Long-term growth of diploid human fibroblasts in low serum media

Hayflick and Moorhead demonstrated that diploid human fibroblasts have a limited life span when grown in media containing 10% bovine calf sera. Recent experiments have suggested that antigrowth factors in serum may be a potential contributor to the limited proliferative capacity of normal diploid cells. To reduce the concentration of inhibitory serum factors 10-fold, MRC-5 diploid fibroblasts were cultured in media with only 1% serum. Long-term culture in 1% serum requires the addition of purified growth factors to sustain proliferation. Although there are dramatic changes in cell morphology, we find that the long-term division potential of MRC-5 cells cultured in media containing 1% serum and growth factors differs little from that found with cells cultured in 10% serum. In contrast, MRC-5 cells cultured in 10% serum and added growth factors have a somewhat extended life span. These results suggest that negative growth factors are not responsible for the limited proliferative capacity of in vitro cultured human fibroblasts. Moreover, the evidence that human fibroblasts can undergo major changes in cell morphology and still retain a normal life span raises questions about the validity of using morphological changes as indicators of cellular senescence.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/28800/1/0000634.pd

Decline in histone H5 phosphorylation during erythroid senescence in chick embryos

Previous studies have implicated histone H5 dephosphorylation as a casual factor in genetic inactivation and chromatin condensation during erythroid senescence in adult chickens. We show that histone H5 phosphorylation declines in two stages as various cohorts of erythroid cells senesce in chick embryos. The first decline occurs between 5 and 6 days and coincides with the senescence of primitive erythrocytes. The second decline in H5 phosphorylation occurs between 17 and 19 days of chicken development, when the definitive erythrocytes undergo senescence and chromatin condensation. These results point to a role for histone dephosphorylation during the programed senescence of erythroid cells.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/29282/1/0000341.pd

Characterization of a topoisomerase-like activity at specific hypersensitive sites in the Drosophila histone gene cluster

It is well known that treatment of DNA-topoisomerase complexes with SDS induces cleavage of the DNA by trapping a reactive intermediate in which the topoisomerase is covalently linked to the terminal phosphates of the cut DNA. I have used this technique to examine potential topoisomerase binding sites in the histone gene chromatin of Drosophila Kc cells. Treatment of Kc nuclei with SDS induces Mg++-dependent DNA cleavage near the borders of two nuclease-hypersensitive sites located 5' and 3' of histone H4. It is likely that the SDS-induced cleavage at these hypersensitive sites is due to a topoisomerase because protein becomes tightly bound to the ends of the cleaved DNA fragments. Preliminary experiments suggest that a type II topoisomerase may be responsible for the cleavage.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/27848/1/0000259.pd

Functional characterization and developmental regulation of mouse telomerase RNA

Telomerase synthesizes telomeric DNA repeats onto chromosome ends de novo. The mouse telomerase RNA component was cloned and contained only 65 percent sequence identity with the human telomerase RNA. Alteration of the template region in vivo generated altered telomerase products. The shorter template regions of the mouse and other rodent telomerase RNAs could account for the shorter distribution of products (processivity) generated by the mouse enzyme relative to the human telomerase. Amounts of telomerase RNA increased in immortal cells derived from primary mouse fibroblasts. RNA was detected in all newborn mouse tissues tested but was decreased during postnatal development

TMEM8 – a non-globin gene entrapped in the globin web

For more than 30 years it was believed that globin gene domains included only genes encoding globin chains. Here we show that in chickens, the domain of α-globin genes also harbor the non-globin gene TMEM8. It was relocated to the vicinity of the α-globin cluster due to inversion of an ∼170-kb genomic fragment. Although in humans TMEM8 is preferentially expressed in resting T-lymphocytes, in chickens it acquired an erythroid-specific expression profile and is upregulated upon terminal differentiation of erythroblasts. This correlates with the presence of erythroid-specific regulatory elements in the body of chicken TMEM8, which interact with regulatory elements of the α-globin genes. Surprisingly, TMEM8 is not simply recruited to the α-globin gene domain active chromatin hub. An alternative chromatin hub is assembled, which includes some of the regulatory elements essential for the activation of globin gene expression. These regulatory elements should thus shuttle between two different chromatin hubs

Lysosome-mediated processing of chromatin in senescence

Cellular senescence is a stable proliferation arrest, a potent tumor suppressor mechanism, and a likely contributor to tissue aging. Cellular senescence involves extensive cellular remodeling, including of chromatin structure. Autophagy and lysosomes are important for recycling of cellular constituents and cell remodeling. Here we show that an autophagy/lysosomal pathway processes chromatin in senescent cells. In senescent cells, lamin A/C–negative, but strongly γ-H2AX–positive and H3K27me3-positive, cytoplasmic chromatin fragments (CCFs) budded off nuclei, and this was associated with lamin B1 down-regulation and the loss of nuclear envelope integrity. In the cytoplasm, CCFs were targeted by the autophagy machinery. Senescent cells exhibited markers of lysosomal-mediated proteolytic processing of histones and were progressively depleted of total histone content in a lysosome-dependent manner. In vivo, depletion of histones correlated with nevus maturation, an established histopathologic parameter associated with proliferation arrest and clinical benignancy. We conclude that senescent cells process their chromatin via an autophagy/lysosomal pathway and that this might contribute to stability of senescence and tumor suppression

High-resolution analysis of c-fos chromatin accessibility using a novel DNase I-PCR assay

In our previous study, c-fos chromatin accessibility was assayed using DNase I digestion and Southern blot analysis. This low-resolution mapping of c-fos chromatin accessibility demonstrated that serum stimulation of the c-fos enhancer induces a reversivle increase in c-fos DNase I sensitivity and suggested that a 5' to 3' gradient of DNase I sensitivity may form downstream from the c-fos enhancer. To confirm the existence of a 5' to 3' gradient of accessibility, we have recently developed a high-resolution polymerase chain reaction (PCR) assay for DNase I sensitivity. Using this novel DNase I assay, we have reliably detected position- and time-dependent gradients of chromatin accessibility around the c-fos enhancer. These data confirm our earlier results and further support the hypothesis that the changes in c-fos chromatin accessibility originate near the 5' enhancer. As a technique for future examinations of gene structure, our data demonstrate the value of the DNase I-PCR assay for rapidly preparing comprehensive and high-resolution maps of chromatin accessibility for any sequenced genomic region.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/30107/1/0000479.pd

Transcription forms and remodels supercoiling domains unfolding large-scale chromatin structures

DNA supercoiling is an inherent consequence of twisting DNA and is critical for regulating gene expression and DNA replication. However, DNA supercoiling at a genomic scale in human cells is uncharacterized. To map supercoiling we used biotinylated-trimethylpsoralen as a DNA structure probe to show the genome is organized into supercoiling domains. Domains are formed and remodeled by RNA polymerase and topoisomerase activities and are flanked by GC-AT boundaries and CTCF binding sites. Under-wound domains are transcriptionally active, enriched in topoisomerase I, “open” chromatin fibers and DNaseI sites, but are depleted of topoisomerase II. Furthermore DNA supercoiling impacts on additional levels of chromatin compaction as under-wound domains are cytologically decondensed, topologically constrained, and decompacted by transcription of short RNAs. We suggest that supercoiling domains create a topological environment that facilitates gene activation providing an evolutionary purpose for clustering genes along chromosomes

Long-Term Functional Side-Effects of Stimulants and Sedatives in Drosophila melanogaster

Background: Small invertebrate animals, such as nematodes and fruit flies, are increasingly being used to test candidate drugs both for specific therapeutic purposes and for long-term health effects. Some of the protocols used in these experiments feature such experimental design features as lifelong virginity and very low densities. By contrast, the ability of both fruit flies and nematodes to resist stress is frequently correlated with their longevity and other functional measures, suggesting that low-stress assays are not necessarily the only useful protocol for testing the long-term effects of drugs. Methodology/Principal Findings: Here we report an alternative protocol for fruit fly drug-testing that maximizes reproductive opportunities and other types of interaction, with moderately high population densities. We validate this protocol using two types of experimental tests: 1. We show that this protocol detects previously well-established genetic differences between outbred fruit fly populations. 2. We show that this protocol is able to distinguish among the long-term effects of similar types of drugs within two broad categories, stimulants and tranquilizers. Conclusions: Large-scale fly drug testing can be conducted using mixed-sex high-density cage assays. We find that the commonly-used stimulants caffeine and theobromine differ dramatically in their chronic functional effects, theobromine being more benign. Likewise, we find that two generic pharmaceutical tranquilizers, lithium carbonate and valproic acid, differ dramatically in their chronic effects, lithium being more benign. However, these findings do not necessarily apply t