histone modifications and DNA methylation, are utilized under hypoxia. However, whether DNA demethylation mediated by TET1, a DNA dioxygenase converting 5-methylcytosine, 5mC, into 5-hydroxymethylcytosine, 5hmC, plays a role in hypoxia-induced EMT is largely unknown.ResultsWe show that TET1 regulates hypoxia-responsive gene expression. Hypoxia/HIF-2α regulates the expression of TET1. Knockdown of TET1 mitigates hypoxia-induced EMT. RNA sequencing and 5hmC sequencing identified the set of TET1-regulated genes. Cholesterol metabolic process genes are among the genes that showed high prevalence and statistical significance. We characterize one of the genes, INSIG1 (insulin induced gene 1), to confirm its expression and the 5hmC levels in its promoter. Knockdown of INSIG1 also mitigates hypoxia-induced EMT. Finally, TET1 is shown to be a transcriptional co-activator that interacts with HIF-1α and HIF-2α to enhance their transactivation activity independent of its enzymatic activity. TET1 acts as a co-activator to further enhance the expression of INSIG1 together with HIF-2α. We define the domain in HIF-1α that interacts with TET1 and map the domain in TET1 that confers transactivation to a 200 amino acid region that contains a CXXC domain. The TET1 catalytically inactive mutant is capable of rescuing hypoxia-induced EMT in TET1 knockdown cells.ConclusionsThese findings demonstrate that TET1 serves as a transcription co-activator to regulate hypoxia-responsive gene expression and EMT, in addition to its role in demethylating 5mC.Electronic supplementary materialThe online version of this article (doi:10.1186/s13059-014-0513-0) contains supplementary material, which is available to authorized users.

Intratumoural hypoxia induces HIF-1α and promotes tumour progression, metastasis and treatment resistance. HIF-1α stability is regulated by VHL-E3 ligase-mediated ubiquitin-dependent degradation; however, the hypoxia-regulated deubiquitinase that stabilizes HIF-1α has not been identified. Here we report that HAUSP (USP7) deubiquitinase deubiquitinates HIF-1α to increase its stability, induce epithelial-mesenchymal transition and promote metastasis. Hypoxia induces K63-linked polyubiquitinated HAUSP at lysine 443 to enhance its functions. Knockdown of HAUSP decreases acetylation of histone 3 lysine 56 (H3K56Ac). K63-polyubiquitinated HAUSP interacts with a ubiquitin receptor CBP to specifically mediate H3K56 acetylation. ChIP-seq analysis of HAUSP and HIF-1α binding reveals two motifs responsive to hypoxia. HectH9 is the E3 ligase for HAUSP and a prognostic marker together with HIF-1α. This report demonstrates that hypoxia-induced K63-polyubiquitinated HAUSP deubiquitinates HIF-1α and causes CBP-mediated H3K56 acetylation on HIF-1α target gene promoters to promote EMT/metastasis, further defining HAUSP as a therapeutic target in hypoxia-induced tumour progression.