Telomere Shortening Impairs Regeneration of the Olfactory Epithelium in Response to Injury but Not Under Homeostatic Conditions

Atrophy of the olfactory epithelium (OE) associated with impaired olfaction and dry nose represents one of the most common phenotypes of human aging. Impairment in regeneration of a functional olfactory epithelium can also occur in response to injury due to infection or nasal surgery. These complications occur more frequently in aged patients. Although age is the most unifying risk factor for atrophic changes and functional decline of the olfactory epithelium, little is known about molecular mechanisms that could influence maintenance and repair of the olfactory epithelium. Here, we analyzed the influence of telomere shortening (a basic mechanism of cellular aging) on homeostasis and regenerative reserve in response to chemical induced injury of the OE in late generation telomere knockout mice (G3 mTerc−/−) with short telomeres compared to wild type mice (mTerc+/+) with long telomeres. The study revealed no significant influence of telomere shortening on homeostatic maintenance of the OE during mouse aging. In contrast, the regenerative response to chemical induced injury of the OE was significantly impaired in G3 mTerc−/− mice compared to mTerc+/+ mice. Seven days after chemical induced damage, G3 mTerc−/− mice exhibited significantly enlarged areas of persisting atrophy compared to mTerc+/+ mice (p = 0.031). Telomere dysfunction was associated with impairments in cell proliferation in the regenerating epithelium. Deletion of the cell cycle inhibitor, Cdkn1a (p21) rescued defects in OE regeneration in telomere dysfunctional mice. Together, these data indicate that telomere shortening impairs the regenerative capacity of the OE by impairing cell cycle progression in a p21-dependent manner. These findings could be relevant for the impairment in OE function in elderly people

Der Verlust von p53 verhindert das Entfernen von chromosomal-intestinalen Stammzellen in Mäusen mit dysfunktionellen Telomeren

Deletion of components of the DNA-damage signaling pathway can rescue the maintenance and the function of stem cells in aging telomere dysfunctional mice (Choudhury et al; Schätzlein et al.). p53 is activated in response to DNA damage leading to induction of apoptosis or p21-dependent cell cycle arrest. Depletion of intestinal stem cells and crypt atrophy represent major phenotypes in aging telomere dysfunctional mice. To analyze the in vivo role of p53 in this context, Terc-/- mice were crossed to conditional, intestine-specific Trp53-knock out mice. In aging IF1 Terc+/- Trp53 deletion did not induce an obvious intestinal phenotype until 18 months. In contrast Trp53 deletion significantly shortened the lifespan of iG4 Terc-/- Trp53-/- animals compared to iG4 Terc-/- Trp53+/+. This reduction correlated with earlier appearance of crypt atrophy and weight loss but was not associated with tumor formation. Deletion of the Trp53 gene resulted in complete abrogation of p21 activation, accumulation of DNA damage and an increase in apoptosis in the intestinal epithelium of these mice. Moreover this correlated with an accumulation of intestinal stem cells carrying high levels of DNA damage. Our results provide first experimental evidence that p53 induction has a protective role preventing accumulation of damaged stem cells and organ degeneration induced in aging in the context of telomere dysfunction

A Subset of Histone H2B Genes Produces Polyadenylated mRNAs under a Variety of Cellular Conditions

<div><p>Unlike other metazoan mRNAs, replication-dependent histone gene transcripts are not polyadenylated but instead have a conserved stem-loop structure at their 3′ end. Our previous work has shown that under certain conditions replication-dependent histone genes can produce alternative transcripts that are polyadenylated at the 3′ end and, in some cases, spliced. A number of microarray studies examining the expression of polyadenylated mRNAs identified changes in the levels of histone transcripts e.g. during differentiation and tumorigenesis. However, it remains unknown which histone genes produce polyadenylated transcripts and which conditions regulate this process. In the present study we examined the expression and polyadenylation of the human histone H2B gene complement in various cell lines. We demonstrate that H2B genes display a distinct expression pattern that is varies between different cell lines. Further we show that the fraction of polyadenylated <i>HIST1H2BD</i> and <i>HIST1H2AC</i> transcripts is increased during differentiation of human mesenchymal stem cells (hMSCs) and human fetal osteoblast (hFOB 1.19). Furthermore, we observed an increased fraction of polyadenylated transcripts produced from the histone genes in cells following ionizing radiation. Finally, we show that polyadenylated transcripts are transported to the cytoplasm and found on polyribosomes. Thus, we propose that the production of polyadenylated histone mRNAs from replication-dependent histone genes is a regulated process induced under specific cellular circumstances.</p></div

Radiation induced elevated expression of spliced histone transcripts.

<p>A549 cells were exposed to gamma-irradiation (6 Gy) and incubated for 24 hours. RNA was extracted and analyzed by qRT-PCR for (A) spliced and (B) total <i>HIST1H2BD</i> and <i>HIS1H2AC</i>. Values were normalized to <i>RPLP0</i>. Mean±SD, n = 3. (C) Expression of spliced <i>HIST1H2BD</i> and <i>HIST1H2AC</i> transcripts was normalized to the total <i>HIST1H2BD</i> and <i>HIST1H2AC</i> levels. P-values were calculated and statistical significance is represented as follows (*P≤0.05; **P≤0.01).</p

Expression of normal and PolyA⁺<i>HIST1H2BD</i> and <i>HIST1H2AC</i> transcripts in HCT116 cells.

<p>(A) Cells were treated with Nutlin-3a as in Fig. 2. RNA was reverse transcribed into cDNA using both random and poly-T primers to check the mRNA levels of <i>HIST1H2BD</i> and <i>HIST1H2AC</i>, total and polyadenylated transcripts respectively. Values were normalized to <i>RPLP0</i> expression. Mean±SD, n = 3. (B) Enrichment for polyadenylated histone transcripts using PolyATtract® mRNA Isolation System III. Total RNA was used to isolate polyadenylated RNA and reverse transcribed using poly-T primers. Expression of total and polyA⁺<i>HIST1H2BD</i> and <i>HIST1H2AC</i> transcripts was analyzed by qRT-PCR. Values were normalized to <i>RPLP0</i> expression. Mean±SD, n = 3. P-values were calculated and statistical significance was represented as follows (**P≤0.01; ***P≤0.001).</p

Nutlin-3a treatment down-regulates the expression of normal replication-dependent histone H2B genes and up-regulates the expression polyA⁺ transcripts.

<p>(A) Total expression of different replication-dependent histone H2B genes in control and Nutlin-3a treated HCT116 cells. Total RNA was reverse transcribed using random primers and analyzed by qRT-PCR for H2B genes as in Fig. 1. Mean±SD, n = 3. (B) Expression of polyA⁺ histone H2B transcripts in HCT116 cells upon Nutlin-3a treatment. PolyA⁺ mRNA was purified as described in materials and methods and reverse transcribed using random primers. Transcript levels of polyA⁺ H2B genes were analyzed by qRT-PCR. Mean±SD, n = 3. (C) Levels of polyA⁺ H2B transcripts normalized to the total H2B levels from (B). Mean±SD, n = 3.</p

Comparison of stem-loop sequences in H2B genes.

<p>Alignment of the stem-loop sequences of H2B genes performed with ClustalW2 multiple sequence alignments tools (<a href="http://www.ebi.ac.uk/Tools/msa/clustalw2/" target="_blank">http://www.ebi.ac.uk/Tools/msa/clustalw2/</a>). Highly expressed histones are marked in grey. Stem loop sequence is shown in orange, bases that are different from canonical are underlined.</p

Expression of the histone H2B gene complement in different cell lines.

<p>Expression of different H2B genes in the indicated cell lines was analyzed by qRT-PCR. Relative expression values between the individual genes were normalized using diploid genomic DNA (see materials and methods) and indicated as “Rel. gDNA units”. Mean±SD, n = 3.</p

Expression of spliced histone transcripts are increased during committed osteoblast differentiation.

<p>(A) hFOB 1.19 cells were differentiated into osteoblasts for 7 days. Alkaline phosphatase (<i>ALPL</i>) expression in undifferentiated (undiff.) and differentiated (OB) cells was analyzed by qRT-PCR. Values were normalized to <i>HNRNPK</i> expression. Mean±SD, n = 3. (B, C) Samples shown in (A) were examined for (B) spliced <i>HIST1H2BD</i> and (C) <i>HIST1H2AC</i> expression. Values were normalized to total <i>HISTH2BD</i> and <i>HISTH2AC</i> respectively. Mean±SD, n = 3. P-values were calculated and statistical significance is represented as follows (*P≤0.05; **P≤0.01).</p