Identification, characterization, and cell specificity of a human LINE-1 promoter.

Abstract

A constructed human LINE-1 (LlHs) element containing intact 5' and 3' untranslatable regions and an in-frame fusion between the LlHs open reading frame 1 and the bacterial lacZ gene (plLZ) was found to promote the expression of I8-galactosidase in a variety of transiently transfected cell types in tissue culture. Full-length RNA was detected in the transfected cells. Most of the RNA transcripts initiated at or near the beginning of the LlHs segment. Sequences within the LlHs segment of plLZ were sufficient for expression of the reporter gene; however, modulation of the transcriptional regulatory region by upstream sequences was not ruled out. Deletion analysis revealed that the sequences most critical for transcription were located within the first 100 bp of LlHs. Other sequences within the first 668 bp of LlHs also contributed to overall expression. Expression of plLZ was high in human teratocarcinoma cells and low in all other cell types. This pattern of cell-type-specific expression matches the known pattern of endogenous LlHs transcription in cultured cells.LINE-1 elements (Lls) constitute a family of long, repetitive, interspersed sequences that are found in all mammalian genomes (13,23,33 (35). The mothers of both patients have two normal factor VIII genes. Also, a patient with adenocarcinoma of the breast has an LlHs insertion into one myc allele in diseased but not in normal tissues (40).The most commonly proposed mechanism of Li transposition involves (i) synthesis of full-length, polyadenylated transcripts, (ii) reverse transcription of the RNA by an Li-encoded enzyme, and (iii) insertion into staggered chromosomal breaks. Diverse human and monkey cell lines contain abundant RNA that anneals with Li probes (see references in reference 56). These transcripts are predominantly nuclear, heterogeneous in size, and nonpolyadenylated, and they emanate from both strands of the LlHs sequence; they probably do not represent specific LlHs transcription. Skowronski and Singer (57) undertook an extensive search for specific LlHs transcription. Of the many cell types that they examined, only the human teratocarcinoma cell line NTera2D1 contained full-length, sensestrand, cytoplasmic polyadenylated LlHs RNA. Primer extension studies aligned the 5' end of the RNA with the consensus left end of genomic LlHs (56). Each of 19 LlHs cDNAs cloned from the NTera2D1 RNA were unique, indicating that many genomic Lls are transcribed in these cells. No specific LlHs transcripts were detected in HeLa and many other cell types; JEG-3 cells were negative by Northern (RNA) blot and positive by primer extension analysis.The mechanism by which full-length LlHs RNA is produced is not understood. A typical upstream RNA polymerase II promoter would be lost during a cycle of transcription and reverse transcription. Retroviruses and other class I retrotransposons utilize long terminal repeats to synthesize complete cDNAs (13) and thereby maintain their promoters. LINEs and other class II retrotransposons do not possess long terminal repea...

Antisense Promoter of Human L1 Retrotransposon Drives Transcription of Adjacent Cellular Genes

“…21,2001 TRANSCRIPTION OF HUMAN GENES BY L1 ANTISENSE PROMOTERpromotorless vector or sense construct 4S, suggesting that some of the genomic L1Hs (sense) promoters may have retained their activity. This result may explain the discrepancy between the results obtained by two different groups (22,34). Further transfection and analysis of nine additional constructs containing the L1Hs 5ЈUTR in the antisense orientation and a similar construct containing the genomic region of P5 cDNA (derived from BAC) showed low activity (relative luciferase activity [RLA] ϭ 2 to 5) for five constructs and high activity (RLA ϭ 7 to 52) for the remaining constructs (see Fig.…”

“…Direction of transcription for each gene is indicated by a thin arrow above the gene. Transcriptions driven by the L1a and L1b internal promoters (34) and ASPs, driving in opposite direction, are indicated by thin and thick arrows, respectively. Note that gene Y has the same direction of transcription as the L1a ASP but is located Ͼ60 kb further downstream.…”

“…Low-level transcription of L1Hs in other cell lines (HeLa, HL60, and 293) has been indirectly revealed by the presence of ORF1-specific antiserum-positive products (19). Critical sequences necessary for the transcriptional initiation of L1Hs were located in the first 100 bp of the 5ЈUTR (22,34). The region (ϩ13 to ϩ21) contains a binding site for the ubiquitous transcription factor YY1.…”

DNA methylation and expression of LINE-1 and HERV-K provirus sequences in urothelial and renal cell carcinomas

“…The situation is different with the LINE-1 promoter which comprises the CpG rich 5′-end of the elements. The first 200 bp act as the actual promoter whose function is supported by the adjacent downstream 500 bp (Swergold, 1990). The results of Southern analysis and LM-PCR indicate that sites M1-M3 located within the basal promoter and site M4 located in the supporting region are demethylated in carcinomas with DNA hypomethylation in many LINE-1 elements allowing transcription.…”

Repetitive sequence environment distinguishes housekeeping genes

“…Long transposons (Lyon, 1998;Marahrens, 1999;Bailey et al, 2000;Allen et al, 2003;Lippman et al, 2004;Sun et al, 2004) and long tracts of tandem repeats (Pieretti et al, 1991;Hansen et al, 1997;Saveliev et al, 2003) have been implicated in gene silencing via the spread of heterochromatin. Another reason why LINE-1 and other long transposons might be scarce around housekeeping genes is that the euchromatin could spread into the transposons and activate their internal promoters (Swergold, 1990;Minakami et al, 1992;Leib-Mösch and Seifarth, 1995;Speek, 2001;Athanikar et al, 2004). This could cause gene over-expression as has been reported for and IAP element insertion in the 5′ upstream region of the mouse Agouti gene (Whitelaw and Martin, 2001) or down-regulate genes if the transcription proceeds through the gene in the antisense direction (Whitelaw and Martin, 2001).…”