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.

Summary 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 sec of gene activation and 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 PolyADP-Ribose (PAR) Polymerase activity is required to maintain the transcription compartment. We propose that PAR polymers locally retain factors in a transcription compartment.

Whereas the regulation of a gene is uniquely tailored to respond to specific biological needs, general transcriptional mechanisms are used by diversely regulated genes within and across species. The primary mode of regulation is achieved by modulating specific steps in the transcription cycle of RNA polymerase II (Pol II). Pol II “pausing” has recently been identified as a prevalent rate-limiting and regulated step in the transcription cycle. Many sequence-specific transcription factors (TFs) modulate the duration of the pause by directly or indirectly recruiting positive transcription elongation factor b (P-TEFb) kinase, which promotes escape of Pol II from the pause into productive elongation. These specialized TFs find their target-binding sites by discriminating between DNA sequence elements based on the chromatin context in which these elements reside and can result in productive changes in gene expression or nonfunctional “promiscuous” binding. The binding of a TF can precipitate drastic changes in chromatin architecture that can be both dependent and independent of active Pol II transcription. Here, we highlight heat-shock-mediated gene transcription as a model system in which to study common mechanistic features of gene regulation.

The role of the RAP74 ␣1 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 ␣1 helix, and RAP74(1-158), in which the ␣1 helix is deleted, were compared. Analysis of TFIIF RAP74-RAP30 complexes carrying the RAP74(1-158) deletion reveals the role of the ␣1 helix because this mutant has indistinguishable activity compared to TFIIF 74(W164A), which carries a critical point mutation in ␣1. 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 ␣1 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.Transient-state (or pre-steady-state) kinetic analysis allows an enzyme reaction to be tracked through individual catalytic events (21,22). Using rapid chemical quench-flow technology, our laboratory analyzed the formation of multiple specific phosphodiester bonds during elongation by human RNA polymerase II (RNAP II) (27,28,43,44). The RNAP II reaction is tracked with millisecond precision, allowing characterization of all but the very highest rates of bond formation. These studies provide significant insight into active site isomerization, phosphodiester bond formation, translocation, processive elongation, transcriptional stalling, pausing, RNA cleavage, and restart from a shortened RNA 3Ј end.In recent studies of poliovirus RNA-dependent RNA polymerase, Cameron and colleagues (1, 2) demonstrated that EDTA and HCl quenched elongation at different reaction stages, when the reaction was run in the presence of Mn 2ϩ as the divalent cation. This result indicated that the elongation complex (EC) isomerized to sequester two active-site Mn 2ϩ atoms from EDTA chelation and that, after EDTA addition, the isomerized EC continued on the forward pathway to complete bond formation. HCl, by contrast, was expected to quench the reaction instantly, demonstrating the time of chemistry. The human RNAP II elongation mechanism can similarly be divided into stages, according to the time of Mg 2ϩ sequestration (isomerization; EDTA quench) and the time of phosphodiester bond synthesis (chemistry; HCl quench) (43). Applying this double-quench approach, we obtain a detailed view of the human RNAP II mechanism.Transcription factor IIF (TFIIF) is a heterodimer (or heterotetramer) of RAP74 and RAP30 subunits (4,7,8,37,38). In this work, we compared RNAP II elongation in the presence o...

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) imaging. 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.

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HaloPlex is an amplicon based method for targeted sequencing. The protocol utilizes specificity gained from restriction enzyme recognition, hybridization and DNA ligation to capture molecules originating from the target region to be sequenced. The target region is fully customizable from a single gene up to several thousand discrete regions. Amplicon based methods for multiplex target enrichment are, in general, convenient methods for capturing a wide range of target region sizes. However, in contrast to hybridization capture methods where random shearing is deployed, it is not possible for Haloplex or other amplicon based techniques to use the start point of paired end reads to identify duplicate reads. Duplicate read information can be useful for improving base calling accuracy and to monitor sampling to determine the degree of confidence to assign calls at different presumed allelic fractions. For somatic variants, which are generally present at a lower than 50% allelic fraction, it is even more advantageous to know how many molecules have been sampled when calling a particular base. To enable identification of duplicate reads from libraries prepared with Haloplex, we have added a molecular barcode to the introduced primer cassette. The molecular barcode consists of ten degenerate bases allowing for over one million unique sequences to be present for tagging of molecules. Using information derived from the molecular barcode sequences we demonstrate observation of variants down to 5% allelic fraction in multiple molecules tagged with different molecular barcodes. The new protocol has, besides the introduction of molecular barcodes, been optimized in a few additional aspects. Due to improved reagent formulations and streamlining of workflow, complete target enrichment can now be completed in less than 5 hours. Using 50 ng input we demonstrate >85% specificity and above 90% of target regions being covered at >10% of average depth. Citation Format: Charmian Cher, Henrik Johansson, Javelin Chi, Katie Zobeck, Linus Forsmark, Magnus Isaksson, Holly Hogrefe. Accurate variant detection using molecular barcodes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 4897. doi:10.1158/1538-7445.AM2015-4897