Nicholas D Allen

Duncan M Baird
email: bairddm@cardiff.ac.uk

Telomere length maintenance in stem cell populations

Keywords: Telomere, Telomerase, senescence, genome stability, Stem cell

The maintenance of telomere length is essential for upholding the integrity of the genome. There is good evidence to suggest that telomere length maintenance in stem cell populations is important to facilitate the cell division required for tissue homeostasis. This is balanced against the requirement in long lived species for proliferative life span barriers for tumour suppression; the gradual erosion of telomeres provides one such barrier. The dynamics of telomeres in stem cell populations may thus be crucial in the balance between tumour suppression and tissue homeostasis. Here we briefly discuss our current understanding of telomere dynamics in stem cell populations, and provide some data to indicate that telomeres in human embryonic stem cells may be more stable and less prone to large-scale stochastic telomeric deletion.

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The importance of telomere length in stem cell populations.

The regenerative capacity of human tissues declines with age and the incidence of cancers increases; both these processes may be driven by a decline in stem cell function [START_REF] Maslov | Neural stem cell detection, characterization, and age-related changes in the subventricular zone of mice[END_REF][START_REF] Morrison | The aging of hematopoietic stem cells[END_REF][START_REF] Chen | Genetic regulation of primitive hematopoietic stem cell senescence[END_REF][START_REF] De Haan | Mouse strain-dependent changes in frequency and proliferation of hematopoietic stem cells during aging: correlation between lifespan and cycling activity[END_REF][START_REF] Kuhn | Neurogenesis in the dentate gyrus of the adult rat: age-related decrease of neuronal progenitor proliferation[END_REF]. There has to be a balance, in long-lived organisms such as humans, between maintaining regenerative potential on one hand, and tumour suppression on the other. Several studies focusing on p16 INK4A , a key mediator of replicative senescence, suggest that this may be the case. In the bone marrow of mice, p16 INK4A levels increase with age in haematopoietic stem cells, but not in other cell types [START_REF] Janzen | Stem-cell ageing modified by the cyclindependent kinase inhibitor p16INK4a[END_REF]; this correlated with a decline as a function of age, in the ability of these cells to reconstitute the immune system of irradiated mice, whereas p16 INK4A -/-mice had more stem cells and were better able to reconstitute an immune system. Similar observations were made in neurons from the forebrain, where again p16 INK4A levels increased with age, which correlated with a decline in proliferation, this was to some extent ameliorated in p16 INK4A -/-mice [START_REF] Molofsky | Increasing p16INK4a expression decreases forebrain progenitors and neurogenesis during ageing[END_REF]. As p16 INK4A is a tumour suppressor, the deficiency of protein leads to an increased frequency of cancer [START_REF] Sharpless | Loss of p16Ink4a with retention of p19Arf predisposes mice to tumorigenesis[END_REF], and yet these data show that the absence of p16 INK4A can slow most aspects of stem cell ageing, thus highlighting the balance between tumour suppression and regenerative capacity. This is likely to result from multiple factors such as Bmi1 that promote proliferation, balanced against tumour suppression for example by p16 INK4A [START_REF] Jacobs | The oncogene and Polycomb-group gene bmi-1 regulates cell proliferation and senescence through the ink4a locus[END_REF][START_REF] Molofsky | Bmi-1 promotes neural stem cell self-renewal and neural development but not mouse growth and survival by repressing the p16Ink4a and p19Arf senescence pathways[END_REF][START_REF] Molofsky | Bmi-1 dependence distinguishes neural stem cell self-renewal from progenitor proliferation[END_REF]. One mechanism that may contribute to fine tune this balance may be telomere length, whereby stem cells may need to maintain telomeres at a length that provides sufficient replicative capacity for tissue homeostasis, verses the requirement to minimise telomere length and replicative capacity as a tumour suppressive mechanism.

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Telomere length is an important determinant of telomeric function, in the presence of a functional DNA damage response short telomeres elicit a DNA damage checkpoint, that leads to either replicative senescence or apoptosis [START_REF] Di Fagagna | A DNA damage checkpoint response in telomereinitiated senescence[END_REF]. Telomere-dependent replicative senescence represents the tumour suppressive function of telomeres [START_REF] Feldser | Short telomeres limit tumor progression in vivo by inducing senescence[END_REF][START_REF] Rodier | Cancer and aging: the importance of telomeres in genome maintenance[END_REF]; the corollary of which may be an age-related accumulation of senescent cells [START_REF] Jeyapalan | Accumulation of senescent cells in mitotic tissue of aging primates[END_REF][START_REF] Herbig | Cellular senescence in aging primates[END_REF]. The presence of senescent cells not only reduces the proportion of mitotically active cells but may also, but by virtue of exhibiting a more catabolic phenotype they can actively degrade the tissue microenvironment [START_REF] Campisi | Senescent cells, tumor suppression, and organismal aging: good citizens, bad neighbors[END_REF]. Interestingly even the presence of a small proportion of senescent cells, may render the tissue microenvironment more permissive for tumour progression [START_REF] Krtolica | Senescent fibroblasts promote epithelial cell growth and tumorigenesis: a link between cancer and aging[END_REF]. The importance of telomere length from the standpoint of ageing and cancer is exemplified by the telomerase knock out mice. Where after several generations telomeres erode sufficiently to confer a phenotype, whereby the presence of short telomeres lead to defects in proliferative tissues that mirror some age-related phenotypes in humans. In addition to a shortened lifespan these include, immunosenescence, alopecia, hair greying and intestinal atrophy and increased rate of tumour formation [START_REF] Blasco | Telomere shortening and tumor formation by mouse cells lacking telomerase RNA[END_REF][START_REF] Herrera | Disease states associated with telomerase deficiency appear earlier in mice with short telomeres[END_REF][START_REF] Lee | Essential role of mouse telomerase in highly proliferative organs[END_REF][START_REF] Rudolph | Longevity, stress response, and cancer in aging telomerase-deficient mice[END_REF]. These phenotypes could be partially rescued in the context of mutations in the DNA damage checkpoint responses, for example the absence of p53 rescued some of the proliferative defects, but this was accompanied by an increase in the rate of tumour formation [START_REF] Chin | p53 deficiency rescues the adverse effects of telomere loss and cooperates with telomere dysfunction to accelerate carcinogenesis[END_REF]. Interestingly in the context of Terc and p21 mutations, the rescue of age related phenotypes was not accompanied by an increase in tumourigenesis, despite rescuing proliferative and self renewal detects in stem cell populations; these observations indicate that, p53/p21 dependent, telomere driven, replicative senescence was driving the age-related phenotypes, and furthermore, that apoptosis may be tumour protective [START_REF] Choudhury | Cdkn1a deletion improves stem cell function and lifespan of mice with dysfunctional telomeres without accelerating cancer formation[END_REF]. Some of the phenotypes exhibited by the telomerase knockout mice mirror those observed in individuals with dyskeratosis congenita

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(DC): a disease characterised by mutations in genes that result in impaired telomere length maintenance, such as components of the telomerase complex that results in haploinsufficiency for telomerase activity [START_REF] Vulliamy | The RNA component of telomerase is mutated in autosomal dominant dyskeratosis congenita[END_REF][START_REF] Vulliamy | Mutations in the telomerase component NHP2 cause the premature ageing syndrome dyskeratosis congenita[END_REF][START_REF] Heiss | X-linked dyskeratosis congenita is caused by mutations in a highly conserved gene with putative nucleolar functions[END_REF], or components of the shelterin complex [START_REF] Walne | TINF2 mutations result in very short telomeres: analysis of a large cohort of patients with dyskeratosis congenita and related bone marrow failure syndromes[END_REF]. DC patients exhibit reduced haematopoiesis, reduced number of haematopoietic progenitors and colony forming ability [START_REF] Marley | Evidence for a continuous decline in haemopoietic cell function from birth: application to evaluating bone marrow failure in children[END_REF][START_REF] Kirwan | Circulating haematopoietic progenitors are differentially reduced amongst subtypes of dyskeratosis congenita[END_REF], and chromosomal instability phenotypes [START_REF] Dokal | Dyskeratosis congenita fibroblasts are abnormal and have unbalanced chromosomal rearrangements[END_REF][START_REF] Dokal | Dyskeratosis congenita is a chromosomal instability disorder[END_REF] In addition, like telomerase knockout mice, DC patients display disease anticipation, whereby the severity and age of onset of the disease, get progressively worse from one generation to the next [START_REF] Vulliamy | Disease anticipation is associated with progressive telomere shortening in families with dyskeratosis congenita due to mutations in TERC[END_REF]. Failure of telomere length maintenance has been implicated in other bone marrow failure syndromes including Fanconi anaemia, Shwachman-Diamond syndrome, Diamond-Blackfan anaemia and aplastic anaemia [START_REF] Thornley | Abnormal telomere shortening in leucocytes of children with Shwachman-Diamond syndrome[END_REF][START_REF] Dokal | Inherited aplastic anaemias/bone marrow failure syndromes[END_REF][START_REF] Yamaguchi | Mutations in TERT, the gene for telomerase reverse transcriptase, in aplastic anemia[END_REF][START_REF] Ball | Progressive telomere shortening in aplastic anemia[END_REF]. Thus clinical data together with mouse models indicate telomere length maintenance is important in the context of human disease, the ageing process and cancer; it appears likely that telomere length maintenance in the stem cells compartments may play a crucial role in these processes.

Telomere length dynamics in human stem cell populations.

Data concerning telomere length and telomerase activity in stem cells is limited. Adult stem cell populations undergo cell division comparatively infrequently [START_REF] Fuchs | Socializing with the neighbors: stem cells and their niche[END_REF] and therefore, based on the end replication problem, stem cells would be predicted to suffer limited telomere erosion, irrespective of telomerase expression. The most extensively studied stem cells are those of the haematopoietic system. Haematopoietic stem cells (HSCs) are known to express telomerase [START_REF] Morrison | Telomerase activity in hematopoietic cells is associated with self-renewal potential[END_REF][START_REF] Broccoli | Telomerase activity in normal and malignant hematopoietic cells[END_REF] yet they appear to be subjected to telomere loss as a function of age [START_REF] Vaziri | Evidence for a mitotic clock in human hematopoietic stem cells: loss of telomeric DNA with age[END_REF]; telomerase activity is insufficient to maintain telomere length in HSCs and thus the proliferative potential of these cells may decline with age. However telomerase is up regulated following immune stimulation of B and T cells [START_REF] Norrback | Telomerase regulation and telomere dynamics in germinal centers[END_REF][START_REF] Norrback | Telomerase activation in normal B lymphocytes and non-Hodgkin's lymphomas[END_REF][START_REF] Hiyama | Activation of telomerase in human lymphocytes and hematopoietic progenitor cells[END_REF][START_REF] Maini | Virus-induced CD8+ T cell clonal expansion is associated with telomerase up-regulation and telomere length preservation: a mechanism for rescue from replicative senescence[END_REF][START_REF] Hodes | Telomeres in T and B cells[END_REF], this is sufficient to reduce the rate of telomere erosion, and

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ACCEPTED MANUSCRIPT some cases result in telomere lengthening [START_REF] Weng | Telomere lengthening and telomerase activation during human B cell differentiation[END_REF][START_REF] Hathcock | Induction of telomerase activity and maintenance of telomere length in virus-specific effector and memory CD8+ T cells[END_REF][START_REF] Plunkett | The flow cytometric analysis of telomere length in antigenspecific CD8+ T cells during acute Epstein-Barr virus infection[END_REF]. Importantly the up regulation of telomerase activity following stimulation, confers a proliferative lifespan extension compared to cells that do not express telomerase, this allows for repeated clonal expansions in response to the antigen [START_REF] Hathcock | Induction of telomerase activity and maintenance of telomere length in virus-specific effector and memory CD8+ T cells[END_REF]. However this is not unlimited and ultimately telomere erosion and the loss of proliferative potential may underlie some aspects of immunosenescence [START_REF] Plunkett | The flow cytometric analysis of telomere length in antigenspecific CD8+ T cells during acute Epstein-Barr virus infection[END_REF][START_REF] Akbar | Will telomere erosion lead to a loss of T-cell memory?[END_REF].

In the epidermis, telomerase is expressed by cells specifically on the basal layer [START_REF] Harle-Bachor | Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes[END_REF], yet telomere length decreases with age [START_REF] Lindsey | In vivo loss of telomeric repeats with age in humans[END_REF]. Spermatogonial stem cells express high levels of telomerase [START_REF] Kolquist | Expression of TERT in early premalignant lesions and a subset of cells in normal tissues[END_REF][START_REF] Wright | Telomerase activity in human germline and embryonic tissues and cells[END_REF][START_REF] Fujisawa | Telomerase activity in the testis of infertile patients with selected causes[END_REF], this is considered to maintain telomere length in the germline for subsequent generations. Indeed to date the male germline is the only tissue that has shown to exhibit telomere lengthening as a function of age [START_REF] Baird | Telomere instability in the male germline[END_REF][START_REF] Allsopp | Telomere length predicts replicative capacity of human fibroblasts[END_REF]. There is evidence to suggest that there is a gradient in telomere length from the stem cell compartments which decreases in more differentiated cell populations. This was exemplified by the lingual mucosa, where in situ hybridisation revealed the longest telomeres in the basel cells [START_REF] Aida | Basal cells have longest telomeres measured by tissue Q-FISH method in lingual epithelium[END_REF]. Further evidence using in situ methods in mice also showed distinct gradients of telomere length, with the longest telomeres observed in the stem cell compartments of the skin, small intestine, cornea, testis and brain [START_REF] Flores | The longest telomeres: a general signature of adult stem cell compartments[END_REF].

Of particular importance, this work included the observation that the telomere length in the stem cell compartments appear to shorten with age [START_REF] Flores | The longest telomeres: a general signature of adult stem cell compartments[END_REF]. Thus the evidence from humans and mice, indicates that stem cells maintain telomeres at a longer length, relative to the other cells within the tissue in which they reside. This is probably a function of a combination of telomerase activity together with a reduced cell turnover. However, with the exception of spermatogonial stem cells, some stem cell compartments have been shown to suffer telomere erosion as a function of age.

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Telomerase activity is likely to be limiting in most cells that express telomerase at physiological levels. Over expression of the RNA and protein components of telomerase individually does not result in significant telomere lengthening [START_REF] Cristofari | Telomere length homeostasis requires that telomerase levels are limiting[END_REF]. However when both components are over expressed simultaneously, these super-telomerase cells exhibit ongoing telomere lengthening with no apparent length control. These data imply that in cells where telomere length is stable telomerase levels are limiting, it is the shortest telomeres in the cells that are preferentially lengthened [START_REF] Cristofari | Telomere length homeostasis requires that telomerase levels are limiting[END_REF][START_REF] Teixeira | Telomere length homeostasis is achieved via a switch between telomerase-extendible and -nonextendible states[END_REF]. This must require a fine balance in the levels of telomerase activity to maintain telomere length; therefore subtly lower levels, such as may be observed in stem cell compartments, could lead to gradual telomere erosion. Whether telomere length would be become limiting in this situation is unclear; telomerase preferentially extends shorter telomeres [START_REF] Teixeira | Telomere length homeostasis is achieved via a switch between telomerase-extendible and -nonextendible states[END_REF] and thus telomeres could erode to a new shorter, but stable state, as observed in many cancer derived cell lines [START_REF] Cristofari | Telomere length homeostasis requires that telomerase levels are limiting[END_REF]. Furthermore mice that are haploinsufficient for either the RNA or protein components of telomerase maintain telomeres at a shorter length but do not exhibit obvious direct telomere defects, such as telomere fusion [START_REF] Hathcock | Haploinsufficiency of mTR results in defects in telomere elongation[END_REF][START_REF] Erdmann | Distinct dosage requirements for the maintenance of long and short telomeres in mTert heterozygous mice[END_REF].

Telomere instability

Telomeres shorten primarily as a consequence of gradual end-replication losses with ongoing cell division [START_REF] Olovnikov | Principle of marginotomy in template synthesis of polynucleotides[END_REF][START_REF] Levy | Telomere endreplication problem and cell aging[END_REF]. However more detailed analysis of telomere dynamics in human cells has indicated the existence of additional mechanisms that generate sporadic large-scale changes in telomere length [START_REF] Baird | Mechanisms of telomeric instability[END_REF]. In normal human cells, rare telomeres have been observed that lack signals from fluorescently labelled telomere repeat containing probes; these may arise as consequence of sporadic large-scale telomeric deletion events [START_REF] Callen | Breaks at telomeres and TRF2-independent end fusions in Fanconi anemia[END_REF][START_REF] Wang | Homologous recombination generates Tloop-sized deletions at human telomeres[END_REF][START_REF] Deng | The origin recognition complex localizes to telomere repeats and prevents telomere-circle formation[END_REF]. Using an experimentally transformed cell line carrying a tagged chromosome end, Murnane and colleagues observed changes in telomere length which appeared to have resulted from large-scale telomere deletion events [START_REF] Murnane | Telomere dynamics in an immortal human cell line[END_REF], other

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types of events included the deletion of the entire telomere and adjacent DNA coupled with healing of the end, possibly by de novo telomere addition, or chromosomal fusion events [START_REF] Fouladi | The relationship between spontaneous telomere loss and chromosome instability in a human tumor cell line[END_REF].

Interestingly these events occur in cells that express telomerase and can maintain telomere length for extended periods in culture, yet they can still suffer telomeric instability. Single molecule PCR analysis of telomere length has shown that in the absence of telomerase, gradual telomere erosion results in a decrease in the mean and an increase in the variance of the distribution [START_REF] Levy | Telomere endreplication problem and cell aging[END_REF][START_REF] Baird | Extensive allelic variation and ultrashort telomeres in senescent human cells[END_REF]. These data are consistent with the telomere dynamics predicted as a consequence of endreplication losses, together with a putative C-strand resection [START_REF] Olovnikov | Principle of marginotomy in template synthesis of polynucleotides[END_REF][START_REF] Levy | Telomere endreplication problem and cell aging[END_REF]. However, superimposed on the gradual erosion of the bulk of the telomere length distribution, were large-scale, apparently sporadic, telomere deletion events. These events have been observed both in the presence or absence of telomerase and can result in telomeres containing less than 20 TTAGGG repeats [START_REF] Baird | Extensive allelic variation and ultrashort telomeres in senescent human cells[END_REF][START_REF] Britt-Compton | Structural stability and chromosome-specific telomere length is governed by cis-acting determinants in humans[END_REF]. The occurrence of these deleted telomeres is sporadic, and they do not accumulate with ongoing cell division. This indicates that, either the cell in which the deletion event occurred exited the cell cycle, or alternatively the deleted telomere was not long enough to confer telomeric function and was subjected to further processing. Such telomeres could be repaired to full length or subjected to telomere fusion. Indeed fusion analysis has revealed that telomeres that had suffered a deletion event are subjected to fusion [START_REF] Capper | The nature of telomere fusion and a definition of the critical telomere length in human cells[END_REF]. Importantly in normal cells that contain a complement of telomeres that are long and fully functional, rare fusion events are detected that involve very short telomeres. These events are consistent with the concept that normal cells can be subjected to stochastic telomeric deletion, and these deleted telomeres can undergo fusion. This implies that cells within normal tissues in vivo may be subjected to telomeric deletion events, that can lead, via anaphase bridging, breakage and fusion events, to large-scale, potentially oncogenic mutation. This cell intrinsic mutational load may be

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particularly significant in the context of cell cycle checkpoint mutations, where a telomere that had suffered a deletion event, can be subjected to fusion, rather than causing the cell to exit the cell cycle. Indeed there are clonal patches of keratinocytes within the epidermis, that exhibit p53 mutations [START_REF] Ziegler | Sunburn and p53 in the onset of skin cancer[END_REF][START_REF] Jonason | Frequent clones of p53-mutated keratinocytes in normal human skin[END_REF][START_REF] Brash | Sunlight and the onset of skin cancer[END_REF] and thus short dysfunctional telomeres in these cells may exhibit a greater propensity to undergo fusion than in keratinocytes with functional p53 . These data indicate that it will be informative to understand the dynamics of telomere length in stem cell populations. To this end, we have some preliminary data to suggest that at least some stem cell populations may exhibit a greater degree of telomere stability compared to other cell types that have been analysed. Figure 1 shows an example of this data, where human embryonic stem (hES) cells have been subjected to a large scale single telomere length analysis. At the 17p and XpYp telomeres the mean telomere length at both ends was broadly similar at 13.0 and12.8 kb respectively, this is within the range observed in the male germline [START_REF] Baird | Telomere instability in the male germline[END_REF]. A bimodal distribution was apparent at the XpYp telomere, which is consistent with the presence of two telomeric alleles that are maintained at different lengths of 6.9 and 15 kb; a telomere length differential of 8.1 kb. These data are consistent with the concept that the telomere length setting of specific alleles, in the presence of telomerase, may be determined by cis acting elements or allele specific epigenetic modifications [START_REF] Britt-Compton | Structural stability and chromosome-specific telomere length is governed by cis-acting determinants in humans[END_REF][START_REF] Garcia-Cao | Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases[END_REF][START_REF] Gonzalo | Role of the RB1 family in stabilizing histone methylation at constitutive heterochromatin[END_REF]. Telomere length of the hES was heterogeneous with telomeres ranging from 3.0 kb to over 25 kb. However it was striking that we could not observe a single telomere out of a total sample of 753 molecules that was less than 3.0 kb (Figure 1). In fibroblast cell cultures sporadic telomere deletion results in telomeres of less than 10 TTAGGG repeats at a frequency of 9x10 -3 ; these are telomeres that have been shown to undergo fusion [START_REF] Capper | The nature of telomere fusion and a definition of the critical telomere length in human cells[END_REF]. Compared to these cells, the paucity of stochastic telomeric deletion events in our sample of human embryonic stem cells is significant (P<0.001, Chi sq.). These data indicate the

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possibility that human embryonic stem cells may exhibit enhanced telomere stability. Consistent with this view, whilst some human ES cells display karyotypic changes following prolonged periods in culture, the predominant aberrations are aneuploidy, specifically gains of chromosomes 17, 12 and X, with less evidence of non-reciprocal translocations that occur as a consequence of telomere dysfunction [START_REF] Baker | Adaptation to culture of human embryonic stem cells and oncogenesis in vivo[END_REF]. The mechanisms underlying telomere deletion events is not clear [START_REF] Baird | Telomere dynamics in human cells[END_REF], but may include mechanisms such as T-loop recombination [START_REF] Wang | Homologous recombination generates Tloop-sized deletions at human telomeres[END_REF][START_REF] Lustig | Clues to catastrophic telomere loss in mammals from yeast telomere rapid deletion[END_REF], unequal sister chromatid exchange [START_REF] Rudd | Elevated rates of sister chromatid exchange at chromosome ends[END_REF], replication slippage, or resolution of stalled replication forks at sites of unrepaired damage or DNA structures, such as G-quartets, that cannot be replicated [START_REF] Lansdorp | Major cutbacks at chromosome ends[END_REF].

Thus the mechanism by which human embryonic stem cells may exhibit enhanced telomeric stability is also not clear, however in this context it is pertinent to note that these cells exhibit enhanced genomic maintenance in humans [START_REF] Maynard | Human embryonic stem cells have enhanced repair of multiple forms of DNA damage[END_REF], and lower levels of reactive oxygen species in mice [START_REF] Saretzki | Stress defense in murine embryonic stem cells is superior to that of various differentiated murine cells[END_REF][START_REF] Saretzki | Downregulation of multiple stress defense mechanisms during differentiation of human embryonic stem cells[END_REF]. DNA damage, including that generated by oxidative stress, may provide an obstacle to telomeric replication, the resolution of which could initiate large-scale changes in telomere length. Adult stem cells may not exhibit the same levels of genomic stability compared to embryonic stem cells; it will therefore be informative to understand how levels of telomeric stability differ between these cell types.

Chromatin structure and genomic organisation in stem cells.

If the apparent stability of telomeres in hES cells, and indeed other stem cell types, is borne out by further analysis, the mechanistic basis of telomeric stability would warrant investigation. In addition to the enhanced levels of DNA repair one could speculate that telomeric stability could be related to differences in chromatin distributions and/or differences in nuclear organisation.

The organisation of loci within the nucleus can regulate transcription, with different cell types displaying distinct nuclear organisation patterns [START_REF] Cremer | Non-random radial higher-order chromatin arrangements in nuclei of diploid human cells[END_REF][START_REF] Kim | Spatial genome organization during T-cell differentiation[END_REF]. Stem cells are no exception, as they

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display a nuclear architecture, such that loci required for the maintenance of pluripotency, display specific nuclear locations; for example NANOG on 12p has a more central location in ES cells compared to differentiated cells, or 6p that maintains its chromosome position but the OCT4 locus on that chromosome is outside of its usual position [START_REF] Wiblin | Distinctive nuclear organisation of centromeres and regions involved in pluripotency in human embryonic stem cells[END_REF]. It is known from studies in yeast that the tethering of telomeres at the nuclear periphery is important to regulate transcription of telomere associated genes such as the mating-type loci [START_REF] Gartenberg | Sir-mediated repression can occur independently of chromosomal and subnuclear contexts[END_REF], which may be mediated by regulating the telomeric chromatin [START_REF] Gartenberg | Sir-mediated repression can occur independently of chromosomal and subnuclear contexts[END_REF]. It also appears that embryonic stem cells exhibit a distinct chromatin structure, that is important for controlling the expression profiles required to maintain pluripotency [START_REF] Meshorer | Hyperdynamic plasticity of chromatin proteins in pluripotent embryonic stem cells[END_REF][START_REF] Meshorer | Chromatin in pluripotent embryonic stem cells and differentiation[END_REF][START_REF] Francia | Telomere and telomerase modulation by the mammalian Rad9/Rad1/Hus1 DNA-damage-checkpoint complex[END_REF][START_REF] Azuara | Chromatin signatures of pluripotent cell lines[END_REF]. ES cells are uniquely characterised by a prevalence of a bivalent chromatin state, in which many developmentally regulated genes simultaneously possess both activating and silencing epigenetic chromatin modifications [START_REF] Bernstein | A bivalent chromatin structure marks key developmental genes in embryonic stem cells[END_REF]. Whether these differences in chromatin extend to the telomeres is not clear. It is becoming apparent that the chromatin status of telomeres, which exhibit epigenetic marks of compacted heterochromatin, can control telomeric stability [START_REF] Garcia-Cao | Epigenetic regulation of telomere length in mammalian cells by the Suv39h1 and Suv39h2 histone methyltransferases[END_REF][START_REF] Gonzalo | Role of the RB1 family in stabilizing histone methylation at constitutive heterochromatin[END_REF][START_REF] Blasco | The epigenetic regulation of mammalian telomeres[END_REF][START_REF] Gonzalo | DNA methyltransferases control telomere length and telomere recombination in mammalian cells[END_REF]. For example in mice the subtelomeric regions are heavily methylated, but mammalian telomere repeats lack the CpG sites for methylation. Interestingly the knockout of DNA methyltransferases results in large-scale changes in telomere length, that may be associated with increased recombinational activity including sister chromatid exchange [START_REF] Gonzalo | DNA methyltransferases control telomere length and telomere recombination in mammalian cells[END_REF]. Thus chromatin structure contributes to telomeric stability, and specific chromatin patterns in stem cells may confer enhanced telomere length stability.

In summary, telomere length in stem cell populations is an important determinant of the replicative potential that is required for tissue homeostasis, yet telomere length may decline as a function of age. The maintenance of telomere length is determined by a combination of stem

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cell turnover and limiting levels of telomerase, possibly also with enhanced telomere stability.

More studies of telomere length dynamics in human stem cells are required for us to fully understand the role that telomeres may play stabilising the genomes of these cells and how this may relate to the age-related deterioration in stem cell function.

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Human ES Cell culture

The hESC line HUES9 (hES facility; Harvard University, Cambridge, MA, USA) derived in accordance with local and national guidelines was used. hESCs were cultured and passaged on a layer of irradiated mouse embryonic fibroblasts at a density 1x10 6 cells / 60mm dish and passaged by enzymatic and mechanical means [START_REF] Joannides | Automated mechanical passaging: a novel and efficient method for human embryonic stem cell expansion[END_REF] DNA extraction and single telomere length analysis.

DNA was extracted by standard phenol chloroform proteinase K method as described previously [START_REF] Baird | Extensive allelic variation and ultrashort telomeres in senescent human cells[END_REF]. Single telomere length analysis was undertaken at the XpYp and 17p telomeres as described previously [START_REF] Capper | The nature of telomere fusion and a definition of the critical telomere length in human cells[END_REF]. With the exception that each telomere was analysed using 18 reactions each containing approximately 21 telomeric molecules.

Figure legend Figure 1 .

 legend1 Figure legend Figure 1. Telomere length analysis of the human embryonic stem cell line, HUES9 . Large scale analysis of telomere length at the XpYp and 17p telomeres using single telomere length analysis (STELA). Telomere length statistics are shown below.

  . To remove contaminating feeder cells prior to DNA extraction, hESCs were first passaged twice onto matrigel coated plates (BD Biosciences; Oxford UK; http://www.bdeurope.com) with feeder conditioned culture medium. Culture medium (KSR) consisted of Knockout Dulbecco.s Modified Eagle.s Medium (KO-DMEM) supplemented with 20% Serum Replacement, 1% Non-Essential Amino Acids, 1mM L-Glutamine, 0.1mM β-mercaptoethanol (all from Invitrogen; Carlsbad, CA, USA; http://www.invitrogen.com) and 10ng/ml human FGF2 (R&D systems; Minneapolis, MN, USA; http://www.rndsystems.com ).

Acknowledgements

DMB is a Cancer Research UK Senior Cancer Research Fellow.

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Damir Krunic

Sharareh Moshir

Karin M Greulich-Bode

Reynel Figueroa

Ana Cerezo

Hermann Stammer

Hans-Jürgen Stark

Steven G Gray

Kirsten Vang Nielsen

Wolfgang Hartschuh

Prof. Dr Petra Boukamp
email: p.boukamp@dkfz-heidelberg.de

Kirsten Vang Nielsen

Tissue context-activated telomerase in human epidermis correlates with little age-dependent telomere loss

Keywords: age-dependent telomere attrition, telomere length heterogeneity, in situ telomere length analysis, organotypic cultures, histone acetylation

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Introduction

The epidermis of the skin is a constantly renewing tissue that requires tightly controlled mechanisms for maintaining tissue integrity, to provide a life-long barrier against "inside out" water loss and "outside in" attacks such as bacterial infection or damage by UV radiation. The epidermis undergoes constant regeneration allowing the elimination of damaged cells. This is achieved by proliferation of the epidermal stem cells, giving rise to the so-called transit amplifying cells, the actual proliferating cells in the basal compartment, i.e. the stratum basale.

After a few rounds of proliferation these cells terminally differentiate and form multiple differentiated cell layers until they turn into dead horn squames that build the stratum corneum.

Additionally, melanocytes are also distributed throughout the basal layer and are likely to play a role in protecting the stem cells and proliferating basal cells.

The protective function of telomerase activity for telomere integrity could be another potential regulator of epidermal integrity. The human telomerase is a ribonucleoprotein reverse transcriptase composed of two major components, the RNA component (hTR) which functions as a template for adding new telomeric repeats, and the catalytic subunit (hTERT). A number of additional proteins are also required for the activation and inactivation of the complex [START_REF] Collins | The biogenesis and regulation of telomerase holoenzymes Nat[END_REF][START_REF] Rosenberger | A novel regulator of telomerase. S100A8 mediates differentiation-dependent and calcium-induced inhibition of telomerase activity in the human epidermal keratinocyte line HaCaT[END_REF][START_REF] Bianchi | Molecular biology. Refined view of the ends[END_REF].

While being active during embryogenic development, telomerase expression is subsequently inhibited in many adult human tissues. However, expression is maintained in regenerative tissues such as the hematopoietic system and the gastrointestinal tract (for review see [START_REF] Zimmermann | Telomere dynamics in hematopoietic stem cells[END_REF]). Similarly in skin, telomerase is only expressed in the regenerative epidermis while the dermal fibroblasts which only proliferate rarely, e.g. upon wounding, are telomerase-deficient [5; 6]. This could imply that telomerase is regulated in a proliferation-dependent manner and would function in counteracting replication-dependent telomere loss. However, it was reported that telomere length in skin declines with age in a statistically significant manner [START_REF] Lindsey | In vivo loss of telomeric repeats with age in humans[END_REF][START_REF] Friedrich | Telomere length in different tissues of elderly patients Mech[END_REF][START_REF] Nakamura | Comparative analysis of telomere lengths and erosion with age in human epidermis and lingual epithelium[END_REF][START_REF] Sugimoto | Telomere length of the skin in association with chronological aging and photoaging[END_REF]. Since this was also in agreement with data from telomerase knock out studies [START_REF] Rudolph | Longevity, stress response, and cancer in aging telomerase-deficient mice[END_REF], it was suggested that the level of

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telomerase observed in normal human epidermis is insufficient and thus functionally inactive.

Intriguingly, however, and despite the fact of a monthly turn-over of the epidermis, the calculated rate of yearly telomere loss was only about 25 bp thus arguing for a contribution of telomerase in telomere length maintenance.

To better address the role of telomerase in human epidermis, we have explored the consequences of telomerase activity on telomere length regulation in the epidermis in situ and in keratinocyte cultures in vitro. In agreement with our previous studies [2; 5; 12] we show that telomerase is not constitutively expressed but that telomerase activity can be detected when the keratinocytes have formed an epidermal tissue (in the epidermis in situ an in organotypic cultures) but is largely absent in monolayer cultures. Furthermore, we provide evidence that histone acetylation is involved in this tissue context-dependent regulation. In agreement with this differential telomerase expression, also telomere length regulation differs in the epidermis in situ and in monolayer culture arguing for telomerase as being important for the life-long replication of the keratinocytes in human skin.

Material and Methods

Culture conditions

Keratinocyte cultures were established from human foreskin (infant and adult) or adult trunk skin (belly, breast, or upper leg, i.e. sites generally not excessively exposed to UV radiation) -approved by the Heidelberg Ethics Commission -and routinely passaged as previously described [START_REF] Boukamp | Mesenchyme-mediated and endogenous regulation of growth and differentiation of human skin keratinocytes derived from different body sites[END_REF]. In addition, foreskin keratinocyte cultures from newborn donors were kindly provided by D. Spandau, Dept. Dermatology, Indiana University, Indianapolis, USA. The cells were propagated on lethally irradiated human feeder fibroblasts [START_REF] Rheinwald | Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells[END_REF] in FAD medium containing 5% FCS and antibiotics [START_REF] Wu | A new small (40 kd) keratin filament protein made by some cultured human squamous cell carcinomas[END_REF] or grown in keratinocyte growth medium (Epilife,

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Cascade Biologics, Clonetics, Walkersville, USA) and split in a ratio of 1:3 to 1:5. The telomerase-positive human HaCaT skin keratinocytes line was used as the control [START_REF] Boukamp | Normal keratinization in a spontaneously immortalized aneuploid human keratinocyte cell line[END_REF]. Dermal human skin fibroblasts were isolated from explant cultures of human skin samples and routinely cultured as described [START_REF] Figueroa | Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors[END_REF]. Peripheral blood leukocytes were isolated from blood by standard procedures from individuals who all gave their consent for isolating DNA and measuring their telomere length.

Growth factors (EGF, TGFα, GM-CSF, G-CSF, IL-6, KGF, all purchased from R & D GmbH, Wiesbaden, Germany) were either dissolved in DMSO or PBS and added to the culture medium at a final concentration of 10 ng/ml medium 24 h after plating. The cells were incubated for 48 and 72 h with the addition of fresh growth factors after 48 h. The deacetylation inhibitor FK228 (NSC630176) was obtained from the Developmental Therapeutics Program (DTP) of the National Cancer Institute (http://dtp.nci.nih.gov), and dissolved as a stock solution in DMSO.

Cells were treated at final concentrations of 0.1, 1, 10, 100 and 250 ng/ml for 3, 6, 12, 24 and 48 hours or 1 week. Control samples were treated with an equivalent amount of DMSO.

Organotypic cultures (OTC)

Organotypic cultures of normal human keratinocytes were prepared as described [START_REF] Stark | Organotypic keratinocyte cocultures in defined medium with regular epidermal morphogenesis and differentiation[END_REF]. After 1 and 2 weeks cultures were prepared for histology or TRAP assay. Histology was performed following standard protocols. For the TRAP assay, the epithelia were separated from the collagen substrate, frozen in liquid nitrogen and processed as described in Bachor et al. [START_REF] Bachor | Telomerase is active in normal gastrointestinal mucosa and not up-regulated in precancerous lesions[END_REF].

Telomerase (TRAP) assays

Cell lysis of cultured cells and tissues and telomerase assay were performed using the TRAPeze kit (Intergen Company, Oxford, UK, now sold by Q-Biogene, Heidelberg, Germany). 50 ng of total protein extract was used for each assay if not specified otherwise, and specificity was verified by RNase-inactivation (RNase, DNase-free, Roche, Mannheim, Germany) for

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selected samples. Products were separated in non-denaturing 10% polyacrylamide gels, visualized by auto radiography and PhosphorImager scanning (Fujifilm Bas-1500), and quantified with TINA 2.0 software.

Southern blotting

Genomic DNA was prepared from peripheral blood monocytes and keratinocytes using standard protocols. Five µg of DNA was digested with RsaI (Roche, Mannheim, Germany) at 37°C overnight and processed as described [START_REF] Figueroa | Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors[END_REF]. Detection was performed using a CDP-Star kit (Roche, Mannheim, Germany), image analysis with AIDA software (version 2.0, Raytest Isotopenmeßgeräte GmbH, Straubenhardt, Germany), and statistical analysis with Graph Pad Prism Version 3.00 (GraphPad Software, Inc., San Diego, CA, USA)

Quantitative Fluorescence in situ Hybridization

Metaphases were prepared from normal keratinocytes and HaCaT cells (control with a stable mean TRFL of 4 kb, [START_REF] Figueroa | Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors[END_REF]) using standard protocols [START_REF] Lee | Cytogenetic methods for the mouse: preparation of chromosomes, karyotyping, and in situ hybridization[END_REF]. Quantitative Fluorescence in situ Hybridization (Q-FISH) was performed with the Cy3-labeled telomeric PNA probe Cat.-No.

K5326 (DakoCytomation, Glostrup, Denmark) as described [START_REF] Figueroa | Telomere erosion varies during in vitro aging of normal human fibroblasts from young and adult donors[END_REF]. Generally, 15 metaphases were analyzed per cell type. Signals were quantified with the Telomere Quantifier Software (DakoCytomation, Glostrup, Denmark). Telomere lengths (in kb) of the keratinocytes were calculated by mathematical equation to the 4 kb mean telomere length of the HaCaT cells.

Western Blot analysis

Cell pellet preparation and further processing were performed as described [START_REF] Lee | Cytogenetic methods for the mouse: preparation of chromosomes, karyotyping, and in situ hybridization[END_REF].

Antibodies used were: anti-acetyl-H3, -acetyl-H4, or -acetyl-Lys (# 06-911, # 06-598, and # 21873, Upstate, Wolverton Mill South, UK), anti-histone H3 (ChIP Grade, ab1791, Abcam), and horseradish peroxidase-coupled secondary antibody (Dianova, Hamburg, Germany). Protein

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bands were detected using the enhanced chemiluminescence detection kit (Amersham-Pharmacia Biotech, Freiburg, Germany).

Chromatin precipitation (CHIP) analysis

CHIP was performed as described by Santoro and Grummt [START_REF] Santoro | Molecular mechanisms mediating methylation-dependent silencing of ribosomal gene transcription[END_REF]. In brief, feeder cells were removed by short-term trypsin treatment, the keratinocytes were fixed with formaldehyde (1%)

for 15 min at 37°C, collected by scraping. and centrifuged. Cell pellets were resuspended in 460 µl Chip-dilution buffer (Upstate), and the DNAs were sheared and sonicated. The cell lysates were then precleared with protein A-agarose beads and incubated with anti-acetyl-histone H4

(Upstate # 06-866) antibody over night at 4°C. The DNA-protein complexes were eluted with elution buffer (Upstate) for 2 h at 37°C, followed by 65°C over night. The complexes were then purified with phenol-chlorophorm, precipitated with ethanol and resuspended in ddH 2 O. PCR was performed with primers: GGC CGG GCT CCC AGT GGA TTC G (forward) and CAG CGG GGA GCG CGC GGC ATC G (reverse) for 5 min at 96°C, 30 cycles of 30 sec 96°C, 30 sec 58°C, and 2 min 68°C and a final 6 min at 68°C. The PCR products were fractionated on a 2% agarose gel. HaCaT cells were used as a positive controls and no antibody and histone 3 antibody (# 06-911, Upstate); not recommended for immunoprecipitation, as negative controls.

To identify human keratin 16 (hK16) the following primers were used: AAA GCT GGG TGG GAA CTC TGA GCC (forward) and GGT GCC AAG GAG GGA GGT GAG C (reverse) for 5 min at 96°C, 25 cycles of 30 se 96°C, 1 min 58°C, 1 min 68°C and a final 6 min at 68°C.

RNA isolation and RT-PCR analysis

Total RNA was isolated from OCT-frozen tissue sections or from dermal equivalents using RNeasy according to manufacturer's instructions (Qiagen Hilden, Germany). One µg of total RNA from each sample was reverse transcribed to cDNA (Omniscript, Qiagen) as described in detail previously [START_REF] Cerezo | Constitutive overexpression of human telomerase reverse transcriptase but not c-myc blocks terminal differentiation in human HaCaT skin keratinocytes[END_REF]. The cDNA template was used in a PCR reaction with the conditions and

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primers for hTERT and GAPDH as described [START_REF] Cerezo | Constitutive overexpression of human telomerase reverse transcriptase but not c-myc blocks terminal differentiation in human HaCaT skin keratinocytes[END_REF]. To analyze telomere shortening on the single cell level passage 4 keratinocytes (~13 pd)

3D FISH analysis on skin sections

Simultaneous immunofluorescence/FISH

were plated in keratinocyte growth medium (Dermalife, Lifeline Cell Technology, USA) in a slide flask (Nunc, Kat.Nr. 170920, Germany). After cell attachment, the flasks were tightly sealed (to keep CO 2 levels constant), and placed in the temperature-controlled incubator (37°C) of the inverted microscope (Axiovert S100 TV, Carl Zeiss MicroImaging Inc., Germany). Live cell microscopy was performed at a low magnification (10X) and phase-contrast images (850x680 µm) were captured every 6 minutes for 92.8 hours. At the end of monitoring, stacks of 929 images were exported to ImageJ program and used for further analysis. Each cell in this sector was labeled with a number (1,2,3….). After the each cell division, the two daughter cells were further labeled (1-1/1-2, 2-1 /2-2 …). Each individual cell was followed for its next mitosis, allowing for the measurement of the number of mitoses and cell cycle times for each individual cell. After 92.8 h the cells were fixed and stained for telomeres (see above). 46 3D images of the monitored region were taken (63x objective) and the mean telomere signal intensity was measured and expressed in kb for each cell from the monitored region. Altogether 403 cells were monitored.

Results

Telomerase activity varies in keratinocytes from different donors

To study telomerase regulation in more detail we established keratinocyte cultures from 23 epidermal isolates (15 trunk skin and 8 foreskin samples) from different-age donors, including 5 samples from newborn foreskin, and determined their telomerase status by TRAP assay. As shown in figure 1a, TRAP activity in the different samples varied significantly from low to high

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levels with a few samples showing an activity comparable to or only slightly less than that of the immortal HaCaT skin keratinocytes, which were used as control for cells with high telomerase activity [START_REF] Cerezo | Constitutive overexpression of human telomerase reverse transcriptase but not c-myc blocks terminal differentiation in human HaCaT skin keratinocytes[END_REF] (Fig. 1b). Most importantly, telomerase activity was not elevated in the newborn foreskin samples (Fig. 1c). Thus, our findings indicate that the expression level of telomerase is independent of donor age.

Telomerase activity is rapidly down-regulated in monolayer culture in vitro

To determine how proliferation influences telomerase activity, six keratinocyte strains were propagated in culture until they reached terminal growth arrest i.e. cellular senescence. In four of these strains which primarily exhibited high levels, telomerase activity declined within ∼10 pd (Fig. 1d). In the other two strains that only exhibited low levels in early passage cultures, telomerase activity became undetectable even more rapidly (data not shown).

To determine how the culture-dependent telomerase inhibition was regulated, we next investigated hTERT expression by RT-PCR and found that hTERT mRNA was strongly reduced (Fig. 1e). This suggested that telomerase activity was inhibited in monolayer cultures by transcriptional silencing of the hTERT gene.

Telomerase activity can not be restored by conventional culture conditions

Despite telomerase inhibition, all keratinocytes continued to proliferate in monolayer cultures for another 20 to 30 population doublings (pd), demonstrating that proliferation per se was not causal for telomerase expression. However, it is well established that keratinocyte growth and differentiation in the epidermis in situ is promoted through specific growth factors induced upon mutual interaction of the keratinocytes with the underlying dermal fibroblasts [START_REF] Smola | Mutual induction of growth factor gene expression by epidermal-dermal cell interaction[END_REF][START_REF] Maas-Szabowski | Keratinocyte growth regulation in fibroblast cocultures via a double paracrine mechanism[END_REF][START_REF] Maas-Szabowski | Organotypic cocultures with genetically modified mouse fibroblasts as a tool to dissect molecular mechanisms regulating keratinocyte growth and differentiation[END_REF]. One of these factors is EGF which has been shown earlier to activate hTERT expression upon binding to the ets binding site in the hTERT promoter [START_REF] Maida | Direct activation of telomerase by EGF through Ets-mediated transactivation of TERT via MAP kinase signaling pathway[END_REF]. Interestingly, neither cocultivation of the keratinocytes with fibroblast feeder cells [START_REF] Rheinwald | Serial cultivation of strains of human epidermal keratinocytes: the formation of keratinizing colonies from single cells[END_REF], nor propagating them in

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keratinocyte growth medium containing a set of growth factors (including EGF) was able to maintain telomerase activity. To exclude that specific factors were missing or were present in insufficient amounts, we further propagated three keratinocyte strains in the presence of 10 mM/ ml EGF/TGFα, IL-6, G-CSF, GM-CSF, and KGF, applied either individually or in combination.

Cells were harvested after 48 h and 72 h and assayed for telomerase expression analyses but none of these factors was able to re-stimulate telomerase expression in monolayer cultures (Fig. 2a).

Telomerase is regulated in a tissue context-dependent manner in the human keratinocytes

One major difference between the co-culture of keratinocytes with feeder fibroblasts and growth of the keratinocytes in the epidermis in situ is their three-dimensional organization within the tissue. In addition to the multilayered nature of the epidermis, the basal cells, tightly connected to the basement membrane, exhibit a cuboidal morphology as compared to their largely flattened cell shape observed in monolayer culture. This determines cell polarity and most likely an altered sensitivity towards activating or inhibiting factors provided by the dermal fibroblasts.

To address the role of tissue organization, we performed organotypic cultures (OTCs) with 2 nd and 4 th -passage keratinocytes being plated on a dermal equivalent that consisted of a collagen type I gel containing human dermal fibroblasts. Within two weeks the keratinocytes formed a three-dimensional tissue with all the characteristics of a stratified epidermis (Fig. 2b). Most importantly, telomerase activity was absent or only faintly expressed in monolayer cultures but was clearly detectable in keratinocytes grown in the OTCs (Fig. 2c). This result was confirmed with a second independent keratinocyte strain (data not shown) One possible explanation for the absence of hTERT expression after addition of e.g. EGF in monolayer cultures could be that the hTERT promoter might not be accessible. Epigenetic modulation, i.e. DNA CpG-methylation and histone acetylation, known to modulate the chromatin state, have previously been show to modify hTERT/ telomerase expression [START_REF] Cong | Histone deacetylation is involved in the transcriptional repression of hTERT in normal human cells[END_REF].

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Furthermore, histone deacetylation inhibitors reactivated telomerase in a number of cell types [START_REF] Takakura | Telomerase activation by histone deacetylase inhibitor in normal cells[END_REF][START_REF] Hou | The histone deacetylase inhibitor trichostatin A derepresses the telomerase reverse transcriptase (hTERT) gene in human cells Exp[END_REF][START_REF] Liu | Inhibition of histone deacetylases Methods[END_REF][START_REF] Atkinson | Lack of telomerase gene expression in alternative lengthening of telomere cells is associated with chromatin remodeling of the hTR and hTERT gene promoters[END_REF].

Therefore, we treated the keratinocytes in monolayer cultures with the histone deacetylase inhibitor FK228 (now known as romidepsin) (for reviews see [33; 34] and measured TRAP activity after 3, 6, 12, and 24 h. As shown in figure 3a, telomerase activity recurred in a time-dependent manner with maximal activity at 24 h (Fig. 3a). Neither increasing concentrations of FK228 (here shown for 250 ng/ml) nor combining FK228 with EGF did enhance the level of telomerase activity further (data not shown), suggesting that at 100 ng/ml of FK228 was sufficient to completely inhibit deacetylation and thus maximally stimulate telomerase activity in the keratinocytes. Interestingly, repeated treatment with 100 ng/ml FK228 -in order to maintain telomerase activity for long-term growth in monolayer cultures -caused differentiation of the cells within one to two weeks (data not shown). Even with reduced concentrations (< 0.1 ng/ml) the cultures terminally differentiated within the following weeks.

Thus, long-term treatment with histone deacetylation inhibitors and with that global inhibition of deacetylation is incompatible with continuous proliferation and not a useful experimental approach to maintain telomerase activity in cultured (monolayer) keratinocytes. Dermal fibroblasts, telomerase-negative in monolayer culture as well as in the skin in vivo [START_REF] Harle-Bachor | Telomerase activity in the regenerative basal layer of the epidermis inhuman skin and in immortal and carcinoma-derived skin keratinocytes[END_REF], did not become telomerase-positive under these treatment conditions (see Fig. 3a), indicating that hTERT activation in fibroblasts requires alternative/additional regulatory steps.

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Since the deacteylation inhibitor FK228 was able to reactivate telomerase, we reasoned that histones were predominantly deacetylated in monolayer cultures but became acetylated upon FK228 treatment. To test this, we performed Western Blot analyses with keratinocytes before and 24 h after treatment with FK228 by using antibodies that specifically recognize acetylated histones (anti-acetylated histones H3 and H4 as well as an antibody recognizing the acetylated lysines in all histones). All three antibodies showed little or no acetylation in the untreated keratinocyte cultures but increased acetylation after a 24 h treatment with FK228 (Fig. 3b). This

was not due to changes in histone expression per se, as the overall level did not change (Fig. 3d).

Histone acetylation correlates with tissue-dependent telomerase activity in the epidermis

To determine whether histone acetylation may also be responsible for the shift in telomerase expression in the epidermis in situ versus keratinocytes in monolayer cultures, we compared the acetylation profile of keratinocytes freshly isolated from the epidermis versus 3 rd passage keratinocytes and found a similar difference in histone acetylation as seen with the FK228-treated keratinocytes (Fig. 3c). The freshly isolated keratinocytes showed distinct bands with the three different antibodies while essentially no bands were seen with 3 rd -passage keratinocytes.

To confirm the role of histone deacetylation (and by analogy chromatin compaction) on hTERT promoter inhibition, we performed chromatin precipitation studies (CHIP) with different passage keratinocytes. While hTERT promoter fragments from first-passage keratinocytes precipitated with an antibody specific for the acetylated histone 4 (acetyl H4), the amount of hTERT fragments decreased with further propagation in monolayer culture and could no longer be detected at later passages (Fig. 3e). This result was confirmed with a different keratinocyte strain (data not shown) suggesting that deacetylation-dependent silencing of the hTERT promoter was not unique for a specific keratinocyte population.

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If acetylation/deacetylation was involved in telomerase regulation in the epidermis in situ and in monolayer culture, reactivation of telomerase in organotypic cultures should require hTERT promoter acetylation. Indeed, CHIP analysis demonstrated the presence of acetylated hTERT promoter fragments in the epidermis formed in organotypic cultures (Fig. 3f). As a gene known to be actively transcribed both in the regenerating epidermis in situ and in monolayer culture, we also investigated for keratin 16 promoter acetylation and found no changes (Fig. 3g), further strengthening the specificity of hTERT promoter deacetyation for the inhibition of telomerase activity.

Telomere length declines in a replication-dependent manner during propagation in monolayer cultures

Although it was suggested that telomerase activity of human keratinocytes would be insufficient to counteract telomere erosion, our finding that telomerase was only detectable when keratinocytes they had formed an epidermis but not when they were grown in monolayer cultures prompted us to determine whether telomere shortening in monolayer culture would correlate with a more prominent telomere loss than in the epidermis in situ despite the fact that the epidermis regenerates every three to four weeks and accordingly, also the stem cells should also go through a number of replications per year.

To address this, we first propagated different keratinocyte strains up to cellular senescence and investigated their telomere lengths at different passage levels by determining their telomere restriction fragment lengths (TRFL). Two keratinocyte strains derived from infant foreskins, exhibited an initial mean TRFL of about 10 kilobases (kb) and senesced after >30 pds with a mean TRFL of 6 to 7 kb, giving rise to a calculated loss of 100 to 130 bp per pd (one example is shown in Fig. 4a, left panel). Keratinocytes derived from an adult foreskin or from different sites of trunk skin, on the other hand, varied strongly in their rate of telomere loss, ranging from 18 to 125 bp per pd (one example is shown in Fig. 4a, right panel). A similar variation was observed

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when performing Q-FISH analysis (Fig. 4b andc). Altogether these data argue for a telomeric loss of ~100 bp per pd for the keratinocytes when being propagated in monolayer cultures.

While this average estimate already implicated major variations between different keratinocyte populations, we aimed for establishing the rate of telomere loss in individual keratinocytes. For this, keratinocytes were plated at low density and followed by live cell imaging over a period of 92.8 h. Individual cells were marked and after mitoses the two daughter cells were followed for further replication. Those daughter cells that differed in their subsequent replication number where chosen for telomere length measurement. Telomere length measurements were performed by in situ hybridization of the telomeres with a telomere-specific PNA probe, deconvolution microscopy, 3D-telomere reconstruction [23; 35]. Measurement of the individual signal intensities was performed and the mean of all telomeres per nucleus was then compared for each "daughter pair".

Being able to identify 14 daughter pairs that differed in at least one replication round (2/14 differed in 2 and 1/14 differed in 3) we found an unexpected heterogeneity (Fig. 5). First, most but not all daughter cells remained synchronized in their replication time. Second, the cell cycle time varied from 12 to 35 h between different daughter pairs and third, telomere erosion varied from 45 to 1044 bp with no obvious correlation between the rate of telomere loss and cell cycle time. These results implicate that even within the same keratinocyte population there is no defined rate of replication-dependent telomere loss.

Telomere length heterogeneity rather than an age-dependent decline is characteristic for early-passage human skin keratinocytes

To determine telomere length regulation in human epidermis in situ we measured the mean TRFL from 31 early passage keratinocyte samples from donors ranging from newborn (0 y) to the age of 89. The six newborn foreskin samples showed a homogeneous distribution with a mean TRFL of 9 to 10 kb (Fig. 6a). The samples from the infant and adult donors (3 foreskins

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and 22 trunk skins), on the other hand, differed (Fig. 6b). Instead of a steady age-dependent decline, these samples showed strong inter-individual telomere length heterogeneity with differences ranging from 11 to 5 kb. Interestingly, the largest differences were seen in the group of 30 to 50 years while variations were less pronounced in samples from older donors (>60 years) and the oldest donor (89) actually showed a similar mean TRFL as the youngest donors with 10 kb. From these data we calculated decline of ~25 kb loss per year and this decline was not statistically significant (p value of 0.1027; Fig. 6c).

Telomere length measurement on intact skin sections confirms the minimal agedependent decline

Since establishing keratinocytes in culture may represent selection for specific subpopulations, we next adapted our 3D telomeric FISH analysis for telomere length analysis on tissue sections and combining it with immuno-labelling, we evaluated telomere length of the epidermal keratinocytes in skin samples from 12 different donors (ranging from 2 to 81 years of age). In addition, this technique allowed the distinction between the different cell types, i.e.

keratinocytes, melanocytes, and fibroblasts present within the same skin section (Fig. 7a).

We first investigated the telomere lengths for the epidermal keratinocytes. Their respective mean lengths integrated well into the age-dependent pattern seen by Southern blot analysis (Fig. 7b and see Fig. 6c squares). Accordingly, combining all data and calculating their linear regression, we now obtained a statistically significant (p-value of 0.0181) yearly loss of 25 bp.

Epidermal differentiation does not contribute to telomere loss

To determine whether and how differentiation would contribute to telomere length regulation, we next studied samples from 14 different donors by dividing the epidermis into basal, suprabasal, and upper part (as indicated in figure 7c) and measured telomere length in at least 5 individual areas of each specimen (Fig. 7d). We found one sample with both suprabasal

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und upper part containing shorter telomeres than the basal layer, one sample with the upper layers containing shorter and two where the upper layers contained slightly longer telomeres. In most (10/14) samples, however, telomere lengths were very similar in all planes, indicating that epidermal differentiation does not contribute to telomere loss.

Telomere length maintenance is a characteristic of human skin

Since the in situ technique allowed us to distinguish between different cell types within the same skin sample, we also investigated for telomere length of the non-epidermal cells, the melanocytes, which make up ~10% of the basal layer of the epidermis, and the fibroblasts which Finally and as a control for a different proliferation-active tissue, we also investigated hematopoietic cells, i.e. peripheral blood lymphocytes (PBL). It is well accepted that PBLs exhibit age-dependent telomere attrition [START_REF] Baerlocher | Telomeres in hematopoietic stem cells Ann[END_REF] and in agreement with these previous data, we found a statistically significant reduction with a p value of 0.0010 when establishing the mean TRFL for PBLs from different-age donors (Fig. 7f).

Discussion

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No significant age-dependent telomere loss of keratinocytes in human epidermis in situ

The epidermis, as the outermost barrier of the human body depends on mechanisms that guarantee life-long tissue integrity. One of these mechanisms is its constant regeneration which allows for a steady elimination of "damaged" cells from the basal proliferative compartment by passing through a defined program of differentiation and desquamation as dead horn squames.

This continuous regeneration, however, also bears the risk of constant telomere shortening and, therefore, exhaustion of the proliferative potential of the epidermis in aged skin due to critically short telomeres. Indeed, evidence existed for a significant age-dependent decline in telomere length for peripheral blood lymphocytes ( [START_REF] Frenck | The rate of telomere sequence loss in human leukocytes varies with age[END_REF]; for review see [START_REF] Drummond | Concise review: telomere biology in normal and leukemic hematopoietic stem cells[END_REF]) as well as human skin [START_REF] Lindsey | In vivo loss of telomeric repeats with age in humans[END_REF][START_REF] Friedrich | Telomere length in different tissues of elderly patients Mech[END_REF][START_REF] Nakamura | Comparative analysis of telomere lengths and erosion with age in human epidermis and lingual epithelium[END_REF][START_REF] Sugimoto | Telomere length of the skin in association with chronological aging and photoaging[END_REF].

However, in contrast to blood lymphocytes, the rate of telomere loss in human epidermis or in complete skin was small with a range of 9 to 36 bp/year. Only one study reported on a rate of 75 bp/year [START_REF] Friedrich | Telomere length in different tissues of elderly patients Mech[END_REF]. Our data confirm the significant rate of age-dependent telomere loss in blood lymphocytes. Here it is noteworthy that our PBL samples were derived from members of two related families, thus restricting genetic differences. Our data also support the low rate of telomere loss in human epidermis, thereby arguing for potential differences in the regulation of telomere length maintenance between these two tissues.

We further demonstrate that in the same skin sections where we measured telomere length of keratinocytes, the fibroblasts in the dermis and the melanocytes within the basal layer of the epidermis both exhibit longer telomeres and a similar minor age-dependent regression rate (see Fig. 7b). This finding is in good agreement with TRFL data from Sugimoto et al. [START_REF] Sugimoto | Telomere length of the skin in association with chronological aging and photoaging[END_REF] reporting on longer telomeres in the dermis than the epidermis and a regression rate of 11 bp/y for the dermis and 9 bp/y for the epidermis. Although only a few samples were available from very young donors so far, the difference in telomere length between epidermal keratinocytes versus dermal fibroblasts and melanocytes already seemed to be established early in life. If this is correct and provided that the different cell types start with a similar telomere length, these results implicate a rapid telomere loss during early epidermal development, i.e. a phase when

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even epidermal stem cells are supposed to proliferate heavily. During this time, apparently, telomerase is not sufficient for telomere length maintenance. Thereafter, when proliferation is restricted to maintaining tissue homeostasis, however, the level of telomerase seems adequate to minimizes telomere erosion to a similar rate as seen in the non-regenerating cells, i.e. to only a few bp per year.

A rapid telomere loss was described for PBLs of young children (>1kb per year) followed by an apparent plateau between the age of four and young adulthood and gradual attrition later in life [START_REF] Frenck | The rate of telomere sequence loss in human leukocytes varies with age[END_REF]. The authors proposed that loss of telomeric repeats in hematopoietic cells is a dynamic process that is differentially regulated in young children and adults. Besides a potential rapid telomere loss discussed for the embryogenic development, we did not detect obvious phases of telomere attrition in the human epidermis thereafter. It is intriguing to note that all three skin cell types exhibit a similar rate of telomere loss. Considering the fact that fibroblasts and melanocytes hardly ever proliferate, it is tempting to suggest that the rate of 11 bp/y reported by Sugimoto [START_REF] Sugimoto | Telomere length of the skin in association with chronological aging and photoaging[END_REF] and ~25 bp/y determined here, may not even be proliferation-related. If so, the more this argues for active telomere lengths maintenance throughout life time of the constantly proliferating keratinocytes. Along this line, Counter et al [START_REF] Counter | Telomere shortening in cultured autografts of patients with burns[END_REF] found that in patients with severe burns the epidermal transplants derived from keratinocytes expanded in monolayer cultures had shorter telomeres than in non-cultured skin transplants from the same individuals or those in skin of healthy donors older than 80 years. From this they concluded that due to the great loss of telomeric DNA the engrafted cells might have a shortened lifespan, which could have negative repercussions on the long-term viability of these grafts. Having now shown that telomerase is inhibited in culture but can be reactivated in cells that are allowed to form an epidermal tissue, such concerns may be invalid since in the tissue context telomerase may be reactivated and protect against consecutive severe telomere attrition. This said, it is tempting to suggest that intrinsic skin aging occurs rather independently of telomere shortening. The mechanism how telomere length is maintained, however, seems to differ and to depend on the cell type. We

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postulate a telomerase-based mechanism for the epidermal skin keratinocytes, while dermal fibroblasts and melanocytes maintain their long telomeres due to their low proliferation rate.

Histone acetylation contributes to hTERT/telomerase expression in the epidermal tissue

In line with the differences in telomere length regulation in the epidermis in situ and in keratinocytes in conventional cultures we also find a clear difference in telomerase regulation.

Telomerase is expressed when the keratinocytes grow as an epidermis either in situ or in organotypic cultures but is inhibited in monolayer culture. Since proliferation is ongoing under both conditions, actually, proliferation is increased in monolayer culture as compared to the epidermis in situ, telomerase is not constitutively active in the keratinocytes and accordingly dispensable for keratinocyte growth. But it is obviously crucial in the tissue context. This said, it is noteworthy that the epidermis is a tissue that is highly prone to stress and damage. It is, therefore, tempting to hypothesize that besides counteracting proliferation-dependent telomere loss telomerase may similarly be essential for counteracting damage-dependent telomere loss in order to maintain epidermal tissue integrity and maintenance throughout life time.

Our data suggest that epigenetic remodelling of the hTERT promoter is an important regulatory step in the concert of tissue-dependent telomerase activation in human epidermis.

While histone acetylation correlates with an active hTERT promoter in freshly isolated or early passage keratinocytes, hTERT is silenced upon passaging in monolayer culture and this in turn correlates with its promoter deacetylation. In organotypic cultures the hTERT promoter became reacetylated and accordingly telomerase re-expressed. Furthermore, keratinocytes in monolayer cultures re-expressed telomerase activity when treated with the deacetylation inhibitor FK228. It was recently shown that hTERT was hypoacetylated in human mesenchymal stem cells (hMSC) in culture and that telomerase could be reactivated by the deacetylation inhibitor Trichostatin A [START_REF] Serakinci | Telomerase promoter reprogramming and interaction with general transcription factors in the human mesenchymal stem cell[END_REF]. This may implicate that also in these cells hTERT is acetylated and active in vivo and that

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the observed deacetylation could be a "culture"-dependent trait as we suggest for the human epidermal keratinocytes.

Histone deacetylation coupled with a gradual accumulation of hTERT promoter methylation was reported previously for differentiation-dependent hTERT/telomerase repression in the human HT teratocarcinoma, human HL60 acute myeloid leukaemia cells, and mouse embryonic stem cells [START_REF] Liu | Epigenetic regulation of human telomerase reverse transcriptase promoter activity during cellular differentiation Genes Chromosomes[END_REF]. Our finding that histone acetylation plays a role in the tissue contextdependent telomerase regulation supports the idea that histone acetylation/ deacetylation regulate telomerase activity in physiological processes. Importantly, this type of regulation also allows for downstream regulatory processes. EGF was reported to be a potent telomerase activator, stimulating hTERT transcription through an ets motive in the hTERT promoter [START_REF] Maida | Direct activation of telomerase by EGF through Ets-mediated transactivation of TERT via MAP kinase signaling pathway[END_REF] and that withdrawal of EGF or amphiregulin inhibited telomerase activity in culture [START_REF] Matsui | Influence of aging and cell senescence on telomerase activity in keratinocytes[END_REF]. In our experiments EGF was not able to induce telomerase activity but this can now be explained by hTERT promoter deacetylation which made the hTERT promoter inaccessible for EGF in the monolayer cultures. On the other hand, growth factors such as EGF and TGFα play an important role in stimulating keratinocyte growth and differentiation in the epidermis in situ [25; 26].

Being active and accessible in the tissue context, our data, therefore, do not exclude that EGF may be part of the regulatory network controlling activation and inhibition of hTERT/telomerase activity in the process of epidermal regeneration and tissue homeostasis.

Altogether, we have demonstrated that telomerase is expressed in human epidermal keratinocytes in a tissue organisation-dependent manner and that in the epidermis from skin from different age donors this expression is correlated with an only minor age-dependent telomere loss. This suggests that telomerase is functionally active and acts as a telomere lengths maintenance mechanism in human epidermis. Since the corresponding melanocytes and dermal fibroblasts show a similar low rate of telomere loss, we have to conclude that the overall telomere erosion is obviously not a driving force of skin aging. As we also show that telomerase is inhibited in monolayer cultures, telomere maintenance is not a constitutive trait but is

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extrinsically regulated. We provide evidence that part of this regulatory program is hTERT promoter acetylation, thereby guaranteeing an active hTERT transcription in the epidermis. In monolayer cultures, on the other hand, hTERT is silenced by histone deacetylation causing telomerase inhibition and accordingly a more extensive replication-dependent telomere loss.

Therefore, our results support our hypothesis that telomerase is not required for proliferation per se but is part of the regulatory network required for life-long epidermal tissue regeneration. 4), the second one: intact epidermis (5) freshly isolated keratinocytes (6) and cells from passage 3 (7) primary culture (8) and passages 2 (9) and 4 [START_REF] Sugimoto | Telomere length of the skin in association with chronological aging and photoaging[END_REF]. RNA from HaCaT cells [START_REF] Rudolph | Longevity, stress response, and cancer in aging telomerase-deficient mice[END_REF] served as a positive control. Each curve represents the mean of at least 10 metaphases. In 14 daughter pairs that differed in at least one replication round we found an unexpected heterogeneity, telomere erosion varied from 45 to 1044 bp. The mean telomere length and the error of mean are shown for cells from each replication round as well as the linear regressions between the replication rounds. The differences between the different regression slopes were not statistically significant (p=0.2665), therefore, one slope could be calculated for all 14 cellscorresponding to a telomere loss of 322 bp/division.

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  of p=0.05 (* 0.01<p<0.05, ** p<0.01). For telomere shortening, linear regression and significance of deviation from zero were measured. A control skin sample with known TSI was included in each experiment. Analyses of the same regions on serial skin sections and cohybridization with a centromere 7-specific PNA probe were used to confirm hybridization efficiency. All experiments were repeated at least twice.To recalculate TSI into telomere length in kb, TSI of HaCaT, HaCaT-myc, human-, and mouse A9 fibroblasts were calculated and plotted against the TRFL values determined from the same cell populations by Southern Blot analysis. Chemilumi image exposures were digitized and transferred to Microsoft Excel, and were the basis for calculating the mean TRFL using the formula: TRFL = Σ(ODi)/ Σ(ODi/Li) where ODi is the integrated signal intensity at position i, and Li -the length of DNA fragment in position i. Calibration curves established this way were used for telomere lengths determination in skin sections.

3 . 6

 36 Histone acetylation/deacetylation is part of the regulatory program of telomerase activity in human skin keratinocytes.

  reside in the underlying dermis. It is important to note that different from the epidermal keratinocytes both cell types only rarely proliferate in intact skin. As shown in Fig7b,for each individual tissue sample, telomeres were longer in the fibroblasts and melanocytes as compared to the respective keratinocytes. Actually, both cell types (within the same sample) exhibited highly comparable telomere lengths and unexpectedly, all three cell types (keratinocytes, fibroblast, and melanocytes) revealed very similar and statistically non-significant regression slopes (epidermis: p-values of 0.0907, dermal fibroblasts: p-value of 0.1972, and melanocytes pvalue of 0.0886) (see Fig.7b).

Fig 1 .

 1 Fig 1. Telomerase is expressed in early-passage keratinocytes but down-regulated but downregulated with further propagation in monolayer culture. A) Telomerase activity was determined by TRAPeze assay in first passage keratinocytes from different-age donors all being telomerase-positive though exhibiting different levels of telomerase activity. HaCaT cells (H) serve as positive control and HaCaT cells treated with RNase, fibroblasts, and dilution buffer as negative control. B) In a titration experiment dilutions of 1:50 (50), 1:25 (25), 1:10 (10), 1:5 (5) and undiluted (1) HaCaT extracts were compared with undiluted extracts from two normal keratinocyte strains thereby showing that telomerase levels in the normal cells can be as high as in the immortal keratinocytes. HaCaT extracts were diluted with extracts from telomerasenegative fibroblasts to guarantee similar protein concentrations. C) Also second-passage keratinocytes from five (1 to 5) newborn (N) foreskin exhibit different levels of telomerase activity. HaCaT cells (H) serve as positive and HaCaT cell treated with RNase (H+) as negative control. D) During further propagation in monolayer culture telomerase activity declines with increasing passage number (pc = primary culture, 1, 3, 9, and 11 = passage number). E) Only freshly isolated or primary culture keratinocytes exhibit good levels of hTERT expression (RT-PCR). Expression of GAPDH is used as a loading control. Data from two individual keratinocyte strains are shown. The first represents: freshly isolated cells (1) and passage 3 (2), 5 (3), and 7 cells (4), the second one: intact epidermis (5) freshly isolated keratinocytes (6) and cells from

Fig 2 .Fig 3 .

 23 Fig 2. Telomerase is re-expressed in culture when the keratinocytes are allowed to form an epidemis-like epithelium. A) Growth factors can not overcome the culture-dependent telomerase inhibition. TRAPeze detection of telomerase activity 48 h after growth factor treatment. 1 =

Fig 4 .

 4 Fig 4. Telomere lengths decrease with consecutive propagation of human skin keratinocytes in monolayer culture. A) Telomere-specific Southern blot from keratinocytes isolated from the epidermis of an infant (age 1, left panel) and an adult donor (age 24, right panel) and cultured up to cellular senescence. Different passages were analyzed for TRFL. Mouse A9 cells with a mean TRFL of ∼11 kb were used as a control. B) Fluorescence in situ hybridization (FISH) images of metaphase spreads from an early and late passage of infant keratinocytes (pds 3 and 27, lower panel) and adult (pds 3 and 15, upper panel), respectively. Yellow: telomeric FISH signals and blue: DAPI counterstained chromosomes. C) Quantitative FISH (Q-FISH) analysis from keratinocytes from, foreskin from a 3-year-old donor (pds 3, 12 and 27), trunk skin from a 75year-old donor (pds 3, 10, and 15) and foreskin from a 33-year-old donor (pds 3, 12 and 18).

Fig 5 .

 5 Fig 5. Telomere loss differs between individual cells of the same populationLive cell microscopy combined with 3D-telomere length measurement was used to identify telomere length between different daughter cells A) phase contrast micrographs of the keratinocyte cultures at the start (24 h after plating -time point 0) and B) at the end (time point 92.8 h) of the live-cell-imaging experiment. C) In a given area each mitosis was marked with a specific number and the daughter cells were followed and marked again when they went through the next mitosis. D) Same area after in situ hybridisation with the telomere-specific PNA probe

Fig 6 .

 6 Fig 6. Telomere length heterogeneity not telomere loss characterizes human skin keratinocytes from different-age-donors. A) and B) Telomere-specific Southern blot analysis of six secondpassage foreskin keratinocyte strains from newborn donors (last lane = HaCaT cells) (A), and freshly isolated or first passage culture keratinocytes from different-age-donors (first lane = HaCaT cells) (B). C) Quantification of the mean TRFL with indicated donor age. Linear regression of the 31 samples (black circles) demonstrates lack of statistical significance (p value of 0.1027) for age-dependent telomere attrition in the human skin keratinocytes. Grey squares represent the data obtained from the 3-dimensional telomere length analysis on tissue sections from different-age donors (see fig. 7).

Fig 7 .

 7 Fig 7. Three-dimensional analysis of telomere length on skin sections verifies Southern Blot data. A) Human skin section stained for keratin (green) marking the epidermal keratinocytes (epidermis) and vimentin (blue) marking the fibroblasts in the dermis as well as melanocytes (marked with arrows) in the basal layer of the epidermis. The telomeres, hybridized with a telomere-specific PNA probe, are presented in red. Epidermis and dermis are indicated. B) Graphic presentation of the mean telomere signal intensities of keratinocytes (green), melanocytes (blue), and dermal fibroblasts (grey) in skins from different-age donors, demonstrates that telomeres in the proliferating keratinocytes are generally shorter than in the

3D Image acquisition, Deconvolution microscopy, and image processing were

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	3D Reconstruction of telomeres (Cy3 emission, pseudo-colored in red) was performed by
	using the Amira module that computes an isosurface within a 3D scalar field. In addition, keratin
	(Cy2 and emission, in green), nuclei (DAPI emission, in blue), and vimentin staining (AMCA
	Analysis. Cells directly grown on glass slides or 5µm skin cryostat sections were fixed in 3.7% formaldehyde for 15 min, washed, permeabilized with 0.2% Triton X-100, rinsed in ddH 2 O, and air-dried. Cy3-labeled PNA probe (PNA Telomere FISH Kit K5326; DAKO A/S, Lustrum, Denmark) was added before denaturing the DNA for 3 min at 80°C. After hybridization (2 h at 30°C) the slides were washed at room temperature (rt) with 70% formamide/10 mM Tris-HCl, pH 7.2 (2x20 min), PBS (1 min), 0.1xSSC for 5 min at 55°C, and PBS containing 0.05% Tween-20 (2x5 min at rt), and rinsed in ddH 2 O. The slides were then shortly air dried, and incubated for 2 h at rt with an anti-keratin (DAKO Corporation, Carpinteria, CA, USA, dilution of 1:1000) and anti-vimentin antibody emission, in blue, or DTAF in green) were visualized with the Amira ProjectionView module; M A N U S C R I P T computing shadow projections of 3D images onto the three major planes (xy, xz, yz).
	(Progen, Heidelberg, Germany, dilution 1:50). After washing with PBS, the slides were incubated with Cy2-conjugated anti-rabbit and AMCA-conjugated anti-guinea pig antibodies (Jackson ImmunoResearch Laboratories Inc., USA, at a dilution of 1: 50) 45 min at rt, washed with PBS, and covered with antifade solution (VectaShield, Vector Laboratories Inc., Burlingame, CA). Alternatively, telomere hybridisation was counterstained with DAPI and combined with DTAF-conjugated anti-guinea pig antibody. A C C E P T E D
	principally performed as described [23] with a modification of the automation program
	(OpenLab software package) allowing deconvolution of >30 image stacks over night.
	Deconvolution of Cy3, Cy2, DAPI, AMCA and DTAF signals were performed individually with
	the same conditions for a given fluorochrome. The deconvolved images (stack of TIFF images)
	were further used for 3D reconstruction with Amira 3D visualization software (version 3.0,
	Indeed-Visual ConceptsGmbH, Berlin), and for telomeric signal intensity determination using
	ImageJ software (version 1.33u, Wayne Rasband, National Institutes of Health, USA).

Quantification of the digitized fluorescent telomere signals was

	accomplished by using
	an ImageJ maximum Z Projection of deconvolved images. Projected telomeric signal intensities
	(TSI) with sizes greater than the threshold (background noise) were measured. Intensities for
	each telomeric spot were tabulated and exported to Microsoft Excel (Microsoft Corp.). GraphPad
	Prism (GraphPad Software, San Diego California, USA) was used for further data handling and
	statistical analysis. Statistical significance was determined using t test with a significance level

Acknowledgements

We would like to thank Elke Tomakidi and Iris Martin for excellent technical assistance as well as Dan Spandau, University of Indiana, Indianapolis, for kindly providing the newborn foreskin keratinocytes. We also would like to thank DAKO Cytomation for generously providing us with telomeric PNA probe . We wish to extend our thanks to Magareta Müller for her valuable discussion and advice and Yetunde Odunsi and Angelika Lampe for her help in editing the manuscript. This work was supported by grants from Sander Stiftung, EC (LSHC-CT-2004-502943), Landesstiftung Baden-Württemberg, Deutsche Krebshilfe e.V., and BMBF WOUND and the NAR Verbundprojekt "Stem Cells and Aging"(all to PB).

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