Expression and differential regulation of human TERRA at several chromosome ends.

The telomeric long noncoding RNA TERRA has been implicated in regulating telomere maintenance by telomerase and homologous recombination, and in influencing telomeric protein composition during the cell cycle and the telomeric DNA damage response. TERRA transcription starts at subtelomeric regions resembling the CpG islands of eukaryotic genes extending toward chromosome ends. TERRA contains chromosome-specific subtelomeric sequences at its 5' end and long tracts of UUAGGG-repeats toward the 3' end. Conflicting studies have been published as to whether TERRA is expressed from one or several chromosome ends. Here, we quantify TERRA species by RT-qPCR in normal and several cancerous human cell lines. By using chromosome-specific subtelomeric DNA primers, we demonstrate that TERRA is expressed from a large number of telomeres. Deficiency in DNA methyltransferases leads to TERRA up-regulation only at the subset of chromosome ends that contain CpG-island sequences, revealing differential regulation of TERRA promoters by DNA methylation. However, independently of the differences in TERRA expression, short telomeres were uniformly present in a DNA methyltransferase deficient cell line, indicating that telomere length was not dictated by TERRA expression in cis Bioinformatic analyses indicated the presence of a large number of putative transcription factors binding sites at TERRA promoters, and we identified a subset of them that repress TERRA expression. Altogether, our study confirms that TERRA corresponds to a large gene family transcribed from multiple chromosome ends where we identified two types of TERRA promoters, only one of which is regulated by DNA methylation.

Premature activation of Cdk1 leads to mitotic events in S phase and embryonic lethality.

Cell cycle regulation, especially faithful DNA replication and mitosis, are crucial to maintain genome stability. Cyclin-dependent kinase (CDK)/cyclin complexes drive most processes in cellular proliferation. In response to DNA damage, cell cycle surveillance mechanisms enable normal cells to arrest and undergo repair processes. Perturbations in genomic stability can lead to tumor development and suggest that cell cycle regulators could be effective targets in anticancer therapy. However, many clinical trials ended in failure due to off-target effects of the inhibitors used. Here, we investigate in vivo the importance of WEE1- and MYT1-dependent inhibitory phosphorylation of mammalian CDK1. We generated Cdk1AF knockin mice, in which two inhibitory phosphorylation sites are replaced by the non-phosphorylatable amino acids T14A/Y15F. We uncovered that monoallelic expression of CDK1AF is early embryonic lethal in mice and induces S phase arrest accompanied by γH2AX and DNA damage checkpoint activation in mouse embryonic fibroblasts (MEFs). The chromosomal fragmentation in Cdk1AF MEFs does not rely on CDK2 and is partly caused by premature activation of MUS81-SLX4 structure-specific endonuclease complexes, as well as untimely onset of chromosome condensation followed by nuclear lamina disassembly. We provide evidence that tumor development in liver expressing CDK1AF is inhibited. Interestingly, the regulatory mechanisms that impede cell proliferation in CDK1AF expressing cells differ partially from the actions of the WEE1 inhibitor, MK-1775, with p53 expression determining the sensitivity of cells to the drug response. Thus, our work highlights the importance of improved therapeutic strategies for patients with various cancer types and may explain why some patients respond better to WEE1 inhibitors.