Summary Rapamycin has been shown to extend lifespan in numerous model organisms including mice, with the most dramatic longevity effects reported in females. However, little is known about the functional ramifications of this longevity-enhancing paradigm in mammalian tissues. We treated 24-month-old female C57BL/6J mice with rapamycin for 3 months and determined health outcomes via a variety of noninvasive measures of cardiovascular, skeletal, and metabolic health for individual mice. We determined that while rapamycin has mild transient metabolic effects, there are significant benefits to late-life cardiovascular function with a reversal or attenuation of age-related changes in the heart. RNA-seq analysis of cardiac tissue after treatment indicated inflammatory, metabolic, and antihypertrophic expression changes in cardiac tissue as potential mechanisms mediating the functional improvement. Rapamycin treatment also resulted in beneficial behavioral, skeletal, and motor changes in these mice compared with those fed a control diet. From these findings, we propose that late-life rapamycin therapy not only extends the lifespan of mammals, but also confers functional benefits to a number of tissues and mechanistically implicates an improvement in contractile function and antihypertrophic signaling in the aged heart with a reduction in age-related inflammation.

Transcription activator-like effectors (TALEs) have revolutionized the field of genome engineering. We present here a systematic assessment of TALE DNA recognition, using quantitative electrophoretic mobility shift assays and reporter gene activation assays. Within TALE proteins, tandem 34-amino acid repeats recognize one base pair each and direct sequence-specific DNA binding through repeat variable di-residues (RVDs). We found that RVD choice can affect affinity by four orders of magnitude, with the relative RVD contribution in the order NG > HD ∼ NN ≫ NI > NK. The NN repeat preferred the base G over A, whereas the NK repeat bound G with 103-fold lower affinity. We compared AvrBs3, a naturally occurring TALE that recognizes its target using some atypical RVD-base combinations, with a designed TALE that precisely matches ‘standard’ RVDs with the target bases. This comparison revealed unexpected differences in sensitivity to substitutions of the invariant 5′-T. Another surprising observation was that base mismatches at the 5′ end of the target site had more disruptive effects on affinity than those at the 3′ end, particularly in designed TALEs. These results provide evidence that TALE–DNA recognition exhibits a hitherto un-described polarity effect, in which the N-terminal repeats contribute more to affinity than C-terminal ones.

A decline in skeletal muscle mass and function with aging is well recognized, but remains poorly characterized at the molecular level. Here, we report for the first time a genome-wide study of DNA methylation dynamics in skeletal muscle of healthy male individuals during normal human aging. We predominantly observed hypermethylation throughout the genome within the aged group as compared to the young subjects. Differentially methylated CpG (dmCpG) nucleotides tend to arise intragenically and are underrepresented in promoters and are overrepresented in the middle and 3′ end of genes. The intragenic methylation changes are overrepresented in genes that guide the formation of the junction of the motor neuron and myofibers. We report a low level of correlation of gene expression from previous studies of aged muscle with our current analysis of DNA methylation status. For those genes that had both changes in methylation and gene expression with age, we observed a reverse correlation, with the exception of intragenic hypermethylated genes that were correlated with an increased gene expression. We suggest that a minimal number of dmCpG sites or select sites are required to be altered in order to correlate with gene expression changes. Finally, we identified 500 dmCpG sites that perform well in discriminating young from old samples. Our findings highlight epigenetic links between aging postmitotic skeletal muscle and DNA methylation.

Transcription factor–DNA interactions are some of the most important processes in biology because they directly control hereditary information. The targets of most transcription factor are unknown. In this report, we introduce Bind-n-Seq, a new high-throughput method for analyzing protein–DNA interactions in vitro, with several advantages over current methods. The procedure has three steps (i) binding proteins to randomized oligonucleotide DNA targets, (ii) sequencing the bound oligonucleotide with massively parallel technology and (iii) finding motifs among the sequences. De novo binding motifs determined by this method for the DNA-binding domains of two well-characterized zinc-finger proteins were similar to those described previously. Furthermore, calculations of the relative affinity of the proteins for specific DNA sequences correlated significantly with previous studies (R2 = 0.9). These results present Bind-n-Seq as a highly rapid and parallel method for determining in vitro binding sites and relative affinities.

Zinc-finger nucleases (ZFNs) are important tools for genome engineering. Despite intense interest by many academic groups, the lack of robust noncommercial methods has hindered their widespread use. The modular assembly (MA) of ZFNs from publicly available one-finger archives provides a rapid method to create proteins that can recognize a very broad spectrum of DNA sequences. However, three-and four-finger arrays often fail to produce active nucleases. Efforts to improve the specificity of the one-finger archives have not increased the success rate above 25%, suggesting that the MA method might be inherently inefficient due to its insensitivity to context-dependent effects. Here we present the first systematic study on the effect of array length on ZFN activity. ZFNs composed of six-finger MA arrays produced mutations at 15 of 21 (71%) targeted loci in human and mouse cells. A novel drop-out linker scheme was used to rapidly assess three-to six-finger combinations, demonstrating that shorter arrays could improve activity in some cases. Analysis of 268 array variants revealed that half of MA ZFNs of any array composition that exceed an ab initio B-score cutoff of 15 were active. These results suggest that, when used appropriately, MA ZFNs are able to target more DNA sequences with higher success rates than other current methods.