No abstract

This study examined the function of H3 and H4 tail domains in replication-dependent chromatin assembly. Results show distinct functions of H3 and H4 tails in nuclear import and chromatin assembly. Further investigations show that H4 diacetylation is essential but not sufficient for nuclear import, as preventing Hat1 binding impedes histone transport in nuclei.

Duchenne muscular dystrophy is the most common and severe form of muscular dystrophy, and although the genetic basis of this disease is well defined, the overall mechanisms that define its pathogenesis remain obscure. Alterations in individual signaling pathways have been described, but little information is available regarding their putative implications in Duchenne muscular dystrophy pathogenesis. Here, we studied the status of various major signaling pathways in the Golden Retriever muscular dystrophy dog that specifically reproduces the full spectrum of human pathology. Using antibody arrays, we found that Akt1, glycogen synthase kinase-3␤ (GSK3␤), 70-kDa ribosomal protein S6 kinase (p70S6K), extracellular signal-regulated kinases 1/2, and p38␦ and p38␥ kinases all exhibited decreased phosphorylation in muscle from a 4-month-old animal with Golden Retriever muscular dystrophy, revealing a deep alteration of the phosphatidylinositol 3-kinase (PI3K)/Akt and mitogen-activated protein kinase pathways. Immunohistochemistry analysis revealed the presence of muscle fibers exhibiting a cytosolic accumulation of Akt1, GSK3␤, and phosphatidylinositol-3 ,4 ,5-trisphosphate 3-phosphatase (PTEN) , an enzyme counteracting PI3K-mediated Akt activation. Enzymatic assays established that these alterations in phosphorylation and expression levels were associated with decreased Akt and increased GSK3␤ and PTEN activities. PTEN/GSK3␤-positive fibers were also observed in muscle sections from 3-and 36-month-old animals , indicating long-term PI3K/Akt pathway alteration. Collectively , our data suggest that increased PTEN expression and activity play a central role in PI3K/Akt/GSK3␤ and p70S6K pathway modulation , which could exacerbate the consequences of dystrophin deficiency.

The protein array methodology is used to study DNA-protein and protein-protein interactions governing gene expression from the Bacillus stearothermophilus PargCo promoter-operator region. Using probes labelled with near-infrared fluorescence dyes with exitation characteristics close to 700 or 800 nm, it is possible to detect signals from proteins (purified or non-purified in Escherichia coli cell extracts) immobilised on a nitrocellulose membrane with a high sensitivity (almost 12 amol of a spotted protein for protein-DNA interactions). Protein array data are confirmed by other methods indicating that molecular interactions of the order 10(-7) M can be monitored with the proposed protein array approach. We show that the PargCo region is a target for binding at least three types of regulatory proteins, ArgR repressors from thermophilic bacteria, the E. coli RNA polymerase alpha subunit and cyclic AMP binding protein CRP. We also demonstrate that the high strength of the PargC promoter is related to an upstream element that binds to the E. coli RNA polymerase alpha subunit.