Chromatin organization and transcriptional control of gene expression in Drosophila

It is increasingly clear that the packaging of DNA in nucleosome arrays serves not only to constrain the genome within the nucleus, but also to encode information concerning the activity state of the gene. Packaging limits the accessibility of many regulatory DNA sequence elements and is functionally significant in the control of transcription, replication, repair and recombination. Here, we review studies of the heat-shock genes, illustrating the formation of a specific nucleosome array at an activatable promoter, and describe present information on the roles of DNA-binding factors and energy-dependent chromatin remodeling machines in facilitating assembly of an appropriate structure. Epigenetic maintenance of the activity state within large domains appears to be a key mechanism in regulating homeotic genes during development; recent advances indicate that chromatin structural organization is a critical parameter. The ability to utilize genetic, biochemical and cytological approaches makes Drosophila an ideal organism for studies of the role of chromatin structure in the regulation of gene expression. Keywords: Cellular memory; Chromatin remodeling complexes; Gene silencing; Heat shock genes; PcG and trxG protein

The dual control of TFIIB recruitment by NC2 is gene specific

Negative co-factor 2 (NC2) is a conserved eukaryotic complex composed of two subunits, NC2α (Drap1) and NC2β (Dr1) that associate through a histone-fold motif. In this work, we generated mutants of NC2, characterized target genes for these mutants and studied the assembly of NC2 and general transcription factors on target promoters. We determined that the two NC2 subunits mostly function together to be recruited to DNA and regulate gene expression. We found that NC2 strongly controls promoter association of TFIIB, both negatively and positively. We could attribute the gene-specific repressor effect of NC2 on TFIIB to the C-terminal domain of NC2β, and define that it requires ORF sequences of the target gene. In contrast, the positive function of NC2 on TFIIB targets is more general and requires adequate levels of the NC2 histone-fold heterodimer on promoters. Finally, we determined that NC2 becomes limiting for TATA-binding protein (TBP) association with a heat inducible promoter under heat stress. This study demonstrates an important positive role of NC2 for formation of the pre-initiation complex on promoters, under normal conditions through control of TFIIB, or upon activation by stress via control of TBP

Functional Conservation of Cis-Regulatory Elements of Heat-Shock Genes over Long Evolutionary Distances

Transcriptional control of gene regulation is an intricate process that requires precise orchestration of a number of molecular components. Studying its evolution can serve as a useful model for understanding how complex molecular machines evolve. One way to investigate evolution of transcriptional regulation is to test the functions of cis-elements from one species in a distant relative. Previous results suggested that few, if any, tissue-specific promoters from Drosophila are faithfully expressed in C. elegans. Here we show that, in contrast, promoters of fly and human heat-shock genes are upregulated in C. elegans upon exposure to heat. Inducibility under conditions of heat shock may represent a relatively simple “on-off” response, whereas complex expression patterns require integration of multiple signals. Our results suggest that simpler aspects of regulatory logic may be retained over longer periods of evolutionary time, while more complex ones may be diverging more rapidly

Expression of the Hsp23 chaperone during Drosophila embryogenesis: association to distinct neural and glial lineages

BACKGROUND: In addition to their strong induction following stress, small heat shock proteins (Hsp) are also expressed during development in a wide variety of organisms. However, the precise identity of cell(s) expressing these proteins and the functional contribution of small heat shock proteins in such developmental context remain to be determined. The present study provides a detailed description of the Drosophila small heat shock protein Hsp23 expression pattern during embryogenesis and evaluates its functional contribution to central nervous system development. RESULTS: Throughout embryogenesis, Hsp23 is expressed in a stage-specific manner by a restricted number of neuronal and glial lineages of the central nervous system. Hsp23 is also detected in the amnioserosa and within a single lateral chordotonal organ. Its expression within the MP2 lineage does not require the presence of a functional midline nor the activity of the Notch signaling pathway. Transactivation assays demonstrate that transcription factors implicated in the differentiation of the midline also regulate hsp23 promoter activity. Phenotypic analysis of a transgenic line exhibiting loss of Hsp23 expression in the central nervous system suggests that Hsp23 is not required for development and function of this tissue. Likewise, its overexpression does not cause deleterious effects, as development remains unaffected. CONCLUSIONS: Based on the presented data, we suggest that the tightly regulated developmental expression of Hsp23 is not actively involved in cell differentiation and central nervous system development per se but rather reflects a putative role in preventive "pre-stress" neuroprotection or in non-vital process(es) common to the identified cell lineages

Whole-Genome Analysis Reveals That Active Heat Shock Factor Binding Sites Are Mostly Associated with Non-Heat Shock Genes in Drosophila melanogaster

During heat shock (HS) and other stresses, HS gene transcription in eukaryotes is up-regulated by the transcription factor heat shock factor (HSF). While the identities of the major HS genes have been known for more than 30 years, it has been suspected that HSF binds to numerous other genes and potentially regulates their transcription. In this study, we have used a chromatin immunoprecipitation and microarray (ChIP-chip) approach to identify 434 regions in the Drosophila genome that are bound by HSF. We have also performed a transcript analysis of heat shocked Kc167 cells and third instar larvae and compared them to HSF binding sites. The heat-induced transcription profiles were quite different between cells and larvae and surprisingly only about 10% of the genes associated with HSF binding sites show changed transcription. There were also genes that showed changes in transcript levels that did not appear to correlate with HSF binding sites. Analysis of the locations of the HSF binding sites revealed that 57% were contained within genes with approximately 2/3rds of these sites being in introns. We also found that the insulator protein, BEAF, has enriched binding prior to HS to promoters of genes that are bound by HSF upon HS but that are not transcriptionally induced during HS. When the genes associated with HSF binding sites in promoters were analyzed for gene ontology terms, categories such as stress response and transferase activity were enriched whereas analysis of genes having HSF binding sites in introns identified those categories plus ones related to developmental processes and reproduction. These results suggest that Drosophila HSF may be regulating many genes besides the known HS genes and that some of these genes may be regulated during non-stress conditions

Structure and expression of the yeast heat-shock gene HSP26.

A search was made for genes in Saccharomyces cerevisiae which are controlled by tightly regulated promoters that direct growth phase-specific gene expression during growth in batch culture. HSP26, a gene which encodes the major yeast small heat-shock protein, Hsp26, was found to be transcriptionally repressed during normal exponential growth (log-phase) and strongly induced during entry into stationary-phase on a glucose medium. The close parallel between the steady-state levels of HSP26 mRNA and the synthesis of the Hsp26 polypeptide, indicated that the synthesis of Hsp26 during the fermentative batch growth cycle is regulated primarily at the level of gene transcription. Activation of HSP26 during stationary-phase does not, however, result from the release of carbon catabolite repression due to glucose exhaustion, nor does it require the function of the WHI2 gene, involved in the normal coordination between cell proliferation and nutrient availability. The possible mechanism by which HSP2 6 is regulated during the yeast growth cycle, and the ’signal’ for stationary-phase induction, are discussed in relation to the heat-inducibility of this gene. Determination of the HSP2 6 gene nucleotide sequence, identified a number of sequence motifs in the promoter region which are characteristic of both heat-shock genes, and efficiently expressed genes in yeast. Sequence analysis of the HSP26 protein coding region identified a single open reading frame potentially encoding a 214 amino acid polypeptide of predicted molecular weight 27,630. This correlated well with the size of Hsp26 as estimated by it’s migration on a 2D-SDS PAGE gel. The predicted amino acid sequence of Hsp26 revealed homologies to other eukaryotic small Hsps, and also to bovine cc-crystall in A, homology being particularly strong in a predicted hydrophobic region of the Hsp26 polypeptide. In addition, with the exception of the amino-terminal methionine (Met), Hsp26 contains no Met residues, consistent with a failure to label the protein efficiently, .in vivo, with [3 5 S ] — methionine and demonstrating that the amino-terminal Met is cleaved post-translationally by an aminopeptidase. The biological function of Hsp26 remains unclear. Contrary to evidence that the small Hsps of a number of eukaryotic organisms appear to be responsible for a tolerance of cells to potentially lethal high temperatures, overexpression of the HSP26 gene on a high copy number yeast episomal plasmid, demonstrated that overexpression of the protein following a heat-shock contributes little to acquired thermotolerance. However, overexpression of Hsp26 in stationary-phase cells revealed the presence of 45-55nm diameter cytoplasmic particles as putative Hsp26 aggregates. That the hydrophobicity of Hsp26 results in it’s self aggregation, is presumably central to the function of this protein

R1 Retrotransposons in Drosophila melanogaster are Transcribed by RNA Polymerase I Upon Heat Shock

The ribosomal RNA genes of Drosophila melanogaster reside within centromere-proximal nucleolar organizers on both the X and Y chromosomes. Each locus contains between 200-300 tandem repeat rDNA units that encode 18S, 5.8S, and 28S ribosomal RNAs (rRNAs) for ribosome biogenesis. In arthropods like Drosophila, about 60% of rDNA genes are inserted with R1 and/or R2 retrotransposons at specific sites within the 28S regions; these units likely fail to produce functional 28S rRNA. We showed previously that R2 expression increases upon nucleolar stress caused by the loss of a ribosome assembly factor, the Nucleolar Phosphoprotein of 140 kDa (Nopp140). Here we show that R1 expression is selectively induced by heat shock. Actinomycin D, but not α-amanitin, blocked R1 expression in S2 cells upon heat shock, indicating that R1 is transcribed by Pol I. RT-PCR analysis confirmed read-through transcription by Pol I from the 28S gene region into R1. Using a genome wide precision run-on sequencing (PRO-seq) data set available at NCBI-GEO, we showed that Pol I activity on R1 elements is negligible under the normal non-heat shock condition but increases dramatically upon heat shock. We propose that prior to heat shock, Pol I pauses within ~350 bp of the 5’ end of R1 wherein we find ‘pause button’ like sequence motifs, and that heat shock releases Pol I for read-through transcription into R1

Blue Light Induces A Neuroprotective Open Access Gene Expression Program in Drosophila Photoreceptors

Background: Light exposure induces oxidative stress, which contributes to ocular diseases of aging. Blue light provides a model for light‑induced oxidative stress, lipid peroxidation and retinal degeneration in Drosophila melanogaster. In contrast to mature adults, which undergo retinal degeneration when exposed to prolonged blue light, newly‑eclosed fies are resistant to blue light‑induced retinal degeneration. Here, we sought to characterize the gene expression programs induced by blue light in fies of diferent ages to identify neuroprotective pathways utilized by photoreceptors to cope with light‑induced oxidative stress. Results: To identify gene expression changes induced by blue light exposure, we profled the nuclear transcriptome of Drosophila photoreceptors from one‑ and six‑day‑old fies exposed to blue light and compared these with dark controls. Flies were exposed to 3 h blue light, which increases levels of reactive oxygen species but does not cause retinal degeneration. We identifed substantial gene expression changes in response to blue light only in six‑day‑old fies. In six‑day‑old fies, blue light induced a neuroprotective gene expression program that included upregulation of stress response pathways and downregulation of genes involved in light response, calcium infux and ion transport. An intact phototransduction pathway and calcium infux were required for upregulation, but not downregulation, of genes in response to blue light, suggesting that distinct pathways mediate the blue light‑associated transcriptional response. Conclusion: Our data demonstrate that under phototoxic conditions, Drosophila photoreceptors upregulate stress response pathways and simultaneously, downregulate expression of phototransduction components, ion transporters, and calcium channels. Together, this gene expression program both counteracts the calcium infux resulting from prolonged light exposure, and ameliorates the oxidative stress resulting from this calcium infux. Thus, six‑day‑old fies can withstand up to 3 h blue light exposure without undergoing retinal degeneration. Developmental transitions during the frst week of adult Drosophila life lead to an altered gene expression program in photoreceptors that includes reduced expression of genes that maintain redox and calcium homeostasis, reducing the capacity of six‑day‑old fies to cope with longer periods (8 h) of light exposure. Together, these data provide insight into the neuroprotective gene regulatory mechanisms that enable photoreceptors to withstand light‑induced oxidative stress

The mRNA cap-binding complex stimulates the formation of pre-initiation complex at the promoter via its interaction with Mot1p in vivo

The cap-binding complex (CBC) binds to the cap structure of mRNA to protect it from exonucleases as well as to regulate downstream post-transcriptional events, translational initiation and nonsense-mediated mRNA decay. However, its role in regulation of the upstream transcriptional events such as initiation or elongation remains unknown. Here, using a formaldehyde-based in vivo cross-linking and chromatin immunoprecipitation assay in conjunction with transcriptional, mutational and co-immunoprecipitational analyses, we show that CBC is recruited to the body of yeast gene, and then stimulates the formation of pre-initiation complex (PIC) at several yeast promoters through its interaction with Mot1p (modifier of transcription). Mot1p is recruited to these promoters, and enhances the PIC formation. We find that CBC promotes the recruitment of Mot1p which subsequently stimulates PIC formation at these promoters. Furthermore, the formation of PIC is essential for recruitment of CBC. Thus, our study presents an interesting observation that an mRNA binding factor exhibits a reciprocal synergistic effect on formation of PIC (and hence transcriptional initiation) at the promoter, revealing a new pathway of eukaryotic gene regulation in vivo