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. 2011 Nov 23;12(11):R117.
doi: 10.1186/gb-2011-12-11-r117.

Heat shock factor binding in Alu repeats expands its involvement in stress through an antisense mechanism

Rajesh Pandey  1 , Amit K Mandal, Vineet Jha, Mitali Mukerji
Affiliations

Heat shock factor binding in Alu repeats expands its involvement in stress through an antisense mechanism

Rajesh Pandey et al. Genome Biol. .

Abstract

Background: Alu RNAs are present at elevated levels in stress conditions and, consequently, Alu repeats are increasingly being associated with the physiological stress response. Alu repeats are known to harbor transcription factor binding sites that modulate RNA pol II transcription and Alu RNAs act as transcriptional co-repressors through pol II binding in the promoter regions of heat shock responsive genes. An observation of a putative heat shock factor (HSF) binding site in Alu led us to explore whether, through HSF binding, these elements could further contribute to the heat shock response repertoire.

Results: Alu density was significantly enriched in transcripts that are down-regulated following heat shock recovery in HeLa cells. ChIP analysis confirmed HSF binding to a consensus motif exhibiting positional conservation across various Alu subfamilies, and reporter constructs demonstrated a sequence-specific two-fold induction of these sites in response to heat shock. These motifs were over-represented in the genic regions of down-regulated transcripts in antisense oriented Alus. Affymetrix Exon arrays detected antisense signals in a significant fraction of the down-regulated transcripts, 50% of which harbored HSF sites within 5 kb. siRNA knockdown of the selected antisense transcripts led to the over-expression, following heat shock, of their corresponding down-regulated transcripts. The antisense transcripts were significantly enriched in processes related to RNA pol III transcription and the TFIIIC complex.

Conclusions: We demonstrate a non-random presence of Alu repeats harboring HSF sites in heat shock responsive transcripts. This presence underlies an antisense-mediated mechanism that represents a novel component of Alu and HSF involvement in the heat shock response.

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Figures

Figure 1
Figure 1
Heat shock factor binding motif and preferred binding sites in Alu repeats of heat shock responsive transcripts. The frequency of three preferred binding sites of HSFs mapped on to Alu repeats, in both sense and antisense orientations, of heat shock responsive transcripts is shown. The HSF site at position 221 is common between both sense and antisense Alus. Sites at positions 175 and 91 are unique to sense and antisense Alus, respectively. The inset shows the consensus motif for HSF sites across all Alu subfamilies.
Figure 2
Figure 2
Location of validated heat shock factor sites within Alus in the promoter region of up-regulated genes. A substantial fraction of up-regulated genes harbor high score (≥8.7) HSF sites within Alus in the upstream region. All the validated genes (SPINK6, HIST1H4A and ANKRD33) have their first HSF site within Alus in the promoter region from the transcription start site. Functional validation of such HSF sites through cloning, site-directed mutagenesis, transient transfection in a cell line and chromatin immunoprecipitation (ChIP) followed by PCR confirmed their role in the heat shock response.
Figure 3
Figure 3
Heat shock factor sites in Alu repeats are functionally active. Reporter constructs of the promoter region of three genes, SPN (G1), SPINK6 (G2) and HIST1H4A (G3), containing a HSF site within Alus when cloned downstream of a minimal promoter containing a firefly luciferase construct show more than two-fold induction in response to heat shock. Site-directed mutagenesis (SDM) in the predicted HSF within Alus led to reduced induction of approximately 1.4-fold in response to heat shock stress. Data were normalized using co-transfected renilla luciferase vector. The experiment was repeated three times and in triplicates to analyze standard deviations in all cases. HSPA1A was used as a positive control after an earlier published report of the functional HSF in the promoter region of the gene; the HSF site is in a non-Alu region of the HSPA1A gene promoter. G1, G2 and G3 represent wild type (WT) constructs, whereas G1*, G2* and G3* represent the SDM constructs. The statistical significance (Student's t-test) of the observed expression changes in response to heat shock in WT and SDM constructs as well as between them are shown in the lower panel, where figures in bold represent a significant P-value change. It is worth mentioning that the SDM constructs do not show significant expression changes following heat shock whereas WT and WT versus SDM show significant changes in expression. Error bars represent standard deviation among experimental replicates.
Figure 4
Figure 4
Heat shock factor binds to its cognate site in Alu repeats. The binding of a HSF to the heat shock element sequence present within the Alu repeat of HIST1H4A, SPINK6 and ANKRD33 genes in the promoter region was confirmed by ChIP-PCR after heat shock treatment. HSPA1A, with a HSF binding site in a non-Alu region, was used as a positive control. Input chromatin was used to ascertain that equal amounts of chromatin were applied in each reaction. Enrichment in heat shock treated samples compared to untreated and no antibody (Ab) ChIP samples is clearly visible. The faint band in the untreated HSPA1A sample confirms earlier reports of binding of HSF to this promoter, although this is not induced. As negative controls we considered regions of the genome that do not contain an HSF site. The negative controls do not show any band in the treated/untreated ChIP or 'no Ab' lanes, although the band in the input chromatin lane confirms the genomic presence of the queried region.
Figure 5
Figure 5
Contrasting patterns of HSF distribution in Alu and non-Alu sequences in upstream and genic regions of heat-shock responsive transcripts. In upstream regions, the non-Alu region had significantly higher HSF site density (P-value 2.05 × 10-24 and 1.8 × 10-20 for up-regulated and down-regulated transcripts, respectively). In genic regions, Alu regions have significantly higher HSF sites than non-Alu regions for both up-regulated (P-value 8.97 × 10-13) and down-regulated (P-value 2.58 × 10-3) transcripts.
Figure 6
Figure 6
Presence of Alus containing HSF sites in 5-kb proximal regions of antisense signals. HSF sites are especially enriched in the upstream (approximately 2-kb) region of the antisense transcript signal. Antisense transcripts may be transcribed through HSF binding to Alu repeats, leading to down-regulation of sense transcripts in response to heat shock.
Figure 7
Figure 7
Schematic for exon array antisense signals observed in heat shock down-regulated genes. Exon array probe co-ordinates for antisense signals are within 5-kb of HSF sites in antisense-oriented Alus. This led us to hypothesize that such Alus harboring high score (≥8.7) HSF sites can initiate antisense transcripts, leading to gene down-regulation through either transcriptional interference or the sense-antisense-mediated RNA interference pathway.
Figure 8
Figure 8
HSF occupancy within Alus in the proximal region of antisense transcripts. The possibility that putative high-score HSF sites within Alus drive antisense transcription was confirmed by ChIP-PCR using ChIP DNA made against HSF1. In response to heat shock, we observed enriched binding of HSF1 at its predicted site compared to the untreated state. The absence of a band in the 'no antibody' (No Ab) control points towards conditional and specific binding of HSF1 to the putative site.
Figure 9
Figure 9
Quantitative RT-PCR analyses of antisense transcripts after siRNA-mediated knockdown of antisense transcripts. Elevated levels of antisense transcripts seen in the Affymetrix antisense exon array analysis were confirmed by qRT-PCR for four antisense transcripts. These transcripts showed down-regulation after knockdown of antisense transcripts by specific siRNAs following transient transfection in a cell line. The levels of two control genes (marked by asterisks) unaffected by heat shock and devoid of Alus harboring HSF sites were monitored as experimental controls. These control genes, MGST1 and RPL8, did not show significant expression differences either during heat shock stress or after siRNA-mediated knockdown of the respective antisense transcripts. Error bars represent standard deviation among experimental replicates.
Figure 10
Figure 10
Quantitative RT-PCR analyses of sense transcripts following siRNA-mediated knockdown of antisense transcripts. In response to heat shock, the sense transcripts are down-regulated. These transcripts contain an antisense Alu in the genic region with a predicted HSF site that may drive antisense transcription. Following siRNA-mediated knockdown of the antisense transcripts (signal in Affymetrix antisense Exon array), sense transcripts were up-regulated in response to heat shock. Error bars represent standard deviation among experimental replicates.
Figure 11
Figure 11
Gene Ontology analyses of differentially expressed transcripts containing HSF sites in Alu and non-Alu regions. Heat map of the significantly enriched GO processes (P-value cutoff <0.05, after correction) in differentially expressed transcripts. The categories are in relation to Table 2: 'Both', Alu + Non-Alu; 'Non-Alu', exclusively in non-Alus; 'Alu', exclusively in Alus. Green and red colors denote GO processes for up-regulated and down-regulated transcripts, respectively.
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