Cell division drives DNA methylation loss in late-replicating domains in primary human cells.

DNA methylation undergoes dramatic age-related changes, first described more than four decades ago. Loss of DNA methylation within partially methylated domains (PMDs), late-replicating regions of the genome attached to the nuclear lamina, advances with age in normal tissues, and is further exacerbated in cancer. We present here experimental evidence that this DNA hypomethylation is directly driven by proliferation-associated DNA replication. Within PMDs, loss of DNA methylation at low-density CpGs in A:T-rich immediate context (PMD solo-WCGWs) tracks cumulative population doublings in primary cell culture. Cell cycle deceleration results in a proportional decrease in the rate of DNA hypomethylation. Blocking DNA replication via Mitomycin C treatment halts methylation loss. Loss of methylation continues unabated after TERT immortalization until finally reaching a severely hypomethylated equilibrium. Ambient oxygen culture conditions increases the rate of methylation loss compared to low-oxygen conditions, suggesting that some methylation loss may occur during unscheduled, oxidative damage repair-associated DNA synthesis. Finally, we present and validate a model to estimate the relative cumulative replicative histories of human cells, which we call "RepliTali" (Replication Times Accumulated in Lifetime).

Articular cartilage and sternal fibrocartilage respond differently to extended microgravity.

The effects of spaceflight on cartilaginous structure are largely unknown. To address this deficiency, articular cartilage (AC) and sternal cartilage (SC) from mice exposed to 30 days of microgravity on the BION-M1 craft were investigated for pathological changes. The flight AC showed some evidence of degradation at the tissue level with loss of proteoglycan staining and a reduction in mRNA expression of mechano-responsive and structural cartilage matrix proteins compared to non-flight controls. These data suggest that degradative changes are underway in the AC extracellular matrix exposed to microgravity. In contrast, there was no evidence of cartilage breakdown in SC flight samples and the gene expression profile was distinct from that of AC with a reduction in metalloproteinase gene transcription. Since the two cartilages respond differently to microgravity we propose that each is tuned to the biomechanical environments in which they are normally maintained. That is, the differences between magnitude of normal terrestrial loading and the unloading of microgravity dictates the tissue response. Weight-bearing articular cartilage, but not minimally loaded sternal fibrocartilage, is negatively affected by the unloading of microgravity. We speculate that the maintenance of physiological loading on AC during spaceflight will minimize AC damage.

Authentication of collagen VI antibodies.

BACKGROUND: Collagen VI is a ubiquitously-expressed macromolecule that forms unique microfibrillar assemblies in the extracellular matrix. Mutations in the COL6A1, COL6A2 and COL6A3 genes result in congenital muscular dystrophy, arguing that collagen is critical for skeletal muscle development and function. Antibodies against collagen VI are important clinical and diagnostic tools in muscular dystrophy. They are used to confirm genetic findings by detecting abnormalities in the distribution, organization and overall levels of collagen VI in cells and tissues isolated from patients. METHODS: Many antibodies have been raised against tissue-purified collagen VI and individual collagen VI chains, however few have been properly validated for sensitivity and chain specificity. To address this deficiency, we compared the ability of 23 commercially-available antibodies to detect extracellular collagen VI by immunohistochemistry on frozen tissue sections. To determine chain specificity, immunoblot analyses were conducted on cell lysates isolated from cells transfected with cDNAs for each individual chain and cells expressing all three chains together. RESULTS: Our analyses identified 15 antibodies that recognized tissue collagen VI by immunohistochemistry at varying intensities and 20 that successfully detected collagen VI by immunoblotting. Three antibodies failed to recognize collagen VI by either method under the conditions tested. All chain-specific antibodies that worked by immunoblotting specifically recognized their correct chain, and no other chains. CONCLUSIONS: This series of side-by-side comparisons reveal at least two antibodies specific for each chain that work well for immunohistochemistry on frozen sections. This validation study expands the repertoire of antibodies available for muscular dystrophy studies caused by defects in collagen VI.