Telomere length, telomeric proteins and genomic instability during the multistep carcinogenic process.

Telomeres form specialized structures at the ends of eukaryotic chromosomes, preventing them from being wrongly recognized as DNA damage. The human telomere DNA sequence is a tandem repetition of the sequence TTAGGG. In normal cells, the DNA replication machinery is unable to completely duplicate the telomeric DNA; thus, telomeres are shortened after every cell division. Having reached a critical length, telomeres may be recognized as double strand break DNA lesions, and cells eventually enter senescence. Carcinogenesis is a multistep process involving multiple mutations and chromosomal aberrations. One of the most prevalent aberrations in pre-cancerous lesions is telomere shortening and telomerase activation. We discuss the role and homeostasis of telomeres in normal cells and their implication in the early steps of carcinogenesis. We also discuss various techniques used, and their limitations, in the study of telomeres and genome instability and their role in carcinogenesis and related genomic modifications.

The SWI/SNF subunit/tumor suppressor BAF47/INI1 is essential in cell cycle arrest upon skeletal muscle terminal differentiation.

Myogenic terminal differentiation is a well-orchestrated process starting with permanent cell cycle exit followed by muscle-specific genetic program activation. Individual SWI/SNF components have been involved in muscle differentiation. Here, we show that the master myogenic differentiation factor MyoD interacts with more than one SWI/SNF subunit, including the catalytic subunit BRG1, BAF53a and the tumor suppressor BAF47/INI1. Downregulation of each of these SWI/SNF subunits inhibits skeletal muscle terminal differentiation but, interestingly, at different differentiation steps and extents. BAF53a downregulation inhibits myotube formation but not the expression of early muscle-specific genes. BRG1 or BAF47 downregulation disrupt both proliferation and differentiation genetic programs expression. Interestingly, BRG1 and BAF47 are part of the SWI/SNF remodeling complex as well as the N-CoR-1 repressor complex in proliferating myoblasts. However, our data show that, upon myogenic differentiation, BAF47 shifts in favor of N-CoR-1 complex. Finally, BRG1 and BAF47 are well-known tumor suppressors but, strikingly, only BAF47 seems essential in the myoblasts irreversible cell cycle exit. Together, our data unravel differential roles for SWI/SNF subunits in muscle differentiation, with BAF47 playing a dual role both in the permanent cell cycle exit and in the regulation of muscle-specific genes.

The core binding factor CBF negatively regulates skeletal muscle terminal differentiation.

BACKGROUND: Core Binding Factor or CBF is a transcription factor composed of two subunits, Runx1/AML-1 and CBF beta or CBFbeta. CBF was originally described as a regulator of hematopoiesis. METHODOLOGY/PRINCIPAL FINDINGS: Here we show that CBF is involved in the control of skeletal muscle terminal differentiation. Indeed, downregulation of either Runx1 or CBFbeta protein level accelerates cell cycle exit and muscle terminal differentiation. Conversely, overexpression of CBFbeta in myoblasts slows terminal differentiation. CBF interacts directly with the master myogenic transcription factor MyoD, preferentially in proliferating myoblasts, via Runx1 subunit. In addition, we show a preferential recruitment of Runx1 protein to MyoD target genes in proliferating myoblasts. The MyoD/CBF complex contains several chromatin modifying enzymes that inhibits MyoD activity, such as HDACs, Suv39h1 and HP1beta. When overexpressed, CBFbeta induced an inhibition of activating histone modification marks concomitant with an increase in repressive modifications at MyoD target promoters. CONCLUSIONS/SIGNIFICANCE: Taken together, our data show a new role for Runx1/CBFbeta in the control of the proliferation/differentiation in skeletal myoblasts.

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.

Post-translational modifications of histones H3 and H4 associated with the histone methyltransferases Suv39h1 and G9a.

Specific combinations of post-translational modifications of histones alter chromatin structure, facilitating gene transcription or silencing. Here we have investigated the 'histone code' associated with the histone methyltransferases Suv39h1 and G9a by combining double immunopurification and mass spectrometry. Our results confirm the previously reported histone modifications associated with Suv39h1 and G9a. Moreover, this method allowed us to demonstrate for the first time an association of acetylated histones with the repressor proteins Suv39h1 and G9a.

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.