title: Regulation of Telomere Length and Organisation in Human Skin Cells in vitro and in vivo
creator: Krunic, Damir
subject: 570
subject: 570 Life sciences
description: Telomeres are specialized DNA-protein structures at the ends of the linear chromosomes that form protective caps. They are composed of multi-fold double-stranded 5’-TTAGGG-3’ repeats and a 3’ single stranded overhang that loops back and invades the duplex region. The so called T-loop structure is stabilized by a number of associated proteins that protect the DNA against degradation and hinders the cellular machinery to recognize the ends as broken DNA, thus being essential for chromosomal integrity. Investigating the three-dimensional (3D) telomere distribution we now show that telomeres of the immortal HaCaT keratinocytes are distributed in distinct non-overlapping territories within the inner third of the nuclear space in interphase cells and extend more widely during mitosis. This distinct localization is abrogated in a HaCaT variant that constitutively expresses the c-Myc onco-protein. Telomeres in HaCaT-myc cells form aggregates (TAs) that are accompanied by an overall irregular telomere distribution in interphase. Since this TA formation also leads to clustering of the respective chromosomes and TA formation is present during mitosis, TAs most likely contribute to genomic instability by forcing abnormal chromosome segregation. As a first step to approach the mechanism of TA formation we compared the difference in nuclear protein expression between HaCaT and HaCaT-myc cells by two-dimensional polyacrylamide gel electrophoresis. Out of 30 differentially expressed proteins, the most promising candidate was Matrin 3, a nuclear matrix protein that, being reduced in HaCaT-myc cells, suggests for a mechanism involving incorrect adhesion to the nuclear matrix.  To investigate the role of telomere loss for skin aging in situ, we developed a 3D deconvolution microscopy based Q-FISH/immunofluorescence technique on individual cells in tissue sections. When investigating skin from different-age donors, we found that similar as for dermal fibroblasts and another non-proliferative cell type in the epidermis, the melanocytes, also the epidermal keratinocytes only show a minimal age-dependent telomere decline. Thus age-dependent telomere loss appears largely neglectable. However, we found significant inter-personal differences and most strikingly, intra-personal variations in telomere lengths between similar sites of the epidermis. Moreover, in 10 of 30 samples of normal skin, preferentially from sun exposed sites of elderly donors, we identified regions within otherwise normal looking epidermis with significantly shorter telomeres. Though size and number of these micro-lesions as well as amount of telomere shortening varied, all enclosed various basal and suprabasal differentiated cells. Most importantly, they were all characterized by expression of the p53 tumour suppressor gene and 53BP1 foci co-localizing with telomeres. Since the latter are representative for DNA double strand breaks and when co-localized with telomeres represent critically short uncapped telomeres, these date demonstrate that in these micro-lesions the telomeres are dysfunctional and likely represent stages of genomic instability. Such distinct areas can only be maintained when damage has occurred in a stem cell. We, therefore, postulate that damage did not cause cell death but was repaired and lead to a decreased telomere length. This reduced telomere length was then transmitted to the daughter cells. Thus each micro-lesions most likely represents the space of one stem cell compartment.  We also identified shorter telomeres in skin from heavily sun-exposed individuals and in several skin sections in sites closer to the surface (outside) as compared to more protected areas of the epidermis (deeper parts of the rete ridges, deeper parts of the hair follicles). Since we further show that in skin from 16 volunteers irradiated with UVA, UVB, or a combination of UVA and UVB, distinct telomere shortening was visible already 3 days post irradiation, UV radiation is clearly a responsible factor for accelerated telomere loss in human skin. Another factor may be forced oxidative damage because also chronic and to a lesser extend acute wounds showed distinct telomere shortening. Finally, we demonstrate for the first time a population of rare cells within the epidermis which have the “longest” telomeres and which likely represent the stem cells. These cells are distributed rather randomly throughout the basal layer. Furthermore, when cultured and replated in organotypic cultures they re-establish as stem cells in the newly developing epidermis. Thus, telomere length is a valuable marker for stem cells and it is damage rather than replication-dependent telomere shortening that leads to significant shortening and potential sites of genomic instability in human skin. 
date: 2008
type: Dissertation
type: info:eu-repo/semantics/doctoralThesis
type: NonPeerReviewed
format: application/pdf
identifier: https://archiv.ub.uni-heidelberg.de/volltextserverhttps://archiv.ub.uni-heidelberg.de/volltextserver/8340/1/Damir_Krunic_PhD_Thesis.pdf
identifier: DOI:10.11588/heidok.00008340
identifier: urn:nbn:de:bsz:16-opus-83409
identifier:   Krunic, Damir  (2008) Regulation of Telomere Length and Organisation in Human Skin Cells in vitro and in vivo.  [Dissertation]     
relation: https://archiv.ub.uni-heidelberg.de/volltextserver/8340/
rights: info:eu-repo/semantics/openAccess
rights: http://archiv.ub.uni-heidelberg.de/volltextserver/help/license_urhg.html
language: eng