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TERT promotes epithelial proliferation through transcriptional control of a Myc- and Wnt-related developmental program.

Choi J, Southworth LK, Sarin KY, Venteicher AS, Ma W, Chang W, Cheung P, Jun S, Artandi MK, Shah N, Kim SK, Artandi SE - PLoS Genet. (2007)

Bottom Line: This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood.We show that TERT(ci) retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis.These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stanford School of Medicine, Stanford, California, United States of America.

ABSTRACT
Telomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERT(ci)) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERT(ci) retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

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Conditional Expression of a Catalytically Inactive TERT Protein Induces the Anagen Phase of Hair Follicles(A) Diagram of TERT protein structure shows domains conserved among all RTs. Three conserved aspartates (shown) are required for RT function. D702 in motif A was mutated to alanine, to create the mouse TERTci allele.(B) Mouse TERTci fails to reconstitute telomerase activity when transduced into TERT−/− MEFs and inhibits endogenous telomerase activity in TERT+/+ MEFs by TRAP. TRAP activities were quantified by measuring band intensities, and relative telomerase activity (RTA) is shown below each lane.(C) Hair follicles cycle between an active growth phase (anagen) and a resting phase (telogen). E, epidermis; ORS, outer root sheath; HS, hair shaft; M, matrix cells; DP, dermal papilla; IRS, inner root sheath; B, bulge stem cell niche; S, sebaceous gland.(D) Northern blot shows doxycycline-dependent suppression of TERT mRNA in dorsal skin from iK5-TERT and iK5-TERTci mice.(E) Induction of either TERTci or TERT mRNA in mouse epidermis initiates an anagen cycle between days 50–60. Doxycycline was withdrawn at day 10. Controls including non-transgenic, K5-tTA single transgenic, and tetop-TERTci and tetop-TERT single transgenic mice remained in telogen (asterisks). In contrast, iK5-TERTci and iK5-TERT mice are in anagen (black arrows). H&E, 100×.
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pgen-0040010-g001: Conditional Expression of a Catalytically Inactive TERT Protein Induces the Anagen Phase of Hair Follicles(A) Diagram of TERT protein structure shows domains conserved among all RTs. Three conserved aspartates (shown) are required for RT function. D702 in motif A was mutated to alanine, to create the mouse TERTci allele.(B) Mouse TERTci fails to reconstitute telomerase activity when transduced into TERT−/− MEFs and inhibits endogenous telomerase activity in TERT+/+ MEFs by TRAP. TRAP activities were quantified by measuring band intensities, and relative telomerase activity (RTA) is shown below each lane.(C) Hair follicles cycle between an active growth phase (anagen) and a resting phase (telogen). E, epidermis; ORS, outer root sheath; HS, hair shaft; M, matrix cells; DP, dermal papilla; IRS, inner root sheath; B, bulge stem cell niche; S, sebaceous gland.(D) Northern blot shows doxycycline-dependent suppression of TERT mRNA in dorsal skin from iK5-TERT and iK5-TERTci mice.(E) Induction of either TERTci or TERT mRNA in mouse epidermis initiates an anagen cycle between days 50–60. Doxycycline was withdrawn at day 10. Controls including non-transgenic, K5-tTA single transgenic, and tetop-TERTci and tetop-TERT single transgenic mice remained in telogen (asterisks). In contrast, iK5-TERTci and iK5-TERT mice are in anagen (black arrows). H&E, 100×.

Mentions: To determine if TERT's RT activity is required to promote stem cell proliferation, we inactivated TERT RT function by introducing a point mutation in its active site. TERT's RT domain comprises seven sub-regions of homology conserved among all telomerases and RTs [20]. Within motifs A and C, three aspartate residues are absolutely conserved and essential for catalytic function [20–22]. The aspartate residue in motif A was changed to alanine by site-directed mutagenesis of cDNAs encoding either human TERT or mouse TERT, yielding the TERTci allele (for catalytically inactive) (Figure 1A). Mouse and human TERTci proteins lacked telomerase activity when expressed in TERT−/− MEFs or primary human fibroblasts, respectively, whereas wild-type TERT proteins efficiently reconstituted telomerase enzyme function (Figures 1B and S1A). Consistent with this loss of RT function, human TERTci did not immortalize primary human fibroblasts, whereas wild-type TERT efficiently extended replicative lifespan and prevented replicative senescence (Figure S1B and S1C). TERTci proteins were efficiently expressed based on their ability to inhibit endogenous telomerase activity (Figures 1B and S1A) [20–22] and because TERT and TERTci protein levels were indistinguishable by Western blot (Figure S1D and S1E). Together, these data show that TERTci protein is catalytically inert and accumulates to levels similar to that of the wild-type protein.


TERT promotes epithelial proliferation through transcriptional control of a Myc- and Wnt-related developmental program.

Choi J, Southworth LK, Sarin KY, Venteicher AS, Ma W, Chang W, Cheung P, Jun S, Artandi MK, Shah N, Kim SK, Artandi SE - PLoS Genet. (2007)

Conditional Expression of a Catalytically Inactive TERT Protein Induces the Anagen Phase of Hair Follicles(A) Diagram of TERT protein structure shows domains conserved among all RTs. Three conserved aspartates (shown) are required for RT function. D702 in motif A was mutated to alanine, to create the mouse TERTci allele.(B) Mouse TERTci fails to reconstitute telomerase activity when transduced into TERT−/− MEFs and inhibits endogenous telomerase activity in TERT+/+ MEFs by TRAP. TRAP activities were quantified by measuring band intensities, and relative telomerase activity (RTA) is shown below each lane.(C) Hair follicles cycle between an active growth phase (anagen) and a resting phase (telogen). E, epidermis; ORS, outer root sheath; HS, hair shaft; M, matrix cells; DP, dermal papilla; IRS, inner root sheath; B, bulge stem cell niche; S, sebaceous gland.(D) Northern blot shows doxycycline-dependent suppression of TERT mRNA in dorsal skin from iK5-TERT and iK5-TERTci mice.(E) Induction of either TERTci or TERT mRNA in mouse epidermis initiates an anagen cycle between days 50–60. Doxycycline was withdrawn at day 10. Controls including non-transgenic, K5-tTA single transgenic, and tetop-TERTci and tetop-TERT single transgenic mice remained in telogen (asterisks). In contrast, iK5-TERTci and iK5-TERT mice are in anagen (black arrows). H&E, 100×.
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Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2211538&req=5

pgen-0040010-g001: Conditional Expression of a Catalytically Inactive TERT Protein Induces the Anagen Phase of Hair Follicles(A) Diagram of TERT protein structure shows domains conserved among all RTs. Three conserved aspartates (shown) are required for RT function. D702 in motif A was mutated to alanine, to create the mouse TERTci allele.(B) Mouse TERTci fails to reconstitute telomerase activity when transduced into TERT−/− MEFs and inhibits endogenous telomerase activity in TERT+/+ MEFs by TRAP. TRAP activities were quantified by measuring band intensities, and relative telomerase activity (RTA) is shown below each lane.(C) Hair follicles cycle between an active growth phase (anagen) and a resting phase (telogen). E, epidermis; ORS, outer root sheath; HS, hair shaft; M, matrix cells; DP, dermal papilla; IRS, inner root sheath; B, bulge stem cell niche; S, sebaceous gland.(D) Northern blot shows doxycycline-dependent suppression of TERT mRNA in dorsal skin from iK5-TERT and iK5-TERTci mice.(E) Induction of either TERTci or TERT mRNA in mouse epidermis initiates an anagen cycle between days 50–60. Doxycycline was withdrawn at day 10. Controls including non-transgenic, K5-tTA single transgenic, and tetop-TERTci and tetop-TERT single transgenic mice remained in telogen (asterisks). In contrast, iK5-TERTci and iK5-TERT mice are in anagen (black arrows). H&E, 100×.
Mentions: To determine if TERT's RT activity is required to promote stem cell proliferation, we inactivated TERT RT function by introducing a point mutation in its active site. TERT's RT domain comprises seven sub-regions of homology conserved among all telomerases and RTs [20]. Within motifs A and C, three aspartate residues are absolutely conserved and essential for catalytic function [20–22]. The aspartate residue in motif A was changed to alanine by site-directed mutagenesis of cDNAs encoding either human TERT or mouse TERT, yielding the TERTci allele (for catalytically inactive) (Figure 1A). Mouse and human TERTci proteins lacked telomerase activity when expressed in TERT−/− MEFs or primary human fibroblasts, respectively, whereas wild-type TERT proteins efficiently reconstituted telomerase enzyme function (Figures 1B and S1A). Consistent with this loss of RT function, human TERTci did not immortalize primary human fibroblasts, whereas wild-type TERT efficiently extended replicative lifespan and prevented replicative senescence (Figure S1B and S1C). TERTci proteins were efficiently expressed based on their ability to inhibit endogenous telomerase activity (Figures 1B and S1A) [20–22] and because TERT and TERTci protein levels were indistinguishable by Western blot (Figure S1D and S1E). Together, these data show that TERTci protein is catalytically inert and accumulates to levels similar to that of the wild-type protein.

Bottom Line: This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood.We show that TERT(ci) retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis.These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

View Article: PubMed Central - PubMed

Affiliation: Department of Medicine, Stanford School of Medicine, Stanford, California, United States of America.

ABSTRACT
Telomerase serves a critical role in stem cell function and tissue homeostasis. This role depends on its ability to synthesize telomere repeats in a manner dependent on the reverse transcriptase (RT) function of its protein component telomerase RT (TERT), as well as on a novel pathway whose mechanism is poorly understood. Here, we use a TERT mutant lacking RT function (TERT(ci)) to study the mechanism of TERT action in mammalian skin, an ideal tissue for studying progenitor cell biology. We show that TERT(ci) retains the full activities of wild-type TERT in enhancing keratinocyte proliferation in skin and in activating resting hair follicle stem cells, which triggers initiation of a new hair follicle growth phase and promotes hair synthesis. To understand the nature of this RT-independent function for TERT, we studied the genome-wide transcriptional response to acute changes in TERT levels in mouse skin. We find that TERT facilitates activation of progenitor cells in the skin and hair follicle by triggering a rapid change in gene expression that significantly overlaps the program controlling natural hair follicle cycling in wild-type mice. Statistical comparisons to other microarray gene sets using pattern-matching algorithms revealed that the TERT transcriptional response strongly resembles those mediated by Myc and Wnt, two proteins intimately associated with stem cell function and cancer. These data show that TERT controls tissue progenitor cells via transcriptional regulation of a developmental program converging on the Myc and Wnt pathways.

Show MeSH
Related in: MedlinePlus