Visualizing cellular stress: A hypothesis-driven confocal laboratory exercise to identify compounds that activate heat shock factor binding at Hsp70 loci.

Exposure of organisms to high temperatures and various chemical and physical stressors can cause protein misfolding and aggregation. In turn, this can disrupt the functions of proteins, threatening both development and homeostasis. To overcome this, cells can initiate the highly conserved heat shock (HS) stress response pathway.

Imaging RNA Polymerase II transcription sites in living cells.

Over the past twenty years, exciting developments in optical and molecular imaging approaches have allowed researchers to examine with unprecedented resolution the spatial organization of transcription sites in the nucleus. An attractive model that has developed from these studies is that active genes cluster to preformed transcription factories that contain multiple active RNA polymerases and transcription factor proteins required for efficient mRNA biogenesis. However, this model has been extensively debated in part due to the fact transcription factories and their features have only been documented in fixed cells.

Kinetics of promoter Pol II on Hsp70 reveal stable pausing and key insights into its regulation.

The kinetics with which promoter-proximal paused RNA polymerase II (Pol II) undergoes premature termination versus productive elongation is central to understanding underlying mechanisms of metazoan transcription regulation. To assess the fate of Pol II quantitatively, we tracked photoactivatable GFP-tagged Pol II at uninduced Hsp70 on polytene chromosomes and showed that Pol II is stably paused with a half-life of 5 min. Biochemical analysis of short nascent RNA from Hsp70 reveals that this half-life is determined by two comparable rates of productive elongation and premature termination of paused Pol II.

Fcp1 dephosphorylation of the RNA polymerase II C-terminal domain is required for efficient transcription of heat shock genes.

Fcp1 dephosphorylates the C-terminal domain of the largest subunit of RNA polymerase II (Pol II) to recycle it into a form that can initiate a new round of transcription. Previously, we identified Drosophila Fcp1 as an important factor in optimal Hsp70 mRNA accumulation after heat shock. Here, we examine the role of Fcp1 in transcription of heat shock genes in vivo.

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.

Paradoxical instability-activity relationship defines a novel regulatory pathway for retinoblastoma proteins.

The retinoblastoma (RB) transcriptional corepressor and related family of pocket proteins play central roles in cell cycle control and development, and the regulatory networks governed by these factors are frequently inactivated during tumorigenesis. During normal growth, these proteins are subject to tight control through at least two mechanisms. First, during cell cycle progression, repressor potential is down-regulated by Cdk-dependent phosphorylation, resulting in repressor dissociation from E2F family transcription factors.

Retinoblastoma protein regulation by the COP9 signalosome.

Similar to their human counterparts, the Drosophila Rbf1 and Rbf2 Retinoblastoma family members control cell cycle and developmentally regulated gene expression. Increasing evidence suggests that Rbf proteins rely on multiprotein complexes to control target gene transcription. We show here that the developmentally regulated COP9 signalosome (CSN) physically interacts with Rbf2 during embryogenesis.

Distinct developmental expression of Drosophila retinoblastoma factors.

Retinoblastoma (RB) tumor suppressor proteins are important regulators of the cell cycle and are implicated in a wide variety of human tumors. Genetic analysis of RB mutations in humans and in model systems indicates that individual RB proteins also have distinct functions in development. Specific target genes or mechanisms of action of individual RB proteins in developmental contexts are not well understood, however.

odd-skipped homologs function during gut development in C. elegans.

Genes in the odd-skipped (odd) family encode a discrete subset of C2H2 zinc finger proteins that are widely distributed among metazoan phyla. Although the initial member (odd) was identified as a Drosophila pair-rule gene, various homologs are expressed within each of the three germ layers in complex patterns that suggest roles in many pathways beyond segmentation. To further investigate the evolutionary history and extant functions of genes in this family, we have initiated a characterization of two homologs, odd-1 and odd-2, identified in the genome of the nematode, Caenorhabditis elegans.