Slimane Ait-Si-Ali

DNMT1 forms a complex with Rb, E2F1 and HDAC1 and represses transcription from E2F-responsive promoters

Methylation of CpG islands is associated with transcriptional silencing and the formation of nuclease-resistant chromatin structures enriched in hypoacetylated histones. Methyl-CpG-binding proteins, such as MeCP2, provide a link between methylated DNA and hypoacetylated histones by recruiting histone deacetylase, but the mechanisms establishing the methylation patterns themselves are unknown. Whether DNA methylation is always causal for the assembly of repressive chromatin or whether features of transcriptionally silent chromatin might target methyltransferase remains unresolved. Mammalian DNA methyltransferases show little sequence specificity in vitro, yet methylation can be targeted in vivo within chromosomes to repetitive elements, centromeres and imprinted loci. This targeting is frequently disrupted in tumour cells, resulting in the improper silencing of tumour-suppressor genes associated with CpG islands. Here we show that the predominant mammalian DNA methyltransferase, DNMT1, co-purifies with the retinoblastoma (Rb) tumour suppressor gene product, E2F1, and HDAC1 and that DNMT1 cooperates with Rb to repress transcription from promoters containing E2F-binding sites. These results establish a link between DNA methylation, histone deacetylase and sequence-specific DNA binding activity, as well as a growth-regulatory pathway that is disrupted in nearly all cancer cells.

HATs off: Selective Synthetic Inhibitors of the Histone Acetyltransferases p300 and PCAF

Histone acetyltransferases (HATs) play important roles in the regulation of gene expression. In this report, we describe the design, synthesis, and application of peptide CoA conjugates as selective HAT inhibitors for the transcriptional coactivators p300 and PCAF. Two inhibitors (Lys-CoA for p300 and H3-CoA-20 for PCAF) were found to be potent (IC(50) approximately = 0.5 microM) and selective (approximately 200-fold) in blocking p300 and PCAF HAT activities. These inhibitors were used to probe enzymatic and transcriptional features of HAT function in several assay systems. These compounds should be broadly useful as biological tools for evaluating the roles of HATs in transcriptional studies and may serve as lead agents for the development of novel antineoplastic therapeutics.

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 HP1α, HP1β, and HP1γ 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 HP1α and HP1β isoforms, but not HP1γ, interact with MyoD in myoblasts. This interaction is direct, as shown using recombinant proteins in vitro. A gene reporter assay revealed that HP1α and HP1β, but not HP1γ, 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.

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.

Programming the transcriptional state of replicating methylated dna.

CpG methylation is maintained in daughter chromatids by the action of DNA methyltransferase at the replication fork. An opportunity exists for transcription factors at replication forks to bind their cognate sequences and thereby prevent remethylation by DNA methyltransferase. To test this hypothesis, we injected a linearized, methylated, and partially single-stranded reporter plasmid into the nuclei of Xenopus oocytes and followed changes in the transcriptional activity after DNA replication. We find that dependent on Gal4-VP16, the action of DNA methyltransferase, and replication-coupled chromatin assembly DNA replication provides a window of time in which regulatory factors can activate or repress gene activity. Demethylation in the promoter region near the GAL4 binding sites of the newly synthesized DNA did not occur even though the Gal4 binding sites were occupied and transcription was activated. We conclude that “passive” demethylation at the replication fork is not simply dependent on the presence of DNA binding transcriptional activators.

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry.

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.