Nucleosomal instability and induction of new upstream protein-DNA associations accompany activation of four small heat shock protein genes in Drosophila melanogaster.

Abstract

We investigated in detail the structural changes that occur in nuclear chromatin upon activation of the four small heat shock protein genes in D. melanogaster. Both -the chemical cleavage reagent methidiumpropyl-EDTA-iron(II) [MPE Fe(II)] and the nuclease DNase I revealed a complex pattern of four or five hypersensitive sites upstream of each gene before activation. In addition, MPE-Fe(II) detected a short positioned array of nucleosomes located on each coding region. Upon heat shock activation a number of changes in the patterns occurred. For each gene, at least one of the upstream hypersensitive regions was eliminated or substantially shifted in position. Regions were established which became highly refractile to digestion by either MPE -Fe(II) or DNase I and, as such, appeared as small "footprints" in the pattern. The location of these refractile regions relative to the cap site varied for each gene examined. The coding regions themselves became highly accessible to DNase I. The nucleosomal arrays detected by MPE-Fe(II) were characterized by a considerable loss of detail and significantly enhanced accessibility, the extent of which probably reflected the relative transcription rate of each gene. Careful mapping of the location and extent of each upstream footprint and comparison with the DNA sequence revealed the presence at each location of two (or more) contiguous or overlapping segments that bear high homology to the heat shock consensus sequence C-T-N-G-A-A-N-N-T-T-C-N-A-G. A specific protein factor (or factors) is most likely bound at or near these sequences in heat-shocked Drosophila cells.

Nucleosome positioning and gene regulation

“…In some cases, gene repression by a nucleosome has been shown to require specific interactions of histones with a nonhistone protein, such as the interaction between the a2 repressor and the histone H4 N-terminal tail [Roth et al, 19921. Analysis of specific mutations in the histone H4 gene indicates that di- The sketch of a preset gene is based on the analysis of the Drosophila melanogaster hsp26 gene [Cartwright and Elgin, 1986;Thomas and Elgin, 19881; the sketch of a remodeling gene is based on the analysis of the yeast PH05 gene [Almer et al, 1986;Straka and Horz, 19911 rect interactions occur between histone H4 and other chromosomal proteins to achieve the epigenetic regulation observed in silencing the HML and HMR mating type cassettes ; see references therein]. The involvement of nucleosomes is further substantiated by the observation that alleviation of repression for many genes also requires wild type histones; alteration of the lysine residues in the N-terminal tail of H4, which eliminates the potential for H4 acetylation, can reduce activation of inducible genes [Durrin et al, 19911.…”

“…The Drosophila melamgaster hsp26 gene provides an excellent example of a preset promoter. Prior to activation, the hsp26 promoter contains two prominent DNase I hypersensitive sites (DH sites) that map to the location of the two heat shock elements (HSEs) that are required for heat shock induction; a nucleosome is positioned precisely on sequences between the two DH sites [Cartwright and Elgin, 1986;Thomas and Elgin, 19881. An RNA polymerase I1 molecule is transcriptionally engaged but paused at the promoter [Rougvie and Lis, 19901.…”

Localization of specific topoisomerase I interactions within the transcribed region of active heat shock genes by using the inhibitor camptothecin.

“…The sequences must also be accessible to the enzyme. Measurements with various DNA cleavage reagents indicate that actively transcribed genes are markedly more accessible than the inactive genes in isolated nuclei (6,50,52 (19).…”

“…A change in the compaction of the chromatin fiber as well as a perturbation of the histone-DNA interactions has been shown to occur at active genes (50,52). The perturbation of the nucleosome array appears to correlate with the level of transcription; among the heat shock genes of Drosophila melanogaster, those transcribed at higher levels are more sensitive to cleavage by DNase I and methidiumpropyl-ferrous EDTA than are those transcribed at lower levels (6).…”

Chromatin structure of a P-element-transduced hsp-28 gene in Drosophila melanogaster.

“…This gene was chosen because it is rapidly induced to high levels of expression in a variety of tissues by a brief heat shock and because the chromatin structure of the hsp-28 locus has been extensively characterized in embryos (1,11,14) and tissue culture cells (1). The plasmid used in these transformation experiments (called Ad44) is shown in Fig.…”