Transposable elements (TEs) are major components of eukaryotic genomes. However, the extent of their impact on genome evolution, function, and disease remain a matter of intense interrogation. The rise of genomics and large-scale functional assays has shed new light on the multi-faceted activities of TEs and implies that they should no longer be marginalized. Here, we introduce the fundamental properties of TEs and their complex interactions with their cellular environment, which are crucial to understanding their impact and manifold consequences for organismal biology. While we draw examples primarily from mammalian systems, the core concepts outlined here are relevant to a broad range of organisms.

SIRT6 is a mammalian homolog of the yeast Sir2 deacetylase that promotes longevity in yeast and invertebrates. Mice deficient for SIRT6 exhibit premature aging and genome instability. Here we show that in mammalian cells subjected to oxidative stress SIRT6 is recruited to the sites of DNA double-strand breaks (DSBs) and strongly stimulates both pathways of DSB repair, nonhomologous end joining and homologous recombination. We found that SIRT6 physically associates with PARP1 leading to stimulation of PARP1 poly-ADP-ribose polymerase activity. Mono-ADP-ribosylation activity of SIRT6 is sufficient for the activation of PARP1 in vitro, while both mono-ADP-ribosylation and deacetylation activities are required for the stimulation of DSB repair in vivo. Our results suggest that SIRT6 mono-ADP-ribosylates PARP1 on lysine 521 thereby stimulating PARP1 activity and enhancing DSB repair under oxidative stress. We propose that SIRT6 functions as a regulator integrating oxidative stress signaling and DNA damage response.

Retrotransposable elements (RTEs) are deleterious at multiple levels, and failure of host surveillance systems can thus have negative consequences. However, the contribution of RTE activity to aging and age-associated diseases is not known. Here we show that during cellular senescence LINE-1 elements (L1s) become transcriptionally derepressed and activate a type-I interferon (IFN-I) response. The IFN-I response is a novel phenotype of late senescence and contributes to the maintenance of the senescence associated secretory phenotype (SASP). The IFN-I response is triggered by cytoplasmic L1 cDNA, and is antagonized by nucleoside reverse transcriptase inhibitors (NRTIs) that inhibit the L1 reverse transcriptase (RT). Treatment of aged mice with the NRTI lamivudine downregulated IFN-I activation and age-associated inflammation in several tissues. We propose that RTE activation is an important component of sterile inflammation that is a hallmark of aging, and that L1 RT is a relevant target for the treatment of age-associated disorders.

The naked mole-rat displays exceptional longevity, with a maximum lifespan exceeding 30 years1–3. This is the longest reported lifespan for a rodent species and is especially striking considering the small body mass of the naked mole-rat. In comparison, a similarly sized house mouse has a maximum lifespan of 4 years4,5. In addition to their longevity, naked mole-rats show an unusual resistance to cancer. Multi-year observations of large naked mole-rat colonies did not detect a single incidence of cancer2,6. Here we identify a mechanism responsible for the naked mole-rat’s cancer resistance. We found that naked mole-rat fibroblasts secrete extremely high molecular weight hyaluronan (HA), which is over five times larger than human or mouse HA. This high molecular weight HA accumulates abundantly in naked mole rat tissues due to the decreased activity of HA-degrading enzymes and a unique sequence of hyaluronan synthase 2 (HAS2). Furthermore, the naked mole-rat cells are more sensitive to HA signaling, as the naked mole rat cells have a higher affinity to HA than the mouse or human cells. Perturbation of the signaling pathways sufficient for malignant transformation of mouse fibroblasts fails to transform naked mole-rat cells. However, once high molecular weight HA is removed by either knocking down HAS2 or overexpressing the HA-degrading enzyme, Hyal2, naked mole-rat cells become susceptible to malignant transformation and readily form tumors in mice. We speculate that naked mole-rats have evolved a higher concentration of HA in the skin to provide skin elasticity needed for life in underground tunnels. This trait may have then been co-opted to provide cancer resistance and longevity to this species.

The two major pathways for repair of DNA double-strand breaks (DSBs) are homologous recombination (HR) and nonhomologous end joining (NHEJ). HR leads to accurate repair, while NHEJ is intrinsically mutagenic. To understand human somatic mutation it is essential to know the relationship between these pathways in human cells. Here we provide a comparison of the kinetics and relative contributions of HR and NHEJ in normal human cells. We used chromosomally integrated fluorescent reporter substrates for real-time in vivo monitoring of the NHEJ and HR. By examining multiple integrated clones we show that the efficiency of NHEJ and HR is strongly influenced by chromosomal location. Furthermore, we show that NHEJ of compatible ends (NHEJ-C) and NHEJ of incompatible ends (NHEJ-I) are fast processes, which can be completed in approximately 30 min, while HR is much slower and takes 7h or longer to complete. In actively cycling cells NHEJ-C is twice as efficient as NHEJ-I, and NHEJ-I is three times more efficient than HR. Our results suggest that NHEJ is a faster and more efficient DSB repair pathway than HR.

DNA double-strand breaks (DSBs) are dangerous lesions that can lead to potentially oncogenic genomic rearrangements or cell death. The two major pathways for repair of DSBs are nonhomologous end joining (NHEJ) and homologous recombination (HR). NHEJ is an intrinsically error-prone pathway while HR results in accurate repair. To understand the origin of genomic instability in human cells it is important to know the contribution of each DSB repair pathway. Studies of rodent cells and human cancer cell lines have shown that the choice between NHEJ or HR pathways depends on cell cycle stage. Surprisingly, cell cycle regulation of DSB repair has not been examined in normal human cells with intact cell cycle checkpoints. Here we measured the efficiency of NHEJ and HR at different cell cycle stages in hTERT-immortalized diploid human fibroblasts. We utilized cells with chromosomallyintegrated fluorescent reporter cassettes, in which a unique DSB is introduced by a rare-cutting endonuclease. We show that NHEJ is active throughout the cell cycle, and its activity increases as cells progress from G 1 to G 2 /M (G 1 < S < G 2 /M). HR is nearly absent in G 1 , most active in the S phase, and declines in G 2 /M. Thus, in G 2 /M NHEJ is elevated, while HR is on decline. This is in contrast to a general belief that NHEJ is most active in G 1 , while HR is active in S, G 2 and M. The overall efficiency of NHEJ was higher than HR at all cell cycle stages. We conclude that human somatic cells utilize error-prone NHEJ as the major DSB repair pathway at all cell cycle stages, while HR is used, primarily, in the S phase.

Graphical Abstract Highlights d SIRT6 KO mice accumulate L1 cDNA, triggering interferon response via cGAS pathway d Wild-type aged mice accumulate L1 cDNA and display type I interferon response d Reverse-transcriptase inhibitors rescue type I interferon response and DNA damage d Reverse-transcriptase inhibitors extend lifespan and improve health of SIRT6 KO mice SUMMARYMice deficient for SIRT6 exhibit a severely shortened lifespan, growth retardation, and highly elevated LINE1 (L1) activity. Here we report that SIRT6-deficient cells and tissues accumulate abundant cytoplasmic L1 cDNA, which triggers strong type I interferon response via activation of cGAS.Remarkably, nucleoside reverse-transcriptase inhibitors (NRTIs), which inhibit L1 retrotransposition, significantly improved health and lifespan of SIRT6 knockout mice and completely rescued type I interferon response. In tissue culture, inhibition of L1 with siRNA or NRTIs abrogated type I interferon response, in addition to a significant reduction of DNA damage markers. These results indicate that L1 activation contributes to the pathologies of SIRT6 knockout mice. Similarly, L1 transcription, cytoplasmic cDNA copy number, and type I interferons were elevated in the wild-type aged mice. As sterile inflammation is a hallmark of aging, we propose that modulating L1 activity may be an important strategy for attenuating age-related pathologies. Context and SignificanceMammalian aging is complex and likely reflects accumulated damage to our genes/DNA. Retrotransposons are a special class of parasitic genetic elements that can replicate their DNA within our genes, at times amounting to up to 20% of human DNA. Retrotransposons, such as the commonly occurring L1, have been associated with aging, neurodegeneration, and cancer. University of Rochester scientists uncovered L1 retrotransposons as the culprit in many aspects of accelerated aging in mice, a model for human aging. They also linked these special gene elements to inflammation. Experimentally blocking retrotransposon amplification improved the health and lifespan of mice. Although there is a long road ahead, inhibiting retrotransposon activity, and the related inflammation, could eventually be a therapy for age-related diseases.

The naked mole-rat is the longest living rodent with a maximum lifespan exceeding 28 years. In addition to its longevity, naked mole-rats have an extraordinary resistance to cancer as tumors have never been observed in these rodents. Furthermore, we show that a combination of activated Ras and SV40 LT fails to induce robust anchorage-independent growth in naked mole-rat cells, while it readily transforms mouse fibroblasts. The mechanisms responsible for the cancer resistance of naked mole-rats were unknown. Here we show that naked mole-rat fibroblasts display hypersensitivity to contact inhibition, a phenomenon we termed ''early contact inhibition.'' Contact inhibition is a key anticancer mechanism that arrests cell division when cells reach a high density. In cell culture, naked mole-rat fibroblasts arrest at a much lower density than those from a mouse. We demonstrate that early contact inhibition requires the activity of p53 and pRb tumor suppressor pathways. Inactivation of both p53 and pRb attenuates early contact inhibition. Contact inhibition in human and mouse is triggered by the induction of p27 Kip1 . In contrast, early contact inhibition in naked mole-rat is associated with the induction of p16 Ink4a . Furthermore, we show that the roles of p16 Ink4a and p27 Kip1 in the control of contact inhibition became temporally separated in this species: the early contact inhibition is controlled by p16 Ink4a , and regular contact inhibition is controlled by p27 Kip1 . We propose that the additional layer of protection conferred by two-tiered contact inhibition contributes to the remarkable tumor resistance of the naked mole-rat.longevity ͉ p16Ink4a ͉ p53 ͉ pRb ͉ tumor suppressor