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LINEs are endogenous mobile genetic elements which have dispersed and accumulated in the genomes of most higher eukaryotes via germline transposition, with up to 100 000 copies for the human LINE-1 (L1H) sequences. Although severely repressed in most normal tissues, L1H is still functional, with evidence for both germline and somatic-essentially in tumors-transpositions. Yet, no transcription factor that could regulate their transcription and be responsible for their transposition has hitherto been described. Here we show that factors belonging to the family of the testis-determining factor gene SRY (the SOX family) can modulate L1H promoter activity over a 10-fold range in a transient transfection assay using a luciferase reporter gene. These effects depend on two functional SRY binding sites which can be identified within the L1H promoter via mobility shift assays. Induction of endogenous L1Hs upon ectopic expression of the SOX11 transcription factor is further demonstrated, thus strengthening the physiological relevance of these new-and highly dispersed-target sites for the otherwise unclassical transcription factors of the SRY family.

Murine intracisternal A-particles (IAPs) are reiterated retrovirus-like transposable elements that can act as insertional mutagens. Accordingly, we previously identified a chimeric transcript initiated at an IAP promoter and extending through a 3'-located open reading frame with significant similarity to the C-terminal domain of the yeast CCR4 general transcription factor. In this report, we characterize the corresponding murine gene, mCCR4, and its human homologue, thus providing the first description of CCR4-like factors in mammals. cDNA cloning revealed two mCCR4 mRNAs of 2.7 and 3.1 kilobases, differing by their transcription start sites within the native mCCR4 gene promoter, and encoding a putative 430-amino acid protein. The mCCR4 gene contains three exons and two introns spanning almost 27 kilobases. The IAP insertion, detected only in some laboratory mouse strains, is recent and lies within the first intron. The 5'-region of the gene has features of housekeeping gene promoters. It lacks a TATA box but contains a CpG island and Sp1 sites. This region discloses strong promoter activity in transient transfection assays and also stimulates transcription in the reverse orientation, a feature common to other CpG island-containing promoters. Transcripts were detected in all the organs tested, although at a variable level, and displayed no strain-dependent differences relative to the IAP insertion, suggesting the existence of mechanisms preserving mCCR4 transcription from the usually deleterious effects of intronic transposition. The strong amino acid conservation between the human, murine, and the previously identified Xenopus CCR4-like proteins, is consistent with an important and conserved role for this protein in vertebrates.

We have characterized the regulatory properties of a 72bp sequence located in the 5′ untranslated domain of the Drosophilacopia retrotransposon, 3′ to the left LTR, by transient transfection assays with cell lines derived from either Drosophila hydei(DH33 cells) or Drosophila melanogaster(Schneider II and Kc cells). Reporter plasmids were constructed which contained the lacZ gene under the control of either the entire copia LTR with 5′ untranslated domain, or a minimal heterologous promoter flanked with the identified copia regulatory sequences. Upon transfection into the copia-free DH33 cells, the presence of the 72bp sequence resulted for all reporter plasmids in a 100-700 fold increase in expression level -as well as in reporter gene RNA levels- whereas this sequence had no enhancing effect upon transfection of the same plasmids into the copia-containing Schneider II or Kc cells. Moreover, mobility shift assays with the 72bp enhancer sequence disclosed two specific bands of retarded mobility with whole-cell extracts from DH33 cells, whereas no retarded band could be detected, under identical conditions, with extracts from Schneider II cells. UV crosslinking experiments between the enhancer sequence and DH33 extracts revealed a single protein species -of app. mel. wt. 50kD- for both retarded bands, thus strongly suggesting that they simply correspond to the sequential binding of two identical factor molecules to the enhancer sequence. These data demonstrate that the copia-free D. hydeicells express a strong transcriptional activator for the copia element and possible interpretations for the absence of this factor in the copia-containing D. melanogastercells are discussed in terms of a possible "adaptation" of the "host" (D. melanogaster) to an otherwise highly mutagenic "parasite" (copia with its transcription factor).

The 5'-untranslated region of the Drosophila gypsy retrotransposon contains an "insulator," which disrupts the interactions between enhancer and promoter elements located apart. The insulator effect is dependent on the suppressor of Hairy-wing (su(Hw)) protein, which binds to reiterated sites within the 350 base pairs of the gypsy insulator, whereby it additionally acts as a transcriptional activator of gypsy. Here, we show that the 350-base pair su(Hw) binding site-containing gypsy insulator behaves in addition as a matrix/scaffold attachment region (MAR/SAR), involved in interactions with the nuclear matrix. In vitro experiments using nuclear matrices from Drosophila, murine, and human cells demonstrate specific binding of the gypsy insulator, not observed with any other sequence within the retrotransposon. Moreover, we show that the gypsy insulator, like previously characterized MAR/SARs, specifically interacts with topoisomerase II and histone H1, i.e. with two essential components of the nuclear matrix. Finally, experiments within cells in culture demonstrate differential effects of the gypsy MAR sequence on reporter genes, namely no effect under conditions of transient transfection and a repressing effect in stable transformants, as expected for a sequence involved in chromatin structure and organization. A model for the gypsy insulator, which combines within a short "compacted" retroviral sequence three functional domains (insulator, enhancer, and the presently unraveled MAR/SAR) dispersed within more extended regions in other "boundary" domains, is discussed in relation to previously proposed models for insulation.

Intracisternal A-particle (IAP) sequences are endogenous retrovirus-like mobile elements, or retrotransposons, present at 1,000 copies in the mouse genome. These elements transpose in a replicative manner via an RNA intermediate and its reverse transcription, and their transposition should therefore be tightly controlled by their transcription level. To analyze the in vivo pattern of expression of these retrovirus-like elements, we constructed several independent transgenic mice with either a complete IAP element marked with an intron or with the IAP promoter, or long terminal repeat (LTR), alone controlling the expression of a lacZ reporter gene with a nuclear localization signal. For all transgenic lines analyzed, IAP expression as determined by reverse transcription-PCR analysis was found to be essentially restricted to the male germ line. Furthermore, in situ 5-bromo-4-chloro-3-indolyl-beta-D-galactopyranoside (X-Gal) staining of all organs disclosed specific beta-galactosidase-positive blue cells only within the testis, found as patches along the seminiferous tubules and often organized as assemblies of 2, 4, 8, or 16 cells. Histochemical analyses of tissues from 13.5-day-old embryos to adults demonstrated that this LTR activity is restricted to gonocytes and premeiotic undifferentiated spermatogonia. Finally, analysis of the methylation status of both transgenes and endogenous IAP LTRs demonstrated identical patterns, with methylation in somatic tissues and hypomethylation in the testis. Transgenic mice therefore reveal an intrinsic, highly restricted IAP expression which had escaped detection in previous global Northern (RNA) blot analyses and with possible strong biological relevance, as IAP activation specifically within the germ line might be a way to generate diversity at the evolutionary level without being deleterious to individuals.