Jackson Taylor

Age-related variations in the methylome associated with gene expression in human monocytes and t cells

Age-related variations in DNA methylation have been reported; however, the functional relevance of these differentially methylated sites (age-dMS) are unclear. Here we report potentially functional age-dMS, defined as age- and cis-gene expression-associated methylation sites (age-eMS), identified by integrating genome-wide CpG methylation and gene expression profiles collected ex vivo from circulating T cells (227 CD4+ samples) and monocytes (1,264 CD14+ samples, age range: 55–94 years). None of the age-eMS detected in 227 T cell samples are detectable in 1,264 monocyte samples, in contrast to the majority of age-dMS detected in T cells that replicated in monocytes. Age-eMS tend to be hypomethylated with older age, located in predicted enhancers, and preferentially linked to expression of antigen processing and presentation genes. These results identify and characterize potentially functional age-related methylation in human T cells and monocytes, and provide novel insights into the role age-dMS may play in the aging process.

DNA Methylation of the Aryl Hydrocarbon Receptor Repressor Associations with Cigarette Smoking and Subclinical Atherosclerosis

Background—Tobacco smoke contains numerous agonists of the aryl hydrocarbon receptor (AhR) pathway, and activation of the AhR pathway was shown to promote atherosclerosis in mice. Intriguingly, cigarette smoking is most strongly and robustly associated with DNA modifications to an AhR pathway gene, the AhR repressor (AHRR). We hypothesized that altered AHRR methylation in monocytes, a cell type sensitive to cigarette smoking and involved in atherogenesis, may be a part of the biological link between cigarette smoking and atherosclerosis. Methods and Results—DNA methylation profiles of AHRR in monocytes (542 CpG sites±150 kb of AHRR, using Illumina 450K array) were integrated with smoking habits and ultrasound-measured carotid plaque scores from 1256 participants of the Multi-Ethnic Study of Atherosclerosis (MESA). Methylation of cg05575921 significantly associated (P=6.1×10−134) with smoking status (current versus never). Novel associations between cg05575921 methylation and carotid plaque scores (P=3.1×10−10) were identified, which remained significant in current and former smokers even after adjusting for self-reported smoking habits, urinary cotinine, and well-known cardiovascular disease risk factors. This association replicated in an independent cohort using hepatic DNA (n=141). Functionally, cg05575921 was located in a predicted gene expression regulatory element (enhancer) and had methylation correlated with AHRR mRNA profiles (P=1.4×10−17) obtained from RNA sequencing conducted on a subset (n=373) of the samples. Conclusions—These findings suggest that AHRR methylation may be functionally related to AHRR expression in monocytes and represents a potential biomarker of subclinical atherosclerosis in smokers.

Troponin T nuclear localization and its role in aging skeletal muscle

Troponin T (TnT) is known to mediate the interaction between Tn complex and tropomyosin (Tm), which is essential for calcium-activated striated muscle contraction. This regulatory function takes place in the myoplasm, where TnT binds Tm. However, recent findings of troponin I and Tm nuclear translocation in Drosophila and mammalian cells imply other roles for the Tn–Tm complex. We hypothesized that TnT plays a nonclassical role through nuclear translocation. Immunoblotting with different antibodies targeting the NH2- or COOH-terminal region uncovered a pool of fast skeletal muscle TnT3 localized in the nuclear fraction of mouse skeletal muscle as either an intact or fragmented protein. Construction of TnT3–DsRed fusion proteins led to the further observation that TnT3 fragments are closely related to nucleolus and RNA polymerase activity, suggesting a role for TnT3 in regulating transcription. Functionally, overexpression of TnT3 fragments produced significant defects in nuclear shape and caused high levels of apoptosis. Interestingly, nuclear TnT3 and its fragments were highly regulated by aging, thus creating a possible link between the deleterious effects of TnT3 and sarcopenia. We propose that changes in nuclear TnT3 and its fragments cause the number of myonuclei to decrease with age, contributing to muscle damage and wasting.

Increased CaVβ1a expression with aging contributes to skeletal muscle weakness

Ca2+ release from the sarcoplasmic reticulum (SR) into the cytosol is a crucial part of excitation–contraction (E‐C) coupling. Excitation–contraction uncoupling, a deficit in Ca2+ release from the SR, is thought to be responsible for at least some of the loss in specific force observed in aging skeletal muscle. Excitation–contraction uncoupling may be caused by alterations in expression of the voltage‐dependent calcium channel α1s (CaV1.1) and β1a (CaVβ1a) subunits, both of which are necessary for E‐C coupling to occur. While previous studies have found CaV1.1 expression declines in old rodents, CaVβ1a expression has not been previously examined in aging models. Western blot analysis shows a substantial increase of CaVβ1a expression over the full lifespan of Friend Virus B (FVB) mice. To examine the specific effects of CaVβ1a overexpression, a CaVβ1a‐YFP plasmid was electroporated in vivo into young animals. The resulting increase in expression of CaVβ1a corresponded to decline of CaV1.1 over the same time period. YFP fluorescence, used as a measure of CaVβ1a‐YFP expression in individual fibers, also showed an inverse relationship with charge movement, measured using the whole‐cell patch‐clamp technique. Specific force was significantly reduced in young CaVβ1a‐YFP electroporated muscle fibers compared with sham‐electroporated, age‐matched controls. siRNA interference of CaVβ1a in young muscles reduced charge movement, while charge movement in old was restored to young control levels. These studies imply CaVβ1a serves as both a positive and negative regulator CaV1.1 expression, and that endogenous overexpression of CaVβ1a during old age may play a role in the loss of specific force.

Transcriptomic profiles of aging in purified human immune cells.

BackgroundTranscriptomic studies hold great potential towards understanding the human aging process. Previous transcriptomic studies have identified many genes with age-associated expression levels; however, small samples sizes and mixed cell types often make these results difficult to interpret.ResultsUsing transcriptomic profiles in CD14+ monocytes from 1,264 participants of the Multi-Ethnic Study of Atherosclerosis (aged 55–94 years), we identified 2,704 genes differentially expressed with chronological age (false discovery rate, FDR ≤ 0.001). We further identified six networks of co-expressed genes that included prominent genes from three pathways: protein synthesis (particularly mitochondrial ribosomal genes), oxidative phosphorylation, and autophagy, with expression patterns suggesting these pathways decline with age. Expression of several chromatin remodeler and transcriptional modifier genes strongly correlated with expression of oxidative phosphorylation and ribosomal protein synthesis genes. 17% of genes with age-associated expression harbored CpG sites whose degree of methylation significantly mediated the relationship between age and gene expression (p < 0.05). Lastly, 15 genes with age-associated expression were also associated (FDR ≤ 0.01) with pulse pressure independent of chronological age.Comparing transcriptomic profiles of CD14+ monocytes to CD4+ T cells from a subset (n = 423) of the population, we identified 30 age-associated (FDR < 0.01) genes in common, while larger sets of differentially expressed genes were unique to either T cells (188 genes) or monocytes (383 genes). At the pathway level, a decline in ribosomal protein synthesis machinery gene expression with age was detectable in both cell types.ConclusionsAn overall decline in expression of ribosomal protein synthesis genes with age was detected in CD14+ monocytes and CD4+ T cells, demonstrating that some patterns of aging are likely shared between different cell types. Our findings also support cell-specific effects of age on gene expression, illustrating the importance of using purified cell samples for future transcriptomic studies. Longitudinal work is required to establish the relationship between identified age-associated genes/pathways and aging-related diseases.

The Cavβ1a subunit regulates gene expression and suppresses myogenin in muscle progenitor cells

Cavβ1a acts as a voltage-gated calcium channel-independent regulator of gene expression in muscle progenitor cells and is required for their normal expansion during myogenic development.

Troponin T3 regulates nuclear localization of the calcium channel Cavβ1a subunit in skeletal muscle.

The voltage-gated calcium channel (Cav) β1a subunit (Cavβ1a) plays an important role in excitation-contraction coupling (ECC), a process in the myoplasm that leads to muscle-force generation. Recently, we discovered that the Cavβ1a subunit travels to the nucleus of skeletal muscle cells where it helps to regulate gene transcription. To determine how it travels to the nucleus, we performed a yeast two-hybrid screening of the mouse fast skeletal muscle cDNA library and identified an interaction with troponin T3 (TnT3), which we subsequently confirmed by co-immunoprecipitation and co-localization assays in mouse skeletal muscle in vivo and in cultured C2C12 muscle cells. Interacting domains were mapped to the leucine zipper domain in TnT3 COOH-terminus (160-244 aa) and Cavβ1a NH2-terminus (1-99 aa), respectively. The double fluorescence assay in C2C12 cells co-expressing TnT3/DsRed and Cavβ1a/YFP shows that TnT3 facilitates Cavβ1a nuclear recruitment, suggesting that the two proteins play a heretofore unknown role during early muscle differentiation in addition to their classical role in ECC regulation.

Magnetofection Enhances Adenoviral Vector-based Gene Delivery in Skeletal Muscle Cells

The goal of magnetic field-assisted gene transfer is to enhance internalization of exogenous nucleic acids by association with magnetic nanoparticles (MNPs). This technique named magnetofection is particularly useful in difficult-to-transfect cells. It is well known that human, mouse, and rat skeletal muscle cells suffer a maturation-dependent loss of susceptibility to Recombinant Adenoviral vector (RAd) uptake. In postnatal, fully differentiated myofibers, the expression of the primary Coxsackie and Adenoviral membrane receptor (CAR) is severely downregulated representing a main hurdle for the use of these vectors in gene transfer/therapy. Here we demonstrate that assembling of Recombinant Adenoviral vectors with suitable iron oxide MNPs into magneto-adenovectors (RAd-MNP) and further exposure to a gradient magnetic field enables to efficiently overcome transduction resistance in skeletal muscle cells. Expression of Green Fluorescent Protein and Insulin-like Growth Factor 1 was significantly enhanced after magnetofection with RAd-MNPs complexes in C2C12 myotubes in vitro and mouse skeletal muscle in vivo when compared to transduction with naked virus. These results provide evidence that magnetofection, mainly due to its membrane-receptor independent mechanism, constitutes a simple and effective alternative to current methods for gene transfer into traditionally hard-to-transfect biological models.

Troponin T Nuclear Localization and its Role in Aging Skeletal Muscle

Troponin T (TnT) is known to mediate the interaction between Tn complex and tropomyosin (Tm), which is essential for calcium-activated striated muscle contraction. This regulatory function takes place in the myoplasm, where TnT binds Tm. However, recent findings of troponin I and Tm nuclear translocation in Drosophila and mammalian cells imply other roles for the Tn–Tm complex. We hypothesized that TnT plays a nonclassical role through nuclear translocation. Immunoblotting with different antibodies targeting the NH2- or COOH-terminal region uncovered a pool of fast skeletal muscle TnT3 localized in the nuclear fraction of mouse skeletal muscle as either an intact or fragmented protein. Construction of TnT3–DsRed fusion proteins led to the further observation that TnT3 fragments are closely related to nucleolus and RNA polymerase activity, suggesting a role for TnT3 in regulating transcription. Functionally, overexpression of TnT3 fragments produced significant defects in nuclear shape and caused high levels of apoptosis. Interestingly, nuclear TnT3 and its fragments were highly regulated by aging, thus creating a possible link between the deleterious effects of TnT3 and sarcopenia. We propose that changes in nuclear TnT3 and its fragments cause the number of myonuclei to decrease with age, contributing to muscle damage and wasting.