Proper telomeric chromatin configuration is thought to be essential for telomere homeostasis and stability. Previous studies in mouse suggested that loss of heterochromatin marks at telomeres might favor onset of Alternative Lengthening of Telomeres (ALT) pathway, by promoting homologous recombination. However, analysis of chromatin status at human ALT telomeres has never been reported. Here, using isogenic human cell lines and cellular hybrids, which rely either on telomerase or ALT to maintain telomeres, we show that chromatin compaction is reduced at ALT telomeres and this is associated with a global decrease in telomeric H3K9me3. This, subsequently, leads to upregulation of telomere transcription. Accordingly, restoration of a more condensed telomeric chromatin through telomerase-dependent elongation of short ALT telomeres reduces telomere transcription. We further show that loss of ATRX chromatin remodeler function, a frequent characteristic of ALT cells, is not sufficient to decrease chromatin condensation at telomeres nor to increase the expression of telomeric RNA species. These results offer new insight on telomeric chromatin properties in ALT cells and support the hypothesis that telomeric chromatin decondensation is important for ALT pathway.

In the presence of functional DNA repair pathways, DNA double-strand breaks (DSBs) are mainly repaired by non-homologous end-joining (NHEJ) or homologous recombination (HR), two conserved pathways that protect cells from aberrant chromosomal rearrangements. During the past two decades however, unusual and presumably distinct DNA end-joining repair activities have been unraveled in NHEJ-deficient cells and these are likely to operate in various chromosomal contexts and species. Most alternative DNA end-joining events reported so far appear to involve microhomologous sequences and are likely to rely on a subset of HR enzymes, namely those responsible for the single-strand annealing mechanism of HR, and on DNA Ligase III. Usually, microhomologies are not initially present at DSB ends and thus need to be unmasked through DNA end resection, a process that can lead to extensive nucleotide loss and is therefore highly mutagenic. In addition to microhomology-mediated end-joining events, recent studies in mammalian cells point towards the existence of a distinct and still ill defined alternative end-joining pathway that does not appear to rely on pre-existing microhomologies and may possibly involve DNA Ligase I. Whether dependent on microhomologies or not, alternative DNA end-joining mechanisms are likely to be highly mutagenic in vivo, being able to drive telomere fusion events and cancer-associated chromosomal translocations in mouse models. In the future, it will be important to better characterize the genetic requirements of these mutagenic alternative mechanisms of DNA end-joining.

Methylation of histone H3 on lysine 9 or 27 is crucial for heterochromatin formation. Previously considered hallmarks of, respectively, constitutive and facultative heterochromatin, recent evidence has accumulated in favor of coexistence of these two marks and their cooperation in gene silencing maintenance. H3K9me2/3 ensures anchorage at chromatin of heterochromatin protein 1α (HP1α), a main component of heterochromatin. HP1α chromoshadow domain, involved in dimerization and interaction with partners, has additional but still unclear roles in HP1α recruitment to chromatin. Because of previously suggested links between polycomb repressive complex 2 (PRC2), which catalyzes H3K27 methylation, and HP1α, we tested whether PRC2 may regulate HP1α abundance at chromatin. We found that the EZH2 and SUZ12 subunits of PRC2 are required for HP1α stability, as knockdown of either protein led to HP1α degradation. Similar results were obtained upon overexpression of H3K27me2/3 demethylases. We further showed that binding of HP1α/β/γ to H3K9me3 peptides is greatly increased in the presence of H3K27me3, and this is dependent on PRC2. These data fit with recent proteomic studies identifying PRC2 as an indirect H3K9me3 binder in mouse tissues and suggest the existence of a cooperative mechanism of HP1α anchorage at chromatin involving H3 methylation on both K9 and K27 residues.

Background: Recently, periostin (POSTN), a gene encoding a protein with similarity to the fasciclin family and involved in cell survival and angiogenesis, has emerged as a promising marker for tumor progression in various types of human cancers. There is some controversy regarding both POSTN expression levels and the nature of periostin-producing cells within tumors. In this study, we used quantitative RT-PCR to assess periostin gene expression in normal tissues, primary cell cultures, tumor tissues and tumor cell lines. Results: Periostin expression levels are highly variable in both normal tissues and tumors and strong POSTN overexpression is mostly detected in tumors from pancreas and liver. POSTN is not expressed in blood cancers. In melanoma samples, average periostin expression is not increased in primary tumors whereas POSTN overexpression was detected in about 60% of melanoma metastatic tumors in the liver or lymph nodes. Identification of the cellular source of periostin production in melanoma metastases -cancer cells or stroma- was assessed by comparing periostin expression in 23 newly-established melanoma cell lines and matched tumors. In contrast to the reduction by more than 99% of COL6A3 stromal marker mRNA in all cell lines, significant POSTN transcription was maintained in some melanoma cell lines, suggesting that both stromal cells and melanoma cells express periostin. The high level of periostin expression in primary cultures of skin fibroblasts suggests that fibroblasts may contribute for a large part to periostin production in melanoma-associated stroma. On the other hand, periostin expression in melanoma cells is probably acquired during the tumorigenic process as 1) normal melanocytes do not express POSTN and 2) melanoma cells from distinct metastases of the same patient were associated with very different levels of periostin expression. Conclusion: Our comparative analysis suggests that, although periostin overexpression is clearly detected in some cancers, it is not a general feature of tumors. In melanoma, our study identifies both stromal and melanoma cells as sources of periostin production and correlates POSTN expression levels with increased primary tumor thickness and metastatic process development.

The AMPK/PGC-1α metabolic pathway and nuclear respiratory factor 1 up-regulate human telomere transcription.