We have investigated the spatial relationship between sites containing newly synthesized RNA and domains containing proteins involved in transcription, such as RNA polymerase II and the transcription factors TFIIH, Oct1, BRG1, E2F-1 and glucocorticoid receptors, using dual immunofluorescence labelling followed by confocal microscopy on cultured cells. As expected, a high degree of colocalisation between the RNA polymerase II and sites containing newly synthesised RNA was observed. Like the newly synthesised RNA and the RNA polymerase II, we found that all the transcription factors that we studied are distributed more or less homogeneously throughout the nucleoplasm, occupying numerous small domains. In addition to these small domains, TFIIH was found concentrated in coiled bodies and Oct1 in a single large domain of about 1.5 microm in 30% of the cells in an asynchronous HeLa cell culture. Remarkably, we found little or no relationship between the spatial distribution of the glucocorticoid receptor, Oct1 and E2F-1 on the one hand and RNA polymerase II and transcription sites on the other hand. In contrast, a significant but incomplete overlap was observed between the spatial distributions of transcription sites and BRG1 and TFIIH. These results indicate that many of the transcription factor-rich nuclear domains are not actively involved in transcription. They may represent incomplete transcription initiation complexes, inhibitory complexes, or storage sites.
Identification of sequence-specific promoters driving polycistronic transcription initiation by RNA polymerase II in trypanosomes
