mportant for the activation function of TAF3. The linker deletions indicated that MedChemExpress HC-067047 removal of 150300 residues from the linker still allowed two- to three-fold activation. In contrast, a larger deletion or deleting both the linker region and the PHD completely abrogated TAF3-mediated coactivation. Immunoblot analysis confirmed the efficient expression of the deletion constructs, and immunofluorescence analysis showed that all linker region and linker/PHD deletion constructs of TAF3 induce translocation of TAF10 to the nucleus. Except the TAF3 construct lacking the linker/PHD, all other deletion constructs could efficiently bind to H3K4me3, indicating that the HFD and linker regions are not directly involved in H3K4me3 recognition by the PHD of TAF3. We have previously shown that the mRNA expression of RPL34, RPL31 and RPS10 genes is sensitive to H3K4me3 levels. Hence, we tested the effect of the various TAF3 constructs on the expression of these genes. To this end, we used HeLa cells with inducible expression of haspin for isolation of chromatin. Different fractions from induced and non-induced cells were obtained by salt extraction and micrococcal nuclease digestion of the nuclear pellet, and these fractions were analysed by immunoblotting. TFIID was detected by antibodies against the TBP-binding subunit, TAF1 and the structural subunit, TAF5. chromatin fraction. TBP was also decreased significantly from the chromatin fraction upon haspin expression. The remaining TBP could be part of other complexes like B-TFIID, SL1 or TFIIIB. PubMed ID:http://www.ncbi.nlm.nih.gov/pubmed/19828152 As negative control, we analysed chromatin association of NC2, which is known to interact with free TBP on DNA. As expected, haspin overexpression induces H3T3 phosphorylation, but has no effect on H3S10ph levels which is in line with the specificity of the haspin kinase for H3T3 and the observation that haspin overexpression does not strongly increase the mitotic population of cells. Analysis of H3K4me3 and H4 levels indicate equal chromatin content in extractions from uninduced and induced cells. Taken together, these results indicate that the haspin kinase can influence chromatin association of TFIID. Knockdown of haspin increases TFIID association with mitotic chromosomes H3T3 phosphorylation by haspin occurs during mitosis and coincides with a general inhibition of transcription. Previous & 2010 European Molecular Biology Organization H3T3ph blocks TFIID association with chromatin RA Varier et al immunofluorescence studies showed that the majority of TFIID dissociates from mitotic chromatin. We investigated the involvement of phosphorylation of H3T3 by haspin in TFIID dissociation from mitotic chromosomes by immunofluorescence and live-cell imaging. To this end, we used human U2OS osteosarcoma cells stably expressing GFP-tagged TAF5. The GFP-TAF5 protein in 
these cells is stably integrated into the TFIID complex and is expressed to 25% of the endogenous TAF5 levels. GFP-TAF5 cells were subjected to siRNAmediated knockdown of haspin and the decrease in haspin expression was analysed by immunoblotting. Histone H2B tagged with dsRed was cotransfected to visualize chromatin. Imaging of U2OS-GFP-TAF5 cells by video microscopy indicated that TAF5 was excluded from mitotic chromosomes after nuclear envelope breakdown and the exclusion was best visible when the chromosomes aligned at the metaphase plate. TAF5 swiftly relocalized on the chromosomes at late anaphase when the cells progressed into cytokinesis. This coi
Posted inUncategorized