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. 2009 Nov 5;28(44):3857-65.
doi: 10.1038/onc.2009.246. Epub 2009 Aug 31.

p53 responsive elements in human retrotransposons

C R Harris  1 , A Dewan, A Zupnick, R Normart, A Gabriel, C Prives, A J Levine, J Hoh
Affiliations

p53 responsive elements in human retrotransposons

C R Harris et al. Oncogene. .

Abstract

Long interspersed nuclear elements-1 (L1s) are highly repetitive DNA elements that are capable of altering the human genome through retrotransposition. To protect against L1 retroposition, the cell downregulates the expression of L1 proteins by various mechanisms, including high-density cytosine methylation of L1 promoters and DICER-dependent destruction of L1 mRNAs. In this report, a large number of p53 responsive elements, or p53 DNA binding sites, were detected in L1 elements within the human genome. At least some of these p53 responsive elements are functional and can act to increase the levels of L1 mRNA expression. The p53 protein can directly bind to a short 15-nucleotide sequence within the L1 promoter. This p53 responsive element within L1 is a recent addition to evolution, appearing approximately 20 million years ago. This suggests an interplay between L1 elements, which have a rich history of causing changes in the genome, and the p53 protein, the function of which is to protect against genomic changes. To understand these observations, a model is proposed in which the increased expression of L1 mRNAs by p53 actually increases, rather than decreases, the genomic stability through amplification of p53-dependent processes for genomic protection.

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Conflict of interest statement

Conflict of interest The authors declare no conflict of interest.

Figures

Figure 1
Figure 1
Distributions of p53REs in the human genome. The distributions of the p53REs (a) → ← and (b) ← → in the human genome are shown. Spacer lengths from 0 to 10 kb are on the x axis, and relative frequencies on the y axis. The (red) line represents the exponential density function. Both observed and expected frequencies of spacers > 10 kb are close to 0 and hence are omitted. A full colour version of this figure is available at the Oncogene journal online.
Figure 2
Figure 2
Genomic structure of L1 and its p53 responsive elements. Numbers in black represent gene coordinates in base pairs from the transcriptional start site.
Figure 3
Figure 3
L1 (sense promoter) induction by p53, and mutational analysis. (a) Levels of transcription produced by an L1 (1–802)–firefly luciferase fusion plasmid upon cotransfection with plasmids encoding wild-type or mutant p53 were compared by luciferase assay. Twenty-four hours after cotransfection, cells were lysed and both firefly luciferase, which was expressed by the L1 promoter, and renilla luciferase, which acts as a transfection control, were assayed. Expression from L1–luciferase fusions containing mutations in the ← → ← or ← → sites were also tested. (b) Three-quarter site within the L1 promoter is shown, relative to binding site for the YY-1, RUNX and SOX transcription factor. (c) Effect of placing the three-quarter site from L1 promoter in front of a pdk4–luciferase promoter fusion. Response of the pdk4 promoter fusion to sequences added from p21 is included for comparison. (d) Levels of full-length L1 transcription produced by stable transfectants of HCT116, which has wild-type p53, in comparison with expression in HCT116 (p53–/–), which has both copies of p53 deleted. To activate p53, the cells were treated with 2 μM camptothecin, or mock-treated with dimethyl sulfoxide (DMSO) before harvesting RNA. RNA was collected after 8 and 24 h after camptothecin or DMSO treatments. The fold-increase in L1 mRNA upon camptothecin treatment is plotted on this graph. The plasmids contain either wild-type L1, or an L1 with a mutation in the 34 site. The GGGG site is the mutant site
Figure 4
Figure 4
Electrophoretic mobility shift assay (EMSA) for p53 binding to L1 sequences. EMSA reaction mixtures containing 32P-labeled oligonucleotides with either the p53-binding sites from the GADD45 gene, or putative binding sites from L1. The LINE-1 oligonucleotide includes the ← → 26 bp ← → and the 15-nt three-quarter site (← → ←) found at the 5′-end of L1s (see Figure 2). Radiolabeled oligonucleotides were incubated with 40, 60 or 80 ng of bacterially expressed and purified His-tagged p53 protein (lanes 1–3 and 7–9)). Unlabeled DNA (50× molar excess) of the corresponding sequence was added as a specific competitor (lanes 4 and 10). The p53-specific antibody PAb1801 was added to supershift p53-DNA complexes (lanes 5 and 11). Closed arrows indicate p53–DNA complexes, whereas open arrows indicate p53-PAb 1801-DNA complexes.
Figure 5
Figure 5
Binding of p53 to L1 sequences in whole cells. Cells from the H1299/V138 cancer cell line were incubated at 32 °C for 7 h to induce p53 activity then briefly treated with formaldehyde to crosslink chromatin to DNA-binding proteins. The isogenic H1299 cell line does not express p53 and is included as a negative control. A chromatin immunoprecipitation reaction using p53 or GFP antisera was then performed as described in the ‘Materials and methods’ section. The immunoprecipitated DNA was probed for specific L1 sequences, as well as for a p21waf1/cip1 positive control sequence and negative control sequences using PCR. Accession numbers for each are given in parentheses. (a) 5′-sequence from L1 at chromosomal position 3q13.31 (AC046136); (b) 5′-sequence from L1 at chromosomal position 2q37.2 (AC010148); (c) 5′-sequence from L1 at chromosomal position 20p12.2 (AL078623); (d) 5′-sequence from L1 at chromosomal position 1q43 (AC015901); (e) p21waf1/cip1 sequence containing a known p53 RE; (f) p21waf1/cip1 containing no p53 RE; (g) chromogranin A sequence containing no p53 RE.
Figure 6
Figure 6
Lineage of p53 target DNA sites within the primate L1s. Consensus sequences in the region spanning the L1 34 site are shown for several L1 subfamilies (Khan et al., 2006). Three point mutations occurred in the L1PA3 subfamily to create the L1 34 site.
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