Association of some potential hormone response elements in human genes with the Alu family repeats

Abstract

Short interspersed repeats of the Alu family located in promoters of some human genes contain high-affinity binding sites for thyroid hormone receptor, retinoic acid receptor and estrogen receptor. The standard binding sites for the receptors represent variants of duplicated AGGTCA motif with different spacing and orientation (direct, DR, or inverted, IR), and Alu sequences were found to have functional DR-4, DR-2 or variant IR-3/IR-17 elements. In this study we analyzed distribution and abundance of the elements in a set of human genomic sequences from GenBank and their association with Alu repeats. Our results indicate that a major fraction of potentially active DR-4, DR-2 and variant IR-3/IR-17 elements in the genes is located within Alu repeats. Alu-associated DR-2 elements are conserved in primate evolution. However, very few Alu have potential DR-3 glucocorticoid-response elements. Gel-shift experiments with the probe (AUB) corresponding to the consensus Alu sequence just upstream of the RNA polymerase III promoter B-box and containing duplicated AGGTCA motif indicate that the probe interacts in a sequence-specific manner with human nuclear proteins which bind to standard IR-0, DR-1, DR-4 or DR-5 elements. The AUB sequence was also able to promote thyroid hormone-dependent trans-activation of a reporter gene. The results support the view that Alu retroposons played an important role in evolution of regulation of the primate gene expression by nuclear hormone receptors.

Introduction

Transcription of eukaryotic genes is controlled by short conserved sequence blocks mostly located in 5′-flanking regions of genes, but also in introns and 3′-flanking regions (Koop and Hood, 1994). The blocks interact with components of the obligatory basal transcription machinery and with non-basal transcription factors initiating combinatorial transcriptional modulation of genes in specific cell lineages and in response to environmental signals. Many members of the large and important superfamily of transcription factors called nuclear hormone receptors (NHRs) recognize and bind to the promoter-associated hexamer block AGGTCA, and its duplicated variants with variable spacing and orientation — direct repeat, DR, or inverted repeat, IR (Mangelsdorf et al., 1990, Mangelsdorf et al., 1995, Umesono et al., 1991). For example, estrogen receptor (ER) binds as homodimer to an element IR-3 (inverted repeat with spacing 3 nt), receptors of the 9-cis-retinoic acid (RXRαβγ) preferentially bind to an element DR-1, receptors of the all-trans retinoic acid (RARαβγ) bind as heterodimers with RXR to elements IR-0 and DR-2,5, and thyroid hormone receptors (TRαβ) bind as heterodimers with RXR to elements IR-0 and DR-4 (Mangelsdorf and Evans, 1995, Mangelsdorf et al., 1995).

Many eukaryotic promoters carry the AGGTCA motif-containing DR or IR elements, suggesting that their transcription may be regulated by the NHRs. In most non-primate mammalian species these elements are embedded in variable sequence contexts. However, it has recently been found that in some human promoters functional DR-4 and DR-2 elements binding TR/RXR or RAR/RXR (Piedrafita et al., 1996, Toyoda et al., 1995, Vansant and Reynolds, 1995) and a variant IR-3/IR-17 element with high-affinity binding site for ER (Norris et al., 1995) are located within conserved sequences of Alu repeats which represent the most abundant family of ubiquitous human retroposons (Schmid and Maraia, 1992). Thyroid hormone response element (TRE) in the type I 5′-deiodinase (D1) gene provides a clear example of a functional Alu-associated element in the human gene promoter (Jakobs et al., 1997, Toyoda et al., 1995, Zhang et al., 1998). The promoter has distal (TRE2) and proximal (TRE1) elements, the first of which is a typical DR-4 element located within Alu repeat and the second is a variant DR-10 element. Both of them have been shown to contribute equally to the thyroid hormone (3,3′,5-triiodotyronine) induction of the promoter in a transient expression assay (Toyoda et al., 1995). It has been also shown recently (Nishikawa et al., 1998) that the level of the D1 mRNA in mononuclear blood cells in normal individuals is increased by the thyroid hormone in a dose-dependent manner, indicating that the promoter is directly activated by the hormone in situ.

Estrogen receptor-dependent upregulation of the BRCA1 mRNA in human breast cells in vivo (Romagnolo et al., 1998, Spillman and Bowcock, 1996) was shown to be mediated, at least in part, by an Alu-associated estrogen response element (ERE) (Norris et al., 1995) located within BRCA1 gene promoter β (Xu et al., 1997). The failure of the earlier attempt to reveal direct estrogen responsibility for the BRCA1 gene expression (Marks et al., 1997) was explained by an absence of the BRCA1 gene transcription initiation site in the plasmid constructs used in that attempt (Marks et al., 1997, Xu et al., 1997). A striking difference is observed in the structure of promoters of the mouse and human BRCA1 genes: exon 1B containing Alu-ERE and promoter β, present in the human gene, are absent from the corresponding mouse genomic sequence (Xu et al., 1997). Interestingly, an attempt to clone the most efficient human genomic EREs resulted in the isolation of a subset of Alu sequences (Norris et al., 1995) indicating that most of the EREs in human genes are associated with Alu repeats. Consensus sequence of the major subfamily of Alu repeats (AluSx) representing about 80% of all members of the family in the human genome (Batzer et al., 1996) contains motif AGGTCA located just upstream of the core element (B-box) of the internal RNA polymerase III (Pol III) promoter of the retroposons. The Alu segment covering this motif had previously been shown to contain DR-2 retinoic acid response element which binds RAR/RXR heterodimer (Vansant and Reynolds, 1995).

In this study we analyzed the distribution of potentially active DR-4, DR-2 and variant IR-3/IR-17 elements in a set of human genomic sequences, and their association with Alu repeats. The results of electrophoretic mobility shift assays (EMSA) indicate that the majority of DR-4, DR-2 and variant IR-3/IR-17 elements in human genes are associated with Alu repeats, and that HeLa cells contain Alu-binding proteins also interacting with consensus DR-1, DR-5 and IR-0 elements. An Alu-consensus element is able to support thyroid hormone-dependent trans-activation of luciferase reporter gene in a transient transfection assay.