Cellular Localization Of The Assembly Of P-TEFb Active Complexes

P-TEFb is a critical elongation factor for the control of transcription elongation in eukaryotes. There are two forms of P-TEFb in cells. The first form is inactive7SK snRNP complex containing other factors such as7SK and HEXIM1. The second form is a transcriptionally active complex. The first discovered active complex is formed by P-TEFb and BRD4. Recent studies have identified another transcriptionally active complex, the super elongation complexes (SECs), which contains P-TEFb, AFF4, E112, as well as ENL, AF9, AFF1and other factors. Previous studies in our lab revealed the signaling pathways and molecular mechanisms of how the P-TEFb inactive complex (7SK snRNP) and the active complex (BRD4/P-TEFb, SECs) were converted into each other. But it is not clear where is the integration site of these factors. It was reported that nuclear speckles may function in the storage/assembly/modification of splicing factors. We suspect that nuclear speckles is the integration place of the transcription elogation factors. Hence, the current study mainly focuses on two respects, namely whether the components of the two types of active P-TEFb complexes co-localized in nuclear speckle domains, and what are the mechanisms for their integration in nuclear speckles. Normal HeLa cell and F1C2cell strain with stably expression of Flag-CDK9were used as our research models. With overexpression, knockdown, and immunofluorescence techniques, we found that BRD4/CyclinTl/CDK9/AFF4are present in nuclear speckles. Under stress conditions, these factors will have different degrees of aggregation in the nuclear speckles. Remarkably, we found that CDK9is essential for BRD4’s localization in the speckles by binding the PID domains of BRD4. However, BRD4, CyclinTl are not required for the localization of CDK9into speckles. And CDK9or ELL2is not the critical factor for recruiting AFF4into nuclear speckles. Notably, ELL2is essential the localization of CyclinTl to speckles by binding to the501-530area of Cyclin Tl. In summary, this study showed that speckles are the integration place of the activate P-TEFb complexes, and preliminary results indicate that the integration mechanism of the different complexes are distinct. These results are important for completing the full molecular model of the regulation of P-TEFb’s activity and for an in-depth understanding of the molecular mechanism of gene transcription.