Drosophila heat shock system as a general model to investigate transcriptional regulation

Abstract

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.

Figure 1

Conservation of HSF-targeted DNA sequence element. The consensus HSE is remarkably conserved among Saccharomyces cerevisiae, D. melanogaster, and Homo sapiens (Fernandes et al. 1994; Trinklein et al. 2004; Guertin and Lis 2010).

Figure 2

Chromatin landscape dictates HSF binding to HSEs in vivo. This region of chromosome 3L contains four potential HSF-binding sites. HSE sequences conform to the HSE matrix with the following p values, from left to right: 3.9 × 10⁻⁶, 1.8 × 10⁻⁵, 1.4 × 10⁻⁵, and 4.3 × 10⁻⁵. Although the HSF-free motif conforms to the consensus with the lowest p value, chromatin structure restricts HSF occupancy. HSEs that are enriched for H4 and H3 acetylation during non-HS (Kharchenko et al. 2010a) are preferentially bound by HSF in vivo (Guertin and Lis 2010).

Figure 3

Decondensation of heat-shock- and ecdysone-induced genes. HS genes at cytological loci 87A and 87C and developmental genes at 74EF and 75B decondense dramatically following activation. Hoeschst-stained DNA (blue), serine-5 phosphorylated Pol II (H14 antibody) (red).

Figure 4

Dynamics of Pol II at Hsp70 loci in vivo. FRAP (Fluorescence recovery after photobleaching) was used to measure dynamics of Pol II (using an Rpb3-GFP [green fluorescent protein] fusion protein) after 10- or 60-min following an instantaneous HS. Nonphotobleached Pol II is rapidly recruited to loci after 10 min but more modestly recruited after 60 min. Time (in seconds) along the x axis represents time elapsed after photobleaching.

Figure 5

Gradual recruitment of Pol II to the Hsp70 loci in vivo. Additional Pol II is recruited to Hsp70 loci (white doublet) at ~2 min post-HS (third panel) and accumulates until ~5-min post-HS (seventh panel). After 5 min of HS, Pol II reaches maximal levels and remains high for the duration of the experiment.