Abstract
BackgroundAlu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA. Recent studies, however, suggest that they may contain transcription factor binding sites and hence possibly play a role in regulating gene expression.ResultsHere, we show that binding sites for a highly conserved member of the nuclear receptor superfamily of ligand-dependent transcription factors, hepatocyte nuclear factor 4alpha (HNF4α, NR2A1), are highly prevalent in Alu repeats. We employ high throughput protein binding microarrays (PBMs) to show that HNF4α binds > 66 unique sequences in Alu repeats that are present in ~1.2 million locations in the human genome. We use chromatin immunoprecipitation (ChIP) to demonstrate that HNF4α binds Alu elements in the promoters of target genes (ABCC3, APOA4, APOM, ATPIF1, CANX, FEMT1A, GSTM4, IL32, IP6K2, PRLR, PRODH2, SOCS2, TTR) and luciferase assays to show that at least some of those Alu elements can modulate HNF4α-mediated transactivation in vivo (APOM, PRODH2, TTR, APOA4). HNF4α-Alu elements are enriched in promoters of genes involved in RNA processing and a sizeable fraction are in regions of accessible chromatin. Comparative genomics analysis suggests that there may have been a gain in HNF4α binding sites in Alu elements during evolution and that non Alu repeats, such as Tiggers, also contain HNF4α sites.ConclusionsOur findings suggest that HNF4α, in addition to regulating gene expression via high affinity binding sites, may also modulate transcription via low affinity sites in Alu repeats.
Highlights
Alu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA
HNF4a binds Alu repeats in vitro Since genome-wide location analysis (i.e., chromatin immunoprecipitation (ChIP)-chip/seq) often filters out or cannot distinguish the exact location of transcription factor (TF) binding events in highly repetitive DNA, we took a combined in vitro/in silico approach to determine whether HNF4a binds Alu elements
Since RAR was previously shown to bind DR2-like sequences (AGGTCAxxAGGTCA) in Alu repeats [17] and since we have previously shown that HNF4a, while preferring DR1s, can bind DR2s [43], we put on the protein binding microarrays (PBMs) ~1470 permutations of DR1 and DR2 sequences as well as ~150 random controls and ~2000 additional sequences in the human genome predicted by a support vector machine (SVM) algorithm to bind HNF4a [42]
Summary
Alu repeats, which account for ~10% of the human genome, were originally considered to be junk DNA. Suggest that they may contain transcription factor binding sites and possibly play a role in regulating gene expression. The notion that repetitive DNA may play a regulatory role and be involved in the evolution of gene regulation was postulated early on, it was not until recently that there was evidence to support those ideas [3,4,5]. Alu insertions have been shown to alter the expression of at least six human genes: CD8a (CD8A), keratin 18 (KRT18), parathyroid hormone (PTH), Wilm’s tumor 1 (WT1), receptor for Fc fragment of IgE, high affinity I, gamma polypeptide (FCER1G) and breast cancer 1, early onset (BRCA1) [22]. Alu sequences may regulate the level of transcripts and proteins in the cell, as well as the function of those proteins
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