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Whole-genome analysis reveals that active heat shock factor binding sites are mostly associated with non-heat shock genes in Drosophila melanogaster.

Gonsalves SE, Moses AM, Razak Z, Robert F, Westwood JT - PLoS ONE (2011)

Bottom Line: There were also genes that showed changes in transcript levels that did not appear to correlate with HSF binding sites.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto, Mississauga, Canada.

ABSTRACT
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.

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Overlap of HSF binding sites indentified by ChIP-chip of Kc cells and immunostaining of polytene chromosomes of 3rd instar larvae.(A) The 434 HSF-bound sites indentified by ChIP-chip analysis were mapped to 265 unique cytolocations for this comparison. (B) Zoomed-in view of the region of chromosome 3R where the Hsp70B genes are located highlighting both the absence of tiling-array probes and the repetitive and/or low complexity sequence in this region as indicated by the RepeatMasker track (bottom). (C) Expanded view of (B) to show the location of the nearest HSF-bound (green circle). This image, generated using the UCSC Genome Browser, illustrates the chromosome region represented in bp (according to release 4.2 of the Drosophila genome) as indicated at the top. Genes are depicted as blue boxes with the thick and thin parts representing exons and introns respectively. Arrows (either blue or white) within the gene indicate the direction of transcription. Vertical black lines show the location of each probe on the Agilent 2×244k tiling arrays (Agilent Technologies) in the depicted region.
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pone-0015934-g002: Overlap of HSF binding sites indentified by ChIP-chip of Kc cells and immunostaining of polytene chromosomes of 3rd instar larvae.(A) The 434 HSF-bound sites indentified by ChIP-chip analysis were mapped to 265 unique cytolocations for this comparison. (B) Zoomed-in view of the region of chromosome 3R where the Hsp70B genes are located highlighting both the absence of tiling-array probes and the repetitive and/or low complexity sequence in this region as indicated by the RepeatMasker track (bottom). (C) Expanded view of (B) to show the location of the nearest HSF-bound (green circle). This image, generated using the UCSC Genome Browser, illustrates the chromosome region represented in bp (according to release 4.2 of the Drosophila genome) as indicated at the top. Genes are depicted as blue boxes with the thick and thin parts representing exons and introns respectively. Arrows (either blue or white) within the gene indicate the direction of transcription. Vertical black lines show the location of each probe on the Agilent 2×244k tiling arrays (Agilent Technologies) in the depicted region.

Mentions: In total we identified 434 HSF bound chromatin segments including regions associated with all but one of the known major heat-inducible genes. A selection of targets was confirmed by PCR (Figure 1C). No HSF binding site was detected upstream of any of the Hsp70B genes due to the absence of probes on the tiling array in this highly repetitive region of genome (Figure 2B,C). We next examined regions bound by HSF at RT (non-HS conditions) and found that 81% coincided with segments bound by HSF under HS conditions, however, in every case, the level of HSF binding is substantially less at RT (i.e. 5-fold lower on average) (Figure 3 and Table S1; data also available in GEO under GSE22335). The most highly enriched HSF binding site at RT is located upstream of Hsp83 in one of the only regions specifically occupied by HSF under non-HS conditions [36]. With the possible exception of this site, the observed weak HSF binding in the RT samples may reflect transient HSF binding, HSF binding in a subset of cells, and/or is the result of the induction of a mild HS response brought on by the initial harvesting and fixation of the cells.


Whole-genome analysis reveals that active heat shock factor binding sites are mostly associated with non-heat shock genes in Drosophila melanogaster.

Gonsalves SE, Moses AM, Razak Z, Robert F, Westwood JT - PLoS ONE (2011)

Overlap of HSF binding sites indentified by ChIP-chip of Kc cells and immunostaining of polytene chromosomes of 3rd instar larvae.(A) The 434 HSF-bound sites indentified by ChIP-chip analysis were mapped to 265 unique cytolocations for this comparison. (B) Zoomed-in view of the region of chromosome 3R where the Hsp70B genes are located highlighting both the absence of tiling-array probes and the repetitive and/or low complexity sequence in this region as indicated by the RepeatMasker track (bottom). (C) Expanded view of (B) to show the location of the nearest HSF-bound (green circle). This image, generated using the UCSC Genome Browser, illustrates the chromosome region represented in bp (according to release 4.2 of the Drosophila genome) as indicated at the top. Genes are depicted as blue boxes with the thick and thin parts representing exons and introns respectively. Arrows (either blue or white) within the gene indicate the direction of transcription. Vertical black lines show the location of each probe on the Agilent 2×244k tiling arrays (Agilent Technologies) in the depicted region.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3021535&req=5

pone-0015934-g002: Overlap of HSF binding sites indentified by ChIP-chip of Kc cells and immunostaining of polytene chromosomes of 3rd instar larvae.(A) The 434 HSF-bound sites indentified by ChIP-chip analysis were mapped to 265 unique cytolocations for this comparison. (B) Zoomed-in view of the region of chromosome 3R where the Hsp70B genes are located highlighting both the absence of tiling-array probes and the repetitive and/or low complexity sequence in this region as indicated by the RepeatMasker track (bottom). (C) Expanded view of (B) to show the location of the nearest HSF-bound (green circle). This image, generated using the UCSC Genome Browser, illustrates the chromosome region represented in bp (according to release 4.2 of the Drosophila genome) as indicated at the top. Genes are depicted as blue boxes with the thick and thin parts representing exons and introns respectively. Arrows (either blue or white) within the gene indicate the direction of transcription. Vertical black lines show the location of each probe on the Agilent 2×244k tiling arrays (Agilent Technologies) in the depicted region.
Mentions: In total we identified 434 HSF bound chromatin segments including regions associated with all but one of the known major heat-inducible genes. A selection of targets was confirmed by PCR (Figure 1C). No HSF binding site was detected upstream of any of the Hsp70B genes due to the absence of probes on the tiling array in this highly repetitive region of genome (Figure 2B,C). We next examined regions bound by HSF at RT (non-HS conditions) and found that 81% coincided with segments bound by HSF under HS conditions, however, in every case, the level of HSF binding is substantially less at RT (i.e. 5-fold lower on average) (Figure 3 and Table S1; data also available in GEO under GSE22335). The most highly enriched HSF binding site at RT is located upstream of Hsp83 in one of the only regions specifically occupied by HSF under non-HS conditions [36]. With the possible exception of this site, the observed weak HSF binding in the RT samples may reflect transient HSF binding, HSF binding in a subset of cells, and/or is the result of the induction of a mild HS response brought on by the initial harvesting and fixation of the cells.

Bottom Line: There were also genes that showed changes in transcript levels that did not appear to correlate with HSF binding sites.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.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Cell and Systems Biology, University of Toronto, Mississauga, Canada.

ABSTRACT
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.

Show MeSH
Related in: MedlinePlus