Recruitment timing and dynamics of transcription factors at the Hsp70 loci in living cells.

Chromatin immunoprecipitation (ChIP) studies provide snapshots of factors on chromatin in cell populations. Here, we use live-cell imaging to examine at high temporal resolution the recruitment and dynamics of transcription factors to the inducible Hsp70 loci in individual Drosophila salivary gland nuclei. Recruitment of the master regulator, HSF, is first detected within 20 s of gene activation; the timing of its recruitment resolves from RNA polymerase II and P-TEFb, and these factors resolve from Spt6 and Topo I. Remarkably, the recruitment of each factor is highly synchronous between different cells. In addition, fluorescence recovery after photobleaching (FRAP) analyses show that the entry and exit of multiple factors are progressively constrained upon gene activation, suggesting the gradual formation of a transcription compartment. Furthermore, we demonstrate that poly(ADP-ribose) (PAR) polymerase activity is required to maintain the transcription compartment. We propose that PAR polymers locally retain factors in a transcription compartment.

Drosophila Set1 is the major histone H3 lysine 4 trimethyltransferase with role in transcription.

Histone H3 lysine 4 trimethylation (H3K4me3) is a major hallmark of promoter-proximal histones at transcribed genes. Here, we report that a previously uncharacterized Drosophila H3K4 methyltransferase, dSet1, and not the other putative histone H3K4 methyltransferases (Trithorax; Trithorax-related protein), is predominantly responsible for histone H3K4 trimethylation. Functional and proteomics studies reveal that dSet1 is a component of a conserved H3K4 trimethyltransferase complex and polytene staining and live cell imaging assays show widespread association of dSet1 with transcriptionally active genes. dSet1 is present at the promoter region of all tested genes, including activated Hsp70 and Hsp26 heat shock genes and is required for optimal mRNA accumulation from the tested genes. In the case of Hsp70, the mRNA production defect in dSet1 RNAi-treated cells is accompanied by retention of Pol II at promoters. Our data suggest that dSet1-dependent H3K4me3 is responsible for the generation of a chromatin structure at active promoters that ensures optimal Pol II release into productive elongation.

Imaging transcription dynamics at endogenous genes in living Drosophila tissues.

How transcription of individual genes is regulated in a single, intact, three-dimensionally organized cell nucleus remains mysterious. Recently, live cell imaging has become an essential tool to dissect the in vivo mechanisms of gene transcription. It not only examines functions of transcription factors at their gene targets within the chromatin context, but it also provides a non-disruptive approach for observing the dynamics of a transcription cycle in real time. However, the identification of any endogenous gene loci and their associated transcription factors remains technically difficult. Here, we describe the method of imaging the transcriptional dynamics of heat shock genes in Drosophila polytene chromosomes in living salivary gland tissues by multiphoton microscopy (MPM). This method has provided the experimental capability to visualize the assembly and dynamics of individual transcription factors and regulators and to dissect their functions at their endogenous gene targets in living cells.

Human RNA polymerase II elongation in slow motion: role of the TFIIF RAP74 alpha1 helix in nucleoside triphosphate-driven translocation.

The role of the RAP74 alpha1 helix of transcription factor IIF (TFIIF) in stimulating elongation by human RNA polymerase II (RNAP II) was examined using millisecond-phase transient-state kinetics. RAP74 deletion mutants RAP74(1-227), which includes an intact alpha1 helix, and RAP74(1-158), in which the alpha1 helix is deleted, were compared. Analysis of TFIIF RAP74-RAP30 complexes carrying the RAP74(1-158) deletion reveals the role of the alpha1 helix because this mutant has indistinguishable activity compared to TFIIF 74(W164A), which carries a critical point mutation in alpha1. We report adequate two-bond kinetic simulations for the reaction in the presence of TFIIF 74(1-227) + TFIIS and TFIIF 74(1-158) + TFIIS. TFIIF 74(1-158) is defective because it fails to promote forward translocation. Deletion of the RAP74 alpha1 helix results in increased occupancy of the backtracking, cleavage, and restart pathways at a stall position, indicating reverse translocation of the elongation complex. During elongation, TFIIF 74(1-158) fails to support detectable nucleoside triphosphate (NTP)-driven translocation from a stall position and is notably defective in supporting bond completion (NTP-driven translocation coupled to pyrophosphate release) during the processive transition between bonds.