| Actins are a highly conserved family of proteins, which are widely distributed in many types of cells, and are essential for multiple cellular functions in vivo. A great deal of accumulating data, through biochemical analyses and immunocytochemical experiments, have proved that actins were existed in nuclei and chromosomes, and played many important roles in cytoplasm, cytoskeleton and nuclei. Although actins are crucially important, the role of the actins in gene transcription regulation is still a disputed issue so far. In this thesis, we studied the effects of nuclear actin on gene transcription regulation, with a special focus on the relations between the nuclear actin and RNA polymerase II in concanavalin A(ConA)-stimulated lymphocytes and HeLa cells. Meanwhile, we also discussed the functions of the actin on the mechanism of RNA polymerase II induced transcription regulation. The main results and conclusion are as follows.1. Lymphocytes activated by ConA showed a series of gene activation features, including the size increase in nuclei and the nucleoli, as well as chromatin decondensation. The morphotypes also corresponded closely to specific phases of macromolecular synthesis after activation. The actin content of the cells was studied by flow cytometry, showing no change in resting and ConA-stimulated mouse lymphocytes. But interestingly, we found that the F-actin level were progressively decreased in the ConA-stimulated cell, hi the earlier period of lymphocytes activation, the actin in the nuclei showed both a progressive increase and a close binding with nuclear skeleton detected by Western bolts.2. The distributions of actin and RNA polymerase II were studied by immuno-double labeling with anti-actin antibody and anti-RNA polymerase antibody respectively, and detected under a fluorescence microscope. The results showed that the distribution of actin, besides in cytoplasm, was identical to that of RNA polymerase II both in whole celland isolated nuclei. Nuclear actin was co-localized with RNA polymerase II in the same manner in terms of distribution and content. Further more, actin and RNA polymerase II could be co-immunoprecipitated, indicating that they could interact with each other. Though the F-actin labeled with FITC-phalliondin existed in nuclei, we did not find its co-localization with RNA polymerase II.3. The actin bundle network was induced in the nucleoplasm region when cells were cultured in a medium with a -amanitin, a transcription inhibitor that stimulates the degradation of the large subunit of RNA polymerase II. The co-localization of actin and RNA polymerase II was decreased when cells were treated with cytochalasin B, which blocked the F-actin assembly. The cells underwent a serious of process in which the nuclei divided but the cytoplasm did not divided, when the cells were cultured in a medium with leptomycin B, a specific inhibitor of nuclear export signal-dependent nuclear export They all formed a few larger RNA polymerase II inert aggregates with the above three treatments. These results demonstrated that actin in nuclei changed its forms from globular actin monomer to polymeric when gene transcription activity was inhibited. Blocking of F-actin polymerization or inhibition of actin from nuclear export would influence the distribution and state of RNA polymerase II.4. Interestingly, the co-localization between actin and RNA polymerase II was similar with the co-localization of myosin I isoform and RNA polymerase II in the nuclei. They could also interacte with BRG1, a key subunit in chromatin remodeling complex. Based on our results, we infered that the actin and myosin isoform I in the nuclei co-participated in gene transcription activities induced by RNA polymerase II. The actin might take part in the chromatin remodeling needed by gene transcription, and the myosin isoform I, perhaps together with RNA polymerases in a manner analogous to energy conversion, could potentially facilitate the movement of DNA and...
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