The result includes the counts of both strands of telomeric repeats. Type I A, C, D, G or H indicate newly acquired ALT lines from type I PD350. The hybridisation was carried out overnight at room temperature. Region 1 and region 2 indicate specific regions inside the mTALT sequence. They protect the ends of chromosomes the same way plastic caps protect the ends of shoe laces. In theory, longer telomeres should allow a cell to divide more. The diluted cells were cytocentrifuged at 200 xg for 10 min using the suitably built cytospin funnels. What's more, telomeres represent a marker for how the body will age: After studying telomere length across multiple species, a research team out of Spain found that the rate of telomere decay over time could accurately predict the life span of that species. As a result, type I (mTALT) exhibited more pronounced changes in chromatin structure. (B) Volcano plots showing the PD800 differentially accessible regions (DARs) compared to PD100 of type I ALT mESCs (left) and type II ALT mESCs (right). The researchers used modified messenger RNA to extend the telomeres. From the ATAC sequencing reads, we collected and analysed telomeric repeat-containing reads. The slides were rinsed with PBST and treated for 1 hat room temperature with ABDIL-diluted secondary antibody. Gene-based annotation of the SNVs showed that most variants were located in intergenic regions and introns, and the relative proportions of variants corresponding to each DNA element were similar between ALT types or among the time points when SNVs occurred (Supplementary Figure S10C). This hypothesis may be supported by the increased telomere fragility in type I (mTALT) under SLX4 knock-down conditions. Although the phenotypes examined in each model are not identical, and different methods may have been used, we compared ATRX/DAXX mutation/expression, presence or absence of TERRA, telomere chromatin structure, and expression of genes that may regulate telomere chromatin (Supplementary Table S3). How can you lengthen your telomeres naturally, without dietary s. Is the ketogenic diet right for autoimmune conditions? Type I A-H indicate newly acquired ALT lines from type I PD350. Despite its rare nature, various ALT patterns may exist in mESCs. You can see that there is a range (a small "cloud") of normal values at any age, for both cell types. The exact origin of the telomeric transcript of type I ALT mESCs remains unknown; however, an unexplored regulatory layer between telomere structure and transcription probably exists in each ALT mESC type. Other Stanford co-authors of the paper are postdoctoral scholars Jennifer Brady, PhD, and Moritz Brandt, MD; senior research scientist Stphane Corbel, PhD; research associate Colin Holbrook; and Juan Santiago, PhD, professor of mechanical engineering. However, both the average length and telomere length heterogeneity increased in PD450 cells. One day it may be possible to target muscle stem cells in a patient with Duchenne muscular dystrophy, for example, to extend their telomeres. Telomeres are the ends of our chromosomes and function to protect them from damage. (I) Quantification of signal-free telomeres with metaphase FISH. See here for a list of our favorite vitamin D supplements, in case you need help reaching sufficient levels (spoiler: You probably do). (D) Gene clustering based on RNA expression profile across time points in type II ALT mESCs. For each ChIP reaction, 3 g of primary antibody was added (H3K27me3, abcam #ab192985; H3K9me3, abcam #ab8898). RNA was extracted using Trizol, chloroform and isopropanol precipitation methods. Box plot displays the median (black bar), first and third quartiles (box), minimum and maximum (whisker) and outliers (grey dots). Finally, do your best to exercise when you caneven if you have time for just a quick walk. Telomere Lengthening: Can You Lengthen Your Telomeres? - Longevity By Stanford Medicine is an integrated academic health system comprising the Stanford School of Medicine and adult and pediatric health care delivery systems. Considering the similarity between type II ALT mESCs and ALT cancer cells, we examined well-known molecular markers of the ALT phenotype in type II ALT mESCs. Study shows telomere length in humans can be altered by medical drugs First, WGS reads were filtered to remove duplicated and poorly aligned reads and indexed with SAMTools (version 1.7, samtools view -h -q 1 -F 0400 -f 02 input.bam | samtools view -bS - > output_file, samtools index input) (39,40). Normal telomeres are protected by telomere-binding proteins and have a closed chromatin structure that prevents the DNA damage response (DDR). GO terms of Group 6 in type I (mTALT) included covalent chromatin modification, histone modification and DNA recombination (Supplementary Figure S4A); those of type II included mRNA processing, RNA splicing and RNA transport (Supplementary Figure S4B). 29 polymerase-amplified products were blotted onto a nitrocellulose membrane with a slot-blotter, and the nitrocellulose membrane was crosslinked with UV. There are also implications for treating conditions of aging, such as diabetes and heart disease. Whole genome sequencing, RNA-seq and ATAC-seq data are deposited in the Korean Nucleotide Archive (KoNA, https://kobic.re.kr/kona) with the accession ID PRJKA220347. On the other hand, in type I ALT mESCs, the overall ratio of some variants increased and the composition within the variant changed (Supplementary Figure S2A and B). Genomic DNA for telomere restriction fragment assay was extracted in agarose plugs to get megabase-sized DNA, based on telomere overhang assay protocol that modified previously reported methods (35). However, c-circle and telomeric sister chromatid exchange were not increased in type II ALT mESCs. For example, active promoter regions showed a good coincidence between the directions of change in RNA and chromatin accessibility (type I (mTALT), RNA-increased peaks: 98.83%, RNA-decreased peaks: 96.56%; type II, RNA-increased peaks: 84.61%, RNA-decreased peaks: 99.31%; Figure 4D). (G) Principal component analysis (PCA) plot of transcriptomes, including newly acquired ALT survivor lines. P values correspond to a two-tailed unpaired t-test. Vegetarian dietary patterns may reduce cardiovascular disease risk, Cure for mitochondrial diseases steps closer with stem cell breakthrough, DNA changes that affect human height uncovered, Creating embryos in the lab: How, why, and what the future holds. We have included model illustrations for reference of the telomere structure in each type of ALT mESC (Figure 1E). De novo motif discovery was done by HOMER (Hypergeometric Optimization of Motif EnRichment, version 4.11) with default options (59). Telomeres help protect the ends of your chromosomes from damage or fusing with nearby chromosomes. As the cells went through growth arrest and ALT activation, their positions changed in different directions (Figure 4A and Supplementary Figure S6B). Stemness-related transcription factors seemed to regulate numerous DARs regardless of the ALT type (Supplementary Table S1). However, this synthesis was counteracted by the SLX4SLX1ERCC4 complex, which promotes telomere exchange without extending the telomeres (83). Telomeres are the caps at the end of each chromosome that protect your DNA. Ramunas says the team was pleasantly surprised to find that modified TERT mRNA effectively increased telomere length. The cells were plated and cultured at 4050% confluency. At the level of individual genes, more genes were functionally affected by single nucleotide variants (SNVs) in type I ALT mESCs (1933 genes in type I versus 521 genes in type II). DNA plugs were restricted with AluI and HinfI, 50 units each, in 0.5 ml CutSmart buffer (NEB) overnight at 37C in a shaker. SARS-CoV-2 NSP1 induces mRNA cleavages on the ribosome, The HDAC6-RNF168 axis regulates H2A/H2A.X ubiquitination to enable double-strand break repair, BLM helicase protein negatively regulates stress granule formation through unwinding RNA G-quadruplex structures, Asymmetric dimerization in a transcription factor superfamily is promoted by allosteric interactions with DNA, Chemical Biology and Nucleic Acid Chemistry, Gene Regulation, Chromatin and Epigenetics, https://demarzolab.pathology.jhmi.edu/telometer/, http://www.bioinformatics.babraham.ac.uk/projects/fastqc/, https://github.com/harvardinformatics/ATAC-seq/blob/master/atacseq/removeChrom.py, https://doi.org/10.1101/2022.04.08.487650, https://doi.org/10.1101/2022.03.25.485759, https://creativecommons.org/licenses/by-nc/4.0/, Receive exclusive offers and updates from Oxford Academic. featureCounts was used for counting mapped reads for genomic regions (version 2.0.3, featureCounts -T 10 -a Mus_musculus.GRCm38.102.gtf -s 2 -p -B -C -t exon -g gene_id *sortedByCoord.out.bam) (46). She also loves the Mediterranean diet (which prioritizes omega-3s as well). BLM knock-down significantly reduced the telomere length of type II ALT mESCs. The reaction was terminated by adding 50 ul tagmentation stop buffer (10 mM pH 8.0 TrisCl, 20 mM pH 8.0 EDTA). Computel was used to estimate telomere length from WGS data and ATAC sequencing data (version 1.2, genome length 2730871774, default options), and telomere variant sequences were also identified and counted. The TelG-Cy3 PNA probe was placed on the slides and denatured for 10 min at 70C before being hybridised for 2 h at room temperature in the dark. The research team, led by John Ramunas, PhD, and Eduard Yakubov, PhD, publish their findings in The FASEB Journal. Even in species having telomerase, ALT may appear owing to experimental telomerase deletion. ). Type II B, D or F indicate newly acquired ALT lines from type II PD400. Schematic comparing the two types of ALT mESCs. . So far gene therapy meant to lengthen telomeres is inconclusive, however there are ways to help delay the shortening of these vital life threads and maintain the heathy lengths that you still have. Cell pellet was resuspended in 50 ul transposition mixture (25 ul 2TD buffer, 2.5 ul transposase, 16.5 ul PBS, 0.5 ul 1% digitonin, 0.5 ul 10% Tween-20, 5 ul DW) (2TD buffer=20 mM pH 7.6 Tris-Cl, 10 mM MgCl2, 20% dimethyl formamide). Colino-Sanguino Y., Clark S.J., Valdes-Mora F. Moreno-Andrs D., Yokoyama H., Scheufen A., Holzer G., Lue H., Schellhaus A.K., Weberruss M., Takagi M., Antonin W. Heaphy C.M., DeWilde R.F., Jiao Y., Klein A.P., Edil B.H., Shi C., Bettegowda C., Rodriguez F.J., Eberhart C.G., Hebbar S. Birger Y., West K.L., Postnikov Y.V., Lim J.H., Furusawa T., Wagner J.P., Laufer C.S., Kraemer K.H., Bustin M. Postnikov Y.V., Kurahashi T., Zhou M., Bustin M. Cuddapah S., Schones D.E., Cui K., Roh T.-Y., Barski A., Wei G., Rochman M., Bustin M., Zhao K. Hartlieb S.A., Sieverling L., Nadler-Holly M., Ziehm M., Toprak U.H., Herrmann C., Ishaque N., Okonechnikov K., Gartlgruber M., Park Y.-G. et al. The latter has even been shown to help lengthen and protect telomeres, according to researchers at the University of California: Specifically, they found that meditators have significantly higher telomerase activity than non-meditators. Although most telomere length maintenance problems are solved by telomerase, in some cancer cells, telomere length is maintained by a recombination-mediated mechanism called alternative lengthening of telomeres (ALT) (9,10). Interestingly, PD350 of type I ALT mESCs and PD400 of type II ALT mESCs already diverged in different directions, indicating that each cell group had its own potential for different ALT mechanisms despite having the same origin. Nonetheless, the potential impact of persistent telomere instability on the entire genome needs to be further explored. Krista Conger is a senior science writer in the Office of Communications. (A) Principal component analysis (PCA) plot of transcriptomes of previously stored mESCs in Figure 1A. Moreover, clustering by individual genes (Supplementary Figure S5B), the chromatin remodelling gene set (GO:0006338, Figure 3H), or the DNA repair gene set (GO:0006281, Figure 3I) replicated this pattern. Type II A-L indicate newly acquired ALT lines from type II PD400. (H) Quantification of telomere length with metaphase FISH. Quantitative proteomics identified the gene expression signatures of the two types of ALT mESCs. (H) Hierarchical clustering of transcriptomes of both types of ALT mESCs and newly acquired ALT lines with the chromatin remodelling gene set (GO:0006338). The remaining pre-amplified mixture was amplified with qPCR with the determined additional cycles. The R code used in the study is uploaded to the following git repository: https://github.com/thecalab/NAR_revision(permanent DOI: https://doi.org/10.5281/zenodo.8108844). The analysis was performed in duplicate for PD100 and triplicate for PD800 in both types of ALT. To better examine the correlation between changes in chromatin structure and RNA expression, the DARs of transcription-regulatory regions (active promoter, bivalent promoter, super enhancer and enhancer) were assigned to individual genes. Scale bar, 3 m. These findings highlight the dynamic and complex nature of ALT. SoftWoRx was used to capture the images (version 6.11). However, we found that the chromatin state of telomeres can vary not only among ALT models but also among ALT types within the same model. The numbers next to the segment name indicate number of peaks coinciding with gene expression changes/ total number of peaks. Pleurodeles waltl, a salamander species, is the first vertebrate species that has been shown to rely solely on ALT at the whole-organism level (21). The cells were fixed in 3.7% formaldehyde and permeabilized in KCM buffer (120 mM KCl, 20 mM NaCl, 10 mM TrisCl, pH 7.5, 0.1% Triton X-100). Also, it is optimised to increase the diversity of the cluster, so that the inter-cluster variability is higher than the intra-cluster variability. We explored how the characteristics of the two types of ALT lines reflect their fates using multi-omics approaches. Telomeric probe (10 pmol/ml dig-(CCCTAA)x4) was hybridised with DNA in DIG Easy Hyb buffer (Roche #11603558001) at 42C overnight on a rotator. Roy. 2009 - 2023 mindbodygreen LLC. Counted sequencing reads were normalised and basically analysed (hierarchical clustering and principal component analysis) with R library DESeq2 (1.32.0) (47). 9 Ways Tending Your Telomeres Keep You Younger Longer However, stress can even impact your health on a much deeper levellike DNA level. In case of telomeric recombination, type I resolution leads to interrupted telomere replication and higher crossover frequency, but type II dissolution promotes telomere lengthening. In summary, the effect of SLX4 on single chromatid exchange showed a different direction of change in both types of ALT mESCs. The CNVs of both types of ALT were prominent in the subtelomeric region (distal 200 kbof each q arm), thus indicating that the telomere dysfunction-induced rearrangement can more severely affect the adjacent regions (Figure 5E). Afterward, they were spun down at 16000xg for 20 min in a pre-cooled centrifuge. Although the initiation and overall framework of the ALT pathway remain unclear, the prevailing hypothesis is that one or more DNA repair pathways promote template-mediated telomere maintenance (11). Next, we clustered genes based on the expression profile across time points (Figure 3C andD and Supplementary Figure S4). All rights reserved. Both restriction enzymes can cut whole genomic DNA (4 bp cutters), except in the telomeres. In type I (mTALT) telomeres, H3K9me3 foci decreased during the senescence (PD350) and ALT activation phases (PD450), then increased more than the level of PD100 as ALT stabilised (PD800). Is It Possible to Lengthen Telomeres With Diet & Exercise? It's an enzyme produced by the body, so what research is looking into is getting the body to increase telomerase to keep the telomeres from shortening ().There are drugs that can increase telomerase activity but unfortunately have been shown to increase the rate of growth of some types of cancer. For type I (mTALT), more chromatin modification- and nucleosome assembly-related terms (Figure 2H; green) were included; for type II, DNA repair-related and DNA replication-related terms appeared (Figure 2I; blue). Shelterin density, unrevealed telomere structures, replication mechanism or other dissimilarities might explain these findings. Overall, the DNA repair pathway appeared to be enhanced in type II ALT mESCs. (K) (left) Representative images of ALT-associated PML bodies (APB) of type II ALT mESCs. On the contrary, in type I ALT mESCs, telomere damage signals increased after ALT activation (32). The telomeric transcript-based R-loop structure may be a critical source of telomere instability in type I ALT mESCs. Key points. For induced cell fate switch, various chromatin-related barriers must be overcome. Induction of the alternative lengthening of telomeres pathway by The degPatterns function of the DEGreport (version 1.28.0) library (http://lpantano.github.io/DEGreport/) was used to find common clusters across both types of mESC ALT cell lines by finding gene sets showing a similar expression pattern between sample groups. Scale bar, 100 m. Similar to type I ALT mESCs (8243 proteins quantified), 8360 proteins were quantified in type II ALT mESCs. Identifying which changes occurring in telomerase-deficient cells during telomere shortening activate ALT is challenging. Alternatively, telomeric chromatin could be spatially heterogeneous, containing both euchromatin and heterochromatin (92). Telomere extension turns back aging clock in cultured human cells Lachner M., OCarroll D., Rea S., Mechtler K., Jenuwein T. Rosenfeld J.A., Wang Z., Schones D.E., Zhao K., DeSalle R., Zhang M.Q. One, the homozygous double knock-out (DKO) 741, used the non-telomeric unique sequence, mTALT, of 7.4 kbfrom a subtelomere of chromosome 11 for telomere lengthening, based on reconstruction of its telomere structure. Each dot represents the number of TIF foci of each cell, and the lines represent the means and SDs from304 cells per condition in two biologically independent replicates. Interestingly, telomeric chromatin appeared closed, whereas telomeric transcripts were highly produced in neuroblastoma, mESC (2019), and type I ALT mESCs. As a result, telomere damage persists, thereby leading to genomic instability. Therefore, neither DNA mutations nor decreased gene expression of ATRX/DAXX appeared to contribute to the activation of ALT in mESCs. In particular, recombination-dependent replication mechanisms, such as break-induced replication (BIR), have been reported to be important for ALT telomere synthesis (1315). Previously, the telomeres of ALT cancers were reported to contain more variant telomeric repeats than telomerase-based cells (66,67). Many activities can help you relieve stress, like exercise, breathwork, and meditation. (F) Estimated telomere length using WGS data of type II ALT mESCs. Next, we investigated whether CNVs accumulated in both types of ALT mESCs following telomere crisis and ALT activation. But now, Ramunas, Yakubov and their team say they have found a way to extend the length of telomeres, which could increase the number of human cells available for studying. Eat a nutritious diet. In other words, whether chromatin regulation by ATRX/DAXX (or by their disruption) is required for ALT phenotypes seems to be model dependent. Bar of senescence indicates the number of novel variants in PD350 in type I or PD400 in type II, ALT initiation represents novel variants in PD450 and ALT maintenance represents novel variants in PD800. DNA in gel was denatured (0.5 M NaOH, 1.5 M NaCl), neutralised (0.5 M pH 7.5 TrisHCl, 1.5 M NaCl), and transferred to nitrocellulose membrane. In contrast to type I, type II ALT mESCs did not amplify mTALT sequences and maintained two single nucleotide polymorphisms (SNPs) within the mTALT region that originated from chromosomes 11 and 13 (Supplementary Figure S1D). Support teaching, research, and patient care. Postdoctoral scholar John Ramunas, PhD, of Stanford shares lead authorship with Eduard Yakubov, PhD, of the Houston Methodist Research Institute. ALT activation in telomerase-deficient cells can be a distinct case of cellular plasticity. For batch correction, we used a full quantile normalization with a betweenLaneNormalization function in RUVSeq package (1.26.0) (60) and a varianceStabilizingTransformation function in DESeq2 package (1.32.0). We found that the telomere of type I ALT mESCs exhibited a more closed state, while the telomere of type II ALT mESCs was more open compared to the pre-ALT state, which is not readily available in most ALT models. Differences in the DNA repair process in the two ALT types are likely to impact not only the telomere but the entire genome. Consistently, the levels of H3K9me3 decreased in post-ALT telomeres (PD450 and PD800) compared with those in pre-ALT telomeres (PD100 and PD400) in type II ALT mESCs with immunofluorescence (Supplementary Figure S9A and S9B). But what are they in the first place? in journalism and a minor in womens, gender, and queer studies from California Polytechnic State University, San Luis Obispo. Linear cycle and relative fluorescence were plotted and the cycle number that corresponds to of maximum fluorescent intensity was determined. However, SLX4 knock-down did not affect the telomere length of type I ALT mESCs. "Aging is not, as thought for so long, a slippery slope towards infirmity and decay." On a recent episode of the mindbodygreen podcast, internationally renowned health psychologistElissa Epel, Ph.D, actually studied the effects of omega-3s on telomeres specifically. Fragility can be expressed or suppressed in a complex manner depending on the genetic or epigenetic environment. Previous attempts to deliver mRNA-encoding TERT caused an immune response against telomerase, which could be deleterious. Five Foods That Protect Your Telomeres and Extend Your Life Support Lucile Packard Children's Hospital Stanford and child and maternal health. Terc knock-out mouse embryonic stem cells were generated and provided by H. Niida and have been described in a previous paper (31). To quantify telomeres in the final ChIP DNA, only the primers capable of quantifying telomeric repeats (left: ACACTAAGGTTTGGGTTTGGGTTTGGGTTTGGGTTAGTGT; right: TGTTAGGTATCCCTATCCCTATCCCTATCCCTATCCCTAACA) were employed from a previous study (64).

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