Dynamics of heat shock factor association with native gene loci in living cells

Abstract

Direct observation of transcription factor action in the living cell nucleus can provide important insights into gene regulatory mechanisms. Live-cell imaging techniques have enabled the visualization of a variety of intranuclear activities, from chromosome dynamics to gene expression. However, progress in studying transcription regulation of specific native genes has been limited, primarily as a result of difficulties in resolving individual gene loci and in detecting the small number of protein molecules functioning within active transcription units. Here we report that multiphoton microscopy imaging of polytene nuclei in living Drosophila salivary glands allows real-time analysis of transcription factor recruitment and exchange on specific native genes. After heat shock, we have visualized the recruitment of RNA polymerase II (Pol II) to native hsp70 gene loci 87A and 87C in real time. We show that heat shock factor (HSF), the transcription activator of hsp70, is localized to the nucleus before heat shock and translocates from nucleoplasm to chromosomal loci after heat shock. Assays based on fluorescence recovery after photobleaching show a rapid exchange of HSF at chromosomal loci under non-heat-shock conditions but a very slow exchange after heat shock. However, this is not a consequence of a change of HSF diffusibility, as shown here directly by fluorescence correlation spectroscopy. Our results provide strong evidence that activated HSF is stably bound to DNA in vivo and that turnover or disassembly of transcription activator is not required for rounds of hsp70 transcription. This and previous studies indicate that transcription activators display diverse dynamic behaviours in their associations with targeted loci in living cells. Our method can be applied to study the dynamics of many factors involved in transcription and RNA processing, and in their regulation at native heat shock genes in vivo.

Imaging Drosophila gene activation and polymerase pausing in vivo

“…Computational deconvolution methods were used initially with wide-field microscopy to optically section Drosophila polytene nuclei 19 . Confocal, spinning disk and multi-photon microscopy further enhanced sectioning capabilities, with multi-photon microscopy being particularly effective in minimizing photodamage and providing high effective-resolution of thick biological specimens 3 . By applying these approaches to Drosophila polytene nuclei, it is possible to observe protein interactions with specific endogenous genetic loci in real time (Fig.…”

“…3). This was invaluable in directly tracking heat shock transcriptional activator (HSF) movement from nucleoplasm to specific heat shock gene loci (Hsp70), and for demonstrating that activated HSF remains bound in a non-exchanging state for many dozens of cycles of transcription 3 . These optical strategies should prove to be critical in exploring challenging mechanistic questions concerning transcription regulation, as discussed later.…”

“…Examination of specific, native gene loci requires more sensitivity and higherresolution views of interphase nuclei. The giant interphase nuclei of Drosophila polytene tissue provide both the sensitivity and effective resolution to detect signals from specific chromosomal loci 3 .…”

“…One, currently specific to Drosophila, makes use of state-of-the-art optics combined with the natural amplification of signals in tissue containing polytene chromosomes, allowing investigation of molecular interactions and dynamics at specific loci in real time in living nuclei 3 . The other, although having a history in Drosophila [4][5][6] , is species-general and uses crosslinking in vivo-that is, chromatin immunoprecipitation (ChIP) assays and variants thereof-to produce a 'molecular image' of specific protein interactions and chromatin modifications with particular DNA sequences in the genome 7 .…”

Heat Shock and Caloric Restriction Have a Synergistic Effect on the Heat Shock Response in a sir2.1-dependent Manner in Caenorhabditis elegans

“…HSF-1 is responsible for transcriptionally up-regulating the heat shock protein (HSP) genes. In mammalian cells, flies, nematodes, and plants, this transcription factor exists as an inactive monomer; however, in response to denaturing stress, it will trimerize and bind to heat shock elements located in the promoter regions of the hsp genes (3)(4)(5)(6). Increased expression of HSPs, such as HSP70 family members, results in cellular protection from a variety of stressors, including elevated temperatures, oxidative stress, heavy metals, proteasome inhibitors, and infection (7).…”