Remodeling Of Complexes Research Articles

Remodeling of regulatory nucleoprotein complexes on the Xenopus hsp70 promoter during meiotic maturation of the Xenopus oocyte.

Transcriptional repression occurs during meiotic maturation of Xenopus oocytes. Injection of a DNA template containing an hsp70 promoter into Xenopus oocytes, followed by progesterone-induced maturation has been used to demonstrate a dynamic competition between the assembly of transcription factor-containing nucleoprotein complexes and repressive nucleosomal arrays during the maturation process. In particular, it is shown that increased levels of injected heat shock protein, the transcriptional activator Gal4-VP16 or the DNA template itself all lead to reduced repression of transcription on maturation. Conversely, injection of additional histone increases repression. Repression of transcription is shown to be accompanied by the formation of a more regular array of nucleosomes and by an increase in the efficiency of nucleosome assembly on the injected plasmid. Meiotic maturation is therefore accompanied by replacement of transcription factor complexes by a repressive chromatin environment.

RAG1 and RAG2 Form a Stable Postcleavage Synaptic Complex with DNA Containing Signal Ends in V(D)J Recombination

During V(D)J recombination, RAG1 and RAG2 cleave DNA adjacent to highly conserved recombination signals, but nothing is known about the protein–DNA complexes that exist after cleavage. Using a properly regulated in vitro V(D)J cleavage system, together with nuclease sensitivity, mobility shift, and immunoprecipitation experiments, we provide evidence that a stable complex is formed postcleavage between synapsed recombination signals. This complex includes the proteins RAG1, RAG2, HMG-1 or the closely related HMG-2 protein, and the components of the DNA- dependent protein kinase. The existence of such a stable complex explains a number of in vivo observations and suggests that remodeling of postcleavage synaptic complexes is an important step in the resolution of signal ends in V(D)J recombination.

MAP kinase activation by flow in endothelial cells. Role of beta 1 integrins and tyrosine kinases.

Local alterations in the hemodynamic environment regulate endothelial cell function, but the signal-transduction mechanisms involved in this process remain unclear. We previously demonstrated that mitogen-activated protein (MAP) kinase is rapidly stimulated by flow in bovine aortic endothelial cells. Integrin receptors may act as mechanotransducers, as suggested by rapid remodeling of focal adhesion complexes in response to flow. To study the role of integrins in flow-mediated MAP kinase activation, we compared the effects of beta 1 integrin activation (with 8A2 antibody) and flow in cultured human umbilical vein endothelial cells (HUVECs). Both 8A2 (3 micrograms/mL) and flow (shear stress, 12 dynes/cm2) stimulated MAP kinase, although the flow response was faster and greater. To characterize flow-activated tyrosine kinases, tyrosine-phosphorylated proteins were immunoprecipitated and identified by Western blot. There was a time-dependent increase in phosphotyrosine content in 60- to 80-kD, 110-kD, 125- to 150-kD, and 180- to 190-kD proteins. A 125-kD protein was identified as focal adhesion kinase (FAK), suggesting that flow activates integrins. In comparison with flow, 8A2 caused less tyrosine phosphorylation of fewer proteins, although FAK was tyrosine phosphorylated. Concurrent stimulation of HUVECs with 8A2 and flow caused additive increases in MAP kinase. Antibody 8A2 increased binding of the beta 1 affinity-sensitive antibody, 15/7, while flow failed to increase binding of 15/7. In summary, both a beta 1-activating antibody and flow stimulate tyrosine kinases, leading to activation of FAK and MAP kinase signal-transduction pathways. However, the cellular responses elicited by 8A2 represent only a portion of those stimulated by flow, suggesting that "costimulatory" events such as calcium mobilization, in addition to integrin activation, mediate the HUVEC response to fluid shear stress.