Suv4-20h Abrogation Enhances Telomere Elongation during Reprogramming and Confers a Higher Tumorigenic Potential to iPS Cells

Reprogramming of adult differentiated cells to induced pluripotent stem cells (iPS) cells has been achieved by over-expression of specific transcription factors. Nuclear reprogramming induces a series of profound changes at the telomeres of the parental differentiated cells, including a telomerase-dependent telomere elongation and the remodeling of telomeric chromatin. In particular, iPS cells show a decreased density of H4K20me3 heterochromatic mark at telomeres compared to the parental cells. Suv4-20h1 and Suv4-20h2 histone methytransferases (HMTases) are responsible for the trimethylation of H4K20 at telomeres, as cells deficient for both HMTases show decreased levels of H4K20me3 at telomeric chromatin. Here, we set to address the role of the Suv4-20h enzymes in telomere reprogramming by generating bona-fide iPS cells from mouse embryonic fibroblasts (MEFs) double null for both HMTases (Suv4-20dn MEFs). We found that Suv4-20h deficiency enhances telomere elongation during reprogramming without altering their ability to protect the chromosome ends or the efficiency of reprogramming. Moreover, teratomas generated from Suv4-20dn iPS cells also have elongated telomeres and an increased growth rate when compared to wild-type controls. These results indicate that abrogation of Suv4-20h enzymes and loss of heterochromatic mark H4K20me3 at telomeric heterochromatin facilitates telomere reprogramming and provides an increased tumorigenic potential to the resulting iPS cells

A Subpopulation of Adult Skeletal Muscle Stem Cells Retains All Template DNA Strands after Cell Division

SummarySatellite cells are adult skeletal muscle stem cells that are quiescent and constitute a poorly defined heterogeneous population. Using transgenic Tg:Pax7-nGFP mice, we show that Pax7-nGFPHi cells are less primed for commitment and have a lower metabolic status and delayed first mitosis compared to Pax7-nGFPLo cells. Pax7-nGFPHi can give rise to Pax7-nGFPLo cells after serial transplantations. Proliferating Pax7-nGFPHi cells exhibit lower metabolic activity, and the majority performs asymmetric DNA segregation during cell division, wherein daughter cells retaining template DNA strands express stem cell markers. Using chromosome orientation-fluorescence in situ hybridization, we demonstrate that all chromatids segregate asymmetrically, whereas Pax7-nGFPLo cells perform random DNA segregation. Therefore, quiescent Pax7-nGFPHi cells represent a reversible dormant stem cell state, and during muscle regeneration, Pax7-nGFPHi cells generate distinct daughter cell fates by asymmetrically segregating template DNA strands to the stem cell. These findings provide major insights into the biology of stem cells that segregate DNA asymmetrically

A Subpopulation of Adult Skeletal Muscle Stem Cells Retains All Template DNA Strands after Cell Division

SummarySatellite cells are adult skeletal muscle stem cells that are quiescent and constitute a poorly defined heterogeneous population. Using transgenic Tg:Pax7-nGFP mice, we show that Pax7-nGFPHi cells are less primed for commitment and have a lower metabolic status and delayed first mitosis compared to Pax7-nGFPLo cells. Pax7-nGFPHi can give rise to Pax7-nGFPLo cells after serial transplantations. Proliferating Pax7-nGFPHi cells exhibit lower metabolic activity, and the majority performs asymmetric DNA segregation during cell division, wherein daughter cells retaining template DNA strands express stem cell markers. Using chromosome orientation-fluorescence in situ hybridization, we demonstrate that all chromatids segregate asymmetrically, whereas Pax7-nGFPLo cells perform random DNA segregation. Therefore, quiescent Pax7-nGFPHi cells represent a reversible dormant stem cell state, and during muscle regeneration, Pax7-nGFPHi cells generate distinct daughter cell fates by asymmetrically segregating template DNA strands to the stem cell. These findings provide major insights into the biology of stem cells that segregate DNA asymmetrically

Stem and progenitor cell division kinetics during postnatal mouse mammary gland development.

The cycling properties of mammary stem and progenitor cells is not well understood. To determine the division properties of these cells, we administered synthetic nucleosides for varying periods of time to mice at different stages of postnatal development and monitored the rate of uptake of these nucleosides in the different mammary cell compartments. Here we show that most cell division in the adult virgin gland is restricted to the oestrogen receptor-expressing luminal cell lineage. Our data also demonstrate that the oestrogen receptor-expressing, milk and basal cell subpopulations have telomere lengths and cell division kinetics that are not compatible with these cells being hierarchically organized; instead, our data indicate that in the adult homeostatic gland, each cell type is largely maintained by its own restricted progenitors. We also observe that transplantable stem cells are largely quiescent during oestrus, but are cycling during dioestrus when progesterone levels are high.We thank the members of Stingl lab, Doug Winton, Jason Carroll, Robert Clarke, Phil Jones and Hamid Raza Ali for scientific discussions. We thank the core facilities at the Cancer Research UK-CI for enabling experiments. In particular, Loic Tauzin, Nina Lane and Mateuz Strzelecki for assistance with cell sorting; the Biological Resources Unit for animal husbandry; and Histopathology staff, in particular Leigh-Anne McDuffus and Cara Walters. J. Stingl’s laboratory acknowledges the support of The University of Cambridge, Cancer Research UK (core grant number C14303/A17197) and Hutchison Whampoa Limited. M.A. Blasco’s laboratory is funded by the Spanish Ministry of Economy and Competitiveness Project SAF2013-45111RETOS, the European Union FP7 Project EUROBATS, the European Research Council (ERC) Project TEL STEM CELL (GA#232854), the Regional Government of Madrid project 2+2 ReCaRe, the AXA Research Fund and the Fundación Botín.This is the final version of the article. It was first available from Nature via http://dx.doi.org/10.1038/ncomms948

Altered telomere homeostasis and resistance to skin carcinogenesis in Suv39h1 transgenic mice

The Suv39h1 and Suv39h2 H3K9 histone methyltransferases (HMTs) have a conserved role in the formation of constitutive heterochromatin and gene silencing. Using a transgenic mouse model system we demonstrate that elevated expression of Suv39h1 increases global H3K9me3 levels in vivo. More specifically, Suv39h1 overexpression enhances the imposition of H3K9me3 levels at constitutive heterochromatin at telomeric and major satellite repeats in primary mouse embryonic fibroblasts. Chromatin compaction is paralleled by telomere shortening, indicating that telomere length is controlled by H3K9me3 density at telomeres. We further show that increased Suv39h1 levels result in an impaired clonogenic potential of transgenic epidermal stem cells and Ras/E1A transduced transgenic primary mouse embryonic fibroblasts. Importantly, Suv39h1 overexpression in mice confers resistance to a DMBA/TPA induced skin carcinogenesis protocol that is characterized by the accumulation of activating H-ras mutations. Our results provide genetic evidence that Suv39h1 controls telomere homeostasis and mediates resistance to oncogenic stress in vivo. This identifies Suv39h1 as an interesting target to improve oncogene induced senescence in premalignant lesions

Therapeutic effects of telomerase in mice with pulmonary fibrosis induced by damage to the lungs and short telomeres

Pulmonary fibrosis is a fatal lung disease characterized by fibrotic foci and inflammatory infiltrates. Short telomeres can impair tissue regeneration and are found both in hereditary and sporadic cases. We show here that telomerase expression using AAV9 vectors shows therapeutic effects in a mouse model of pulmonary fibrosis owing to a low-dose bleomycin insult and short telomeres. AAV9 preferentially targets regenerative alveolar type II cells (ATII). AAV9-Tert-treated mice show improved lung function and lower inflammation and fibrosis at 1-3 weeks after viral treatment, and improvement or disappearance of the fibrosis at 8 weeks after treatment. AAV9-Tert treatment leads to longer telomeres and increased proliferation of ATII cells, as well as lower DNA damage, apoptosis, and senescence. Transcriptome analysis of ATII cells confirms downregulation of fibrosis and inflammation pathways. We provide a proof-of-principle that telomerase activation may represent an effective treatment for pulmonary fibrosis provoked or associated with short telomeres.We are indebted to D Megias for microscopy analysis, to J Mun˜ oz and F Garcı´a for hydroxiproline analysis as well as to CNIO Histopathological Unit. The research was funded by project SAF2013- 45111-R of Societal Changes Programme of the Spanish Ministry of Economics and Competitiveness (MINECO) co-financed through the European Fund of Regional Development (FEDER), Fundacio´n Botı´n and Banco Santander (Santander Universities Global Division) and Roche Extending the Innova- tion Network Program (EIN) Academia Partnering Programme.S

Secondary structure of vertebrate telomerase RNA

Telomerase is a ribonucleoprotein enzyme that maintains telomere length by adding telomeric sequence repeats onto chromosome ends. The essential RNA component of telomerase provides the template for telomeric repeat synthesis. To determine the secondary structure of vertebrate telomerase RNA, 32 new telomerase RNA genes were cloned and sequenced from a variety of vertebrate species including 18 mammals, 2 birds, 1 reptile, 7 amphibians, and 4 fishes. Using phylogenetic comparative analysis, we propose a secondary structure that contains four structural domains conserved in all vertebrates. Ten helical regions of the RNA are universally conserved while other regions vary significantly in length and sequence between different classes of vertebrates. The proposed vertebrate telomerase RNA structure displays a strikingly similar topology to the previously determined ciliate telomerase RNA structure, implying an evolutionary conservation of the global architecture of telomerase RNA

Functional characterization and developmental regulation of mouse telomerase RNA

Telomerase synthesizes telomeric DNA repeats onto chromosome ends de novo. The mouse telomerase RNA component was cloned and contained only 65 percent sequence identity with the human telomerase RNA. Alteration of the template region in vivo generated altered telomerase products. The shorter template regions of the mouse and other rodent telomerase RNAs could account for the shorter distribution of products (processivity) generated by the mouse enzyme relative to the human telomerase. Amounts of telomerase RNA increased in immortal cells derived from primary mouse fibroblasts. RNA was detected in all newborn mouse tissues tested but was decreased during postnatal development