ABSTRACT
Cytosine methylation plays an important role in the epigenetic regulation of eukaryotic gene expression. The methyl-CpG binding domain (MBD) is common to a family of eukaryotic transcriptional regulators. How MBD, a stretch of about 80 amino acids, recognizes CpGs in a methylation dependent manner, and as a function of sequence, is only partly understood. Here we show, using an Escherichia coli cell-free expression system, that MBD from the human transcriptional regulator MeCP2 performs as a specific, methylation-dependent repressor in conjunction with the BDNF (brain-derived neurotrophic factor) promoter sequence. Mutation of either base flanking the central CpG pair changes the expression level of the target gene. However, the relative degree of repression as a function of MBD concentration remains unaltered. Molecular dynamics simulations that address the DNA B fiber ratio and the handedness reveal cooperative transitions in the promoter DNA upon MBD binding that correlate well with our experimental observations. We suggest that not only steric hindrance, but also conformational changes of the BDNF promoter as a result of MBD binding are required for MBD to act as a specific inhibitory element. Our work demonstrates that the prokaryotic transcription machinery can reproduce features of epigenetic mammalian transcriptional regulatory elements.
Subject(s)
Gene Expression Regulation , Methyl CpG Binding Domain , Promoter Regions, Genetic , Brain-Derived Neurotrophic Factor/genetics , Brain-Derived Neurotrophic Factor/metabolism , Cell-Free System , Escherichia coli , Humans , Methyl-CpG-Binding Protein 2/genetics , Methyl-CpG-Binding Protein 2/metabolism , Molecular Docking SimulationABSTRACT
The actin cytoskeleton is a dynamic network that is composed of a variety of F-actin structures. To understand how these structures are produced, we tested the capacity of proteins to direct actin polymerization in a bead assay in vitro and in a mitochondrial-targeting assay in cells. We found that human zyxin and the related protein ActA of Listeria monocytogenes can generate new actin structures in a vasodilator-stimulated phosphoprotein-dependent (VASP) manner, but independently of the Arp2/3 complex. These results are consistent with the concept that there are multiple actin-polymerization machines in cells. With these simple tests it is possible to probe the specific function of proteins or identify novel molecules that act upon cellular actin polymerization.
Subject(s)
Actin Cytoskeleton/metabolism , Actins/metabolism , Bacterial Proteins/metabolism , Cytoskeletal Proteins , Membrane Proteins/metabolism , Metalloproteins/metabolism , Polymers/metabolism , Actin Cytoskeleton/drug effects , Actin Cytoskeleton/ultrastructure , Actin-Related Protein 2 , Actin-Related Protein 3 , Animals , Biological Assay , Cell Adhesion Molecules/metabolism , Cell-Free System , Chlorocebus aethiops , Fluorescent Antibody Technique , Glycoproteins , HeLa Cells/cytology , HeLa Cells/drug effects , HeLa Cells/metabolism , Humans , Metalloproteins/genetics , Microfilament Proteins , Microspheres , Mitochondria/metabolism , Mitochondria/ultrastructure , Phosphoproteins/metabolism , Proteins/metabolism , Recombinant Proteins/metabolism , Transfection , Vero Cells/cytology , Vero Cells/drug effects , Vero Cells/metabolism , Wiskott-Aldrich Syndrome Protein , ZyxinABSTRACT
Inspired by the motility of the bacteria Listeria monocytogenes, we have experimentally studied the growth of an actin gel around spherical beads grafted with ActA, a protein known to be the promoter of bacteria movement. On ActA-grafted beads F-actin is formed in a spherical manner, whereas on the bacteria a "comet-like" tail of F-actin is produced. We show experimentally that the stationary thickness of the gel depends on the radius of the beads. Moreover, the actin gel is not formed if the ActA surface density is too low. To interpret our results, we propose a theoretical model to explain how the mechanical stress (due to spherical geometry) limits the growth of the actin gel. Our model also takes into account treadmilling of actin. We deduce from our work that the force exerted by the actin gel on the bacteria is of the order of 10 pN. Finally, we estimate from our theoretical model possible conditions for developing actin comet tails.