Leukocyte telomere length (LTL) is a heritable biomarker of genomic aging. In this study, we perform a genome-wide meta-analysis of LTL by pooling densely genotyped and imputed association results across large-scale European-descent studies including up to 78,592 individuals. We identify 49 genomic regions at a false dicovery rate (FDR) < 0.05 threshold and prioritize genes at 31, with five highlighting nucleotide metabolism as an important regulator of LTL. We report six genome-wide significant loci in or near SENP7 , MOB1B , CARMIL1 , PRRC2A , TERF2, and RFWD3 , and our results support recently identified PARP1, POT1 , ATM, and MPHOSPH6 loci. Phenome-wide analyses in >350,000 UK Biobank participants suggest that genetically shorter telomere length increases the risk of hypothyroidism and decreases the risk of thyroid cancer, lymphoma, and a range of proliferative conditions. Our results replicate previously reported associations with increased risk of coronary artery disease and lower risk for multiple cancer types. Our findings substantially expand current knowledge on genes that regulate LTL and their impact on human health and disease.

Rett syndrome (RS) is a debilitating neurological disorder affecting mostly girls with heterozygous mutations in the gene encoding the methyl-CpG-binding protein MeCP2 on the X chromosome. Because restoration of MeCP2 expression in a mouse model reverses neurologic deficits in adult animals, reactivation of the wild-type copy of MeCP2 on the inactive X chromosome (Xi) presents a therapeutic opportunity in RS. To identify genes involved in MeCP2 silencing, we screened a library of 60,000 shRNAs using a cell line with a MeCP2 reporter on the Xi and found 30 genes clustered in seven functional groups. More than half encoded proteins with known enzymatic activity, and six were members of the bone morphogenetic protein (BMP)/TGF-β pathway. shRNAs directed against each of these six genes down-regulated X-inactive specific transcript (XIST), a key player in X-chromosome inactivation that encodes an RNA that coats the silent X chromosome, and modulation of regulators of this pathway both in cell culture and in mice demonstrated robust regulation of XIST. Moreover, we show that Rnf12, an X-encoded ubiquitin ligase important for initiation of X-chromosome inactivation and XIST transcription in ES cells, also plays a role in maintenance of the inactive state through regulation of BMP/TGF-β signaling. Our results identify pharmacologically suitable targets for reactivation of MeCP2 on the Xi and a genetic circuitry that maintains XIST expression and X-chromosome inactivation in differentiated cells.XIST | X inactivation | MeCP2 | Rett syndrome | BMP/TGF-β

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by using the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed alternative lengthening of telomeres (ALT). Cells that use ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We found that PML is required for the ALT mechanism, and that this necessity stems from APBs' role in localizing the BLM-TOP3A-RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

Sirtuins are a family of NAD+-dependent protein deacetylases that play critical roles in epigenetic regulation, stress responses, and cellular aging in eukaryotic cells. In an effort to identify small molecule inhibitors of sirtuins for potential use as chemotherapeutics as well as tools to modulate sirtuin activity, we previously identified a nonselective sirtuin inhibitor called cambinol (IC50 ≈ 50 μM for SIRT1 and SIRT2) with in vitro and in vivo antilymphoma activity. In the current study, we used saturation transfer difference (STD) NMR experiments with recombinant SIRT1 and 20 to map parts of the inhibitor that interacted with the protein. Our ongoing efforts to optimize cambinol analogues for potency and selectivity have resulted in the identification of isoform selective analogues: 17 with >7.8-fold selectivity for SIRT1, 24 with >15.4-fold selectivity for SIRT2, and 8 with 6.8- and 5.3-fold selectivity for SIRT3 versus SIRT1 and SIRT2, respectively. In vitro cytotoxicity studies with these compounds as well as EX527, a potent and selective SIRT1 inhibitor, suggest that antilymphoma activity of this compound class may be predominantly due to SIRT2 inhibition.

Replication gaps that persist into mitosis likely represent important threats to genome stability, but experimental identification of these gaps has proved challenging. We have developed a technique that allows us to explore the dynamics by which genome replication is completed before mitosis. Using this approach, we demonstrate that excessive allocation of replication resources to origins within repetitive regions, induced by SIR2 deletion, leads to persistent replication gaps and genome instability. Conversely, the weakening of replication origins in repetitive regions suppresses these gaps. Given known age-and cancer-associated changes in chromatin accessibility at repetitive sequences, we suggest that replication gaps resulting from misallocation of replication resources underlie age-and disease-associated genome instability.SIR2 | DNA replication | repetitive sequences | replication gaps | ribosomal DNA S taggered initiation of DNA replication, which is common across eukaryotes from fungi to humans, means that, at any given time, in S phase, only a fraction of replication origins is activated. In recent years, a model has emerged to explain this pattern of DNA replication (1, 2). This model assumes that the pool of initiation factors required to fire licensed origins in S phase is limited, and therefore sufficient to fire only a subset of licensed origins at any given time. Licensed origins differ in their ability to recruit factors required for firing, so origins with higher affinity or accessibility fire earlier than those with lower affinity or accessibility. After activating the initial set of origins, firing factors are released, enabling the next set of origins to fire. This results in successive waves of origin activation. Genome replication eventually finishes when areas with the least accessible origins are replicated.In healthy human cells, the least accessible genomic regions consist of repetitive DNA, which represents about half of the genome and tends to be compacted into heterochromatin. However, recent studies suggest widespread opening of hetrochromatin during carcinogenesis and aging (3-5). Such reorganization of chromatin would expose a new suite of origins within repetitive DNA that could potentially compete initiation factors away from unique portions of the genome, thereby disrupting the normal genome-wide hierarchy of replication timing. Increased origin activity within repetitive DNA could thus compromise replication elsewhere in the genome. Given the limited pool of initiation factors, we propose that an increase in density of active origins within repetitive regions could result in a decreased density of such origins in unique regions of the genome, which, when combined with the stochastic nature of origin firing, may occasionally result in replication gaps, i.e., unique regions of the genome that are unable to complete replication before mitosis. This so-called "Random Replication Gap Problem" (RRGP) is the subject of long-standing speculation, but such gaps have never been experim...

Telomeres consist of TTAGGG repeats bound by protein complexes that serve to protect the natural end of linear chromosomes. Most cells maintain telomere repeat lengths by utilizing the enzyme telomerase, although there are some cancer cells that use a telomerase-independent mechanism of telomere extension, termed Alternative Lengthening of Telomeres (ALT). Cells that employ ALT are characterized, in part, by the presence of specialized PML nuclear bodies called ALT-associated PML-Bodies (APBs). APBs localize to and cluster telomeric ends together with telomeric and DNA damage factors, which led to the proposal that these bodies act as a platform on which ALT can occur. However, the necessity of APBs and their function in the ALT pathway has remained unclear. Here, we used CRISPR/Cas9 to delete PML and APB components from ALT-positive cells to cleanly define the function of APBs in ALT. We find that PML is required for the ALT mechanism, and that this necessity stems from APBs' role in localizing the BLM-TOP3A-RMI (BTR) complex to ALT telomere ends. Strikingly, recruitment of the BTR complex to telomeres in a PML-independent manner bypasses the need for PML in the ALT pathway, suggesting that BTR localization to telomeres is sufficient to sustain ALT activity.

Type I interferon (IFN-I) signaling paradoxically impairs host immune responses during many primary and secondary bacterial infections. Lack of IFN-I receptor reduces bacterial replication and/or bacterial persistence during infection with several bacteria. However, the mechanisms that mediate the adverse IFN-I effect are incompletely understood. Here, we show that Usp18, an interferon-stimulated gene that negatively regulates IFN-I signaling, is primarily responsible for the deleterious effect of IFN-I signaling during infection of mice with Listeria monocytogenes or Staphylococcus aureus. Mechanistically, USP18 promoted bacterial replication by inhibiting antibacterial tumor necrosis factor-α (TNF-α) signaling. Deleting IFNAR1 or USP18 in CD11c-Cre+ cells similarly reduced bacterial titers in multiple organs and enhanced survival. Our results demonstrate that inhibiting USP18 function can promote control of primary and secondary bacterial infection by enhancing the antibacterial effect of TNF-α, which correlates with induction of reactive oxygen species (ROS). These findings suggest that USP18 could be targeted therapeutically in patients to ameliorate disease caused by serious bacterial infections.

Genetic ablation as well as pharmacological inhibition of sirtuin 2 (SIRT2), an NAD+-dependent protein deacylase, have therapeutic effects in various cancers and neurodegenerative diseases. Previously, we described the discovery of a dual SIRT1/SIRT2 inhibitor called cambinol (IC50 56 and 59 µM, respectively), which showed cytotoxic activity against cancer cells in vitro and a marked anti-proliferative effect in a Burkitt lymphoma mouse xenograft model. A number of recent studies have shown a protective effect of SIRT1 and SIRT3 in neurodegenerative and metabolic diseases as well as in certain cancers prompting us to initiate a medicinal chemistry effort to develop cambinol-based SIRT2-specific inhibitors devoid of SIRT1 or SIRT3 modulating activity. Here we describe potent cambinol-based SIRT2 inhibitors, several of which show potency of ~600 nM with >300 to >800-fold selectivity over SIRT1 and 3, respectively. In vitro, these inhibitors are found to be toxic to lymphoma and epithelial cancer cell lines. In particular, compounds 55 (IC50 SIRT2 0.25 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) and 56 (IC50 SIRT2 0.78 µM and <25% inhibition at 50 µM against SIRT1 and SIRT3) showed apoptotic as well as strong anti-proliferative properties against B-cell lymphoma cells.