Transcriptional regulation of early transposon elements, an active family of mouse long terminal repeat retrotransposons

Abstract

While early transposon (ETn) endogenous retrovirus (ERV)-like elements are known to be active insertional mutagens in the mouse, little is known about their transcriptional regulation. ETns are transcribed during early mouse embryogenesis in embryonic stem (ES) and embryonic carcinoma (EC) cell lines. Despite their lack of coding potential, some ETns remain transposition competent through their use of reverse transcriptase encoded by a related group of ERVs-MusD elements. In this study, we have confirmed high expression levels of ETn and MusD elements in ES and EC cells and have demonstrated an increase in the copy number of ETnII elements in the EC P19 cell line. Using transient transfections, we have shown that ETnII and MusD LTRs are much more active as promoters in P19 cells than in NIH 3T3 cells, indicating that genomic context and methylation are not the only factors determining endogenous transcriptional activity of ETns. Three sites in the 5' part of the long terminal repeat (LTR) were demonstrated to bind Sp1 and Sp3 transcription factors and were found to be important for high LTR promoter activity in P19 cells, suggesting that as yet unidentified Sp binding partners are involved in the regulation of ETn activity in undifferentiated cells. Finally, we found multiple transcription start sites within the ETn LTR and have shown that the LTR retains significant promoter activity in the absence of its noncanonical TATA box. These findings lend insight into the transcriptional regulation of this family of mobile mouse retrotransposons.

FIG. 1.

ETn and MusD transcript abundance in undifferentiated and differentiated cell lines. (A) Northern blot. Ten micrograms of RNA isolated from EC P19, ES R1, and fibroblast NIH 3T3 cells was hybridized with ETnI, ETnII-β, MusD, or β-actin probes. ETnII-β and MusD probes produced single bands, and the ETnI probe produced a single major band of 5.6 kb and a minor band of 7.2 kb. The blot was initially hybridized with an ETnII-β oligonucleotide probe, then stripped and rehybridized in turn with the other indicated probes. (B) Structures of ETn elements and locations of probes used in Northern blotting.

FIG. 2.

Promoter activities of ETnII and MusD LTRs in P19 and NIH 3T3 cells. Four ETnII and four MusD LTRs (see Table 1 for accession numbers) cloned into a pGL3B luciferase reporter vector were assayed for promoter efficiency by transient transfections into P19 and NIH 3T3 cells.

FIG. 3.

Promoter activities of 5′-deleted ETnII LTRs in P19 and NIH 3T3 cells. (A) Graphical representation of the promoter efficiencies of 5′-deleted LTR#6 constructs. Numbers indicate the span of the construct, with base pairs corresponding to those in LTR#6. The putative TATA box and the transcription start site identified previously (20) are shown. Ovals represent possible Sp1 binding sites. The Sp1 binding site mutated in the 61(T)-317 construct is crossed. (B) Promoter activities of the LTR deletion constructs in P19 and NIH 3T3 cells as percentages of the promoter activities of full-length LTR#6.

FIG. 4.

Sequence of the ETnII LTR#6, with positions of deletion constructs and EMSA oligonucleotides shown. The transcription start site, as identified in reference , is indicated as +1. The predicted TATA-like sequence and polyadenylation signals (20) are boxed, and U3, R, and U5 regions are indicated. Oligonucleotides used as probes in EMSA (I, II, and III) are underlined. Sp1/Sp3 recognition sequences are highlighted in gray; core regions are specified in white letters on a gray ground.

FIG. 5.

Binding of Sp1 and Sp3 transcription factors to the regions crucial for high LTR promoter activity. γ-³²P-labeled oligonucleotides identical in sequence to the regions essential for high LTR promoter ability in P19 cells (I, II, and III) were incubated with nuclear extracts from P19 and NIH 3T3 cells. Competition assays were performed with a 200-fold excess of wild-type unlabeled oligonucleotide. Antibodies against Sp1 and Sp3 supershifted specific bands.

FIG. 6.

Promoter activities of Sp-binding site mutant LTRs in P19 and NIH 3T3 cells. (A) Graphical representation of the promoter efficiencies of LTR#6 constructs mutated at Sp1/Sp3 recognition sites. The putative TATA box and the transcription start site identified previously (20) are shown. Ovals represent possible Sp1 binding sites. Crossed ovals represent mutated Sp1 binding sites. The cores of the wild-type (w/t) Sp1 recognition sequences are boxed, and the mutated nucleotides are lowercased. (B) Promoter activities of the mutated LTR constructs in P19 and NIH 3T3 cells as percentages of the promoter activities of wild-type LTR#6.

FIG. 7.

Promoter activities of 3′-deleted ETnII LTRs in P19 and NIH 3T3 cells. (A) Graphical representation of the promoter efficiencies of 3′-deleted LTR#6 constructs. The putative TATA box and the transcription start site identified previously (20) are shown. Ovals represent possible Sp1 binding sites. (B) Promoter activities of the LTR deletion constructs in P19 and NIH 3T3 cells as percentages of the promoter activities of full-length LTR#6.

FIG. 8.

Mapping of transcription initiation sites in ETnII LTRs by 5′-RACE. (A) Transcription initiation sites of transfected full-length and 3′-deleted ETnII LTR#6 in P19 cells. Symbols represent 5′ termini of individual RACE clones, and numbers indicate that certain clones were found multiple times. The 3′-deleted construct (bp 1 to 164) is indicated by an arrow. The previously noted TATA-like element (20) is boxed, and a second such motif is underlined. (B) Transcription initiation sites of endogenous ETnII elements in P19 cells. Arrows represent 5′ termini of RACE clones, and numbers indicate that certain clones were found multiple times. The LTR sequence is that of the new ETnII insertion into the Adcy1 locus (25). The previously noted TATA-like element (20) is boxed, and a second such motif is underlined.

FIG. 9.

Amplification of ETnII elements in the P19 cell line. (A) Digestion scheme. One microgram of genomic DNA was digested with restriction endonucleases PstI and XbaI; XbaI cuts inside the LTR, and PstI cuts in the body of the element, resulting in different fragment lengths for ETnII (approximately 1.4 kb) and MusD (approximately 2.1 to 2.35 kb) elements. The probe used is specific for both the ETnII and MusD elements and is located in the common region just 3′ of the 5′ LTR. (B) Genomic Southern blotting and hybridization. Genomic DNAs from the parental mouse strain of the EC cell line P19, C3H/HeJ (lane1), the EC cell line P19 (lane 2), parental mouse strains of the ES cell lines, 129X1/SvJ (lane 3) and 129S1/Sv-+^p +^Tyr-c Kitl^Sl-J/+ (lane 4), the ES cell lines R1 (lane 5), EK.ECC (lane 6), AB-1 (lane 7), and 671 (lane 8), and the fibroblast cell line NIH 3T3 (lane 9) were digested with PstI and XbaI. The blot was hybridized to the probe specific for both the ETnII and MusD elements.