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

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.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.Alu repeats, which occupy more than one-tenth of the human genome, have been shown to harbor a large number of transcription factor binding sites (TFBSs) [1-3], many of which have also been demonstrated to be functionally active. These have been mostly discovered during the course of characterization of regulatory sites in promoter regions of genes [4-15]. Recently, genome wide informatics analyses have revealed substantial distribution of these sites in Alu repeats - for instance, nearly 90% of retinoic acid response element binding sites in human are in Alus [16]. As Alus also provide substrates for non-homologous recombination, they are also enriched in a large number of regions of segmental duplication [17,18]. Through these recombination events, Alus harboring regulatory sites could also create novel regulatory n