Gamma-ray and hydrogen peroxide induction of gene amplification in hamster cells deficient in DNA double strand break repair.

To investigate the role of DNA double strand breaks (DSBs) and of their repair in gene amplification, we analyzed this process in the V3 Chinese hamster cell line and in the parental line AA8, after exposure to gamma-rays and to hydrogen peroxide (H2O2). V3 is defective in DSB repair because of a mutation in the DNA-dependent protein kinase catalytic subunit (DNA-PKcs) gene, a gene involved in the non-homologous end-joining pathway. As a measure of gene amplification we used the frequency of colonies resistant to N-(phosphonacetyl)-L-aspartate (PALA), since in rodent cells PALA resistance is mainly achieved through the amplification of the CAD (carbamyl-P-synthetase, aspartate transcarbamylase, dihydro-orotase) gene. After treatment with different doses of gamma-rays and of H2O2, we found a dose related increase in the frequency of gene amplification and of chromosome aberrations. When the same doses of damaging agents were used, these increments were higher in V3 than in AA8. These results indicate that DSBs that are not efficiently repaired can be responsible for the induction of gene amplification. H2O2 stimulates gene amplification as well as gamma-rays, however, at similar levels of amplification induction, chromosome damage was about 50% lower. This suggests that gene amplification can be induced by H2O2 through pathways alternative to a direct DNA damage. Stimulation of gene amplification by H2O2, which is one of the products of the aerobic metabolism, supports the hypothesis that cellular metabolic products themselves can be a source of genome instability.

Nuclear positioning, gene activity and cancer.

In the interphasic nucleus, chromosomes are non-randomly arranged within the nuclear space. Indeed, chromosomes are thought to be organised into "chromosome territories". The size of a chromosome territory is roughly determined by its DNA content, but is also influenced by other factors, such as their transcriptional status. Chromatin modifications and positioning of genetic loci in the nucleus play a critical role in the control of gene expression. Emerging evidence suggests that the nucleus is structurally and functionally compartimentalized and desorganization of such a structure might play a major role in the emergence of human diseases such as cancer.

Chromatin dynamics and cancer.

Chomatin remodeling multi-protein complexes play a key regulatory role in many physiological processes such proliferation and differentiation. The alteration in the recruitment or in the function of one of these chromatin remodeling factors can lead to a proliferation defect, that might result from the aberrant activation or repression of key genes that regulate cell proliferation. In fact, aberrant chromatin remodeling leads to severe human diseases such as leukemia, epithelial cancers and Rubinstein-Taybi syndrome.

[RNA interference and its possible use in cancer therapy]. / Inhibition de l'expression génique par l'ARN interférence : applications dans le domaine du cancer.

Post-transcriptional gene silencing (PTGS) or RNA interference (RNAi) is a powerful tool for silencing gene expression. This mechanism was initially considered as a strange phenomenon limited to few plant species. It has become clear that PTGS occurs in both plants and animals and has roles in viral defense and transposon silencing mechanisms. However, the use of RNA interference triggered by the introduction of small double-stranded RNA (dsRNA or siRNA) into mammalian cells as a tool to knock down expression of specific genes holds the promise to selectively inhibit expression of disease-associated genes in humans. On the other hand, there are about 40,000 protein-coding genes in the human genome, but the function of most of them remains unknown. RNAi technology has now been developed for systematically deciphering the functions and interactions of these thousands of genes.

Preferential association of irreversibly silenced E2F-target genes with pericentromeric heterochromatin in differentiated muscle cells.

The heterochromatin-associated H3K9 tri-methylase Suv39h1 is involved in the permanent silencing of E2F target genes in differentiating but not in quiescent cells. Here, we tested the hypothesis that permanent silencing of E2F target genes is associated with their subnuclear positioning close to the pericentromeric heterochromatin compartment, enriched in Suv39h1. Using fluorescence in situ hybridization, we analyzed the subnuclear localization of three E2F target genes relative to the pericentromeric heterochromatin, in cycling fibroblasts or differentiating myoblasts. We observed that all three E2F-target genes have a tendency to relocate closer to the pericentromeric heterochromatin, only when cells differentiate and undergo an irreversible cell cycle withdrawal. These data suggest that repression of E2F target genes in cycling or in differentiating cells is achieved through distinct mechanisms. In differentiating cells, permanent silencing is driven by a Suv39h1-dependent H3K9 tri-methylation and positioning close to the heterochromatin compartment, whereas repression in cycling cells seems independent from subnuclear positioning and requires distinct H3K9 methylation levels.

Chromatin: the interface between extrinsic cues and the epigenetic regulation of muscle regeneration.

Muscle regeneration provides a paradigm by which to study how extrinsic signals coordinate gene expression in somatic stem cells (satellite cells) by directing the genome distribution of chromatin-modifying complexes. Understanding the signal-dependent control of the epigenetic events underlying the transition of muscle stem cells through sequential regeneration stages holds the promise to reveal new targets for selective interventions toward repairing diseased muscles. This review describes the latest findings on how regeneration cues are integrated at the chromatin level to build the transcription network that regulates progression of endogenous muscle progenitors throughout the myogenic program. In particular, we describe how specific epigenetic signatures can confer responsiveness to extrinsic cues on discrete regions of the muscle stem cell genome.

Epigenetic drugs in the treatment of skeletal muscle atrophy.

PURPOSE OF REVIEW: A dynamic network of anabolic and catabolic pathways regulates skeletal muscle mass in adult organisms. Muscle atrophy is the detrimental outcome of an imbalance of this network. The purpose of this review is to provide a critical evaluation of different forms of muscle atrophy from a mechanistic and therapeutic point of view. RECENT FINDINGS: The identification and molecular characterization of distinct pathways implicated in the pathogenesis of muscle atrophy have revealed potential targets for therapeutic interventions. However, an effective application of these therapies requires a better understanding of the relative contribution of these pathways to the development of muscle atrophy in distinct pathological conditions. SUMMARY: We propose that the decline in anabolic signals ('passive atrophy') and activation of catabolic pathways ('active atrophy') contribute differently to the pathogenesis of muscle atrophy associated with distinct diseases or unfavorable conditions. Interestingly, these pathways might converge on common transcriptional effectors, suggesting that an optimal intervention should be directed to targets at the chromatin level. We provide the rationale for the use of epigenetic drugs such as deacetylase inhibitors, which target multiple signaling pathways implicated in the pathogenesis of muscle atrophy.

Differential cooperation between heterochromatin protein HP1 isoforms and MyoD in myoblasts.

Mechanisms of transcriptional repression are important during cell differentiation. Mammalian heterochromatin protein 1 isoforms HP1alpha, HP1beta, and HP1gamma play important roles in the regulation of chromatin structure and function. We explored the possibility of different roles for the three HP1 isoforms in an integrated system, skeletal muscle terminal differentiation. In this system, terminal differentiation is initiated by the transcription factor MyoD, whose target genes remain mainly silent until myoblasts are induced to differentiate. Here we show that HP1alpha and HP1beta isoforms, but not HP1gamma, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1alpha and HP1beta, but not HP1gamma, inhibit MyoD transcriptional activity, suggesting a model in which MyoD could serve as a bridge between nucleosomes and chromatin-binding proteins such as HDACs and HP1. Chromatin immunoprecipitation assays show a preferential recruitment of HP1 proteins on MyoD target genes in proliferating myoblasts. Finally, modulation of HP1 protein level impairs MyoD target gene expression and muscle terminal differentiation. Together, our data show a nonconventional interaction between HP1 and a tissue-specific transcription factor, MyoD. In addition, they strongly suggest that HP1 isoforms play important roles during muscle terminal differentiation in an isoform-dependent manner.

Chromatin modification and muscle differentiation.

Skeletal muscle differentiation is a multistep process, which begins with the commitment of multi-potent mesodermal precursor cells to the muscle fate. These committed cells, the myoblasts, then differentiate and fuse into multinucleated myotubes. The final step of muscle differentiation is the maturation of differentiated myotubes into myofibres. Skeletal muscle development requires the coordinated expression of various transcription factors like the members of the myocyte enhancer binding-factor 2 family and the muscle regulatory factors. These transcription factors, in collaboration with chromatin-remodelling complexes, act in specific combinations and within complex transcriptional regulatory networks to achieve skeletal myogenesis. Additional factors involved in the epigenetic regulation of this process continue to be discovered. In this review, the authors discuss the recent discoveries in the epigenetic regulation of myogenesis. They also summarise the role of chromatin-modifying enzymes regulating muscle gene expression. These different factors are often involved in multiple steps of muscle differentiation and have redundant activities. Altogether, the recent findings have allowed a better understanding of myogenesis and have raised new hopes for the pharmacological development of new therapies aimed at muscle degeneration diseases, such as myotonic dystrophy or Duchenne muscular dystrophy.

siRNA as a route to new cancer therapies.

The RNA interference (RNAi) gene-silencing mechanism is induced by double-stranded RNA (dsRNA) and is highly sequence-specific. It is an extremely powerful tool for silencing gene expression in vitro, and might be used as therapy in human pathologies such as cancer, viral infections and genetic disorders. RNAi was initially discovered in plants, but it has become clear that it is also conserved in animal species. Triggering of RNAi by the introduction of small dsRNA (or small interfering RNA) into living cells as a tool to inhibit the expression of specific genes holds the promise to selectively extinguish the expression of disease-associated genes in humans. On the other hand, RNAi technology will serve to elucidate the functions and interactions of the thousands of human genes in high-throughput systems and help in target validation technology.

A Suv39h-dependent mechanism for silencing S-phase genes in differentiating but not in cycling cells.

The Rb/E2F complex represses S-phase genes both in cycling cells and in cells that have permanently exited from the cell cycle and entered a terminal differentiation pathway. Here we show that S-phase gene repression, which involves histone-modifying enzymes, occurs through distinct mechanisms in these two situations. We used chromatin immunoprecipitation to show that methylation of histone H3 lysine 9 (H3K9) occurs at several Rb/E2F target promoters in differentiating cells but not in cycling cells. Furthermore, phenotypic knock-down experiments using siRNAs showed that the histone methyltransferase Suv39h is required for histone H3K9 methylation and subsequent repression of S-phase gene promoters in differentiating cells, but not in cycling cells. These results indicate that the E2F target gene permanent silencing mechanism that is triggered upon terminal differentiation is distinct from the transient repression mechanism in cycling cells. Finally, Suv39h-depleted myoblasts were unable to express early or late muscle differentiation markers. Thus, appropriately timed H3K9 methylation by Suv39h seems to be part of the control switch for exiting the cell cycle and entering differentiation.