Transcriptional Activation of Mouse Retrotransposons in Vivo: Specific Expression in Ster.Oidogenic Cells in Response to Trophlc Hormones
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Downloaded from genesdev.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Transcriptional activation of mouse retrotransposons in vivo: specific expression in ster.oidogenic cells in response to trophlc hormones Rachel Schiff, Ahuva Itin, and Eli Keshet Department of Virology, The Hebrew University, Hadassah Medical School, Jerusalem 91010 Israel Transcription of cellular retrotransposons is induced by a variety of physiological stimuli. We have used in situ hybridization analysis to determine the cell types in which mouse retrotransposons are transcriptionally activated in vivo under physiological conditions. Here, we report that VL30 retrotransposons are specifically expressed in steroidogenic cells within all four endocrine tissues engaged in synthesis of steroid hormones in response to the respective pituitary-derived trophic hormones. These tissues include ovarian steroidogenic theca cells and lutein cells of the corpus luteum, testosterone-producing Leydig cells of the testis, steroidogenic cells confined to the zona reticularis of the adrenal cortex, and progesterone-producing cells of the placenta. In the course of preovulatory follicular development and maturation, the profile of cells expressing the retrotransposon shifted in parallel to the changing profiles of the leutinizing hormone (LH)-induced steroidogenic output of the respective cells. Expression of VL30 in both male and female gonads was shown to be greatly stimulated by external administration of gonadotropins. In vitro studies using a LH-responsive Leydig cell line have confirmed that expression of the resident retrotransposons is gonadotropin dependent. Run-off transcription assays have indicated that activation is at the transcriptional level. To allow molecular access to gonadatropin-activated transcription units, the long terminal repeat (LTR) regulatory domains were cloned from VL30 cDNAs of LH-induced ovaries. Through the use of reporter gene constructs and transfection experiments it was shown that expression of these elements in steroidogenic cells is LH dependent. Furthermore, cAMP, a known mediator of trophic hormone responses, could replace the hormone for inducibility. Transfection studies have also shown that the retrotransposon LTRs may function as hormone-activated enhancers conferring a LH-dependent phenotype on a surrogate transcription unit. These studies have thus demonstrated that the transcriptional activation of resident retrotransposons in vivo is a dynamic process that can be modulated by gonadotropins and have the potential of imposing this phenotype on adjacent cellular genes. [Key Words: Retrotransposonsl VL301 in situ hybridizationl steroidogenesisl gonadotropins] Received August 31, 1990; revised version accepted January 15, 1991. The mammalian genome harbors a large number of en- The tissue- and cell-type specificities of retrotransposon dogenous proviruses that are genetically related to irdec- LTRs therefore determine the nature of cells in which tious retroviruses. In addition, mammalian chromo- retrotranspositions or retrotransposon-mediated recom- somes contain other types of genetic elements, termed binations may take place. Retrotransposons may also af- retrotransposons, that are genetically unrelated, or only fect the expression of nearby cellular genes, from their distantly related, to known infectious retroviruses but sites of residence through the activity of their transcrip- possess, nevertheless, structural hallmarks of proviruses. tional enhancers. Hence, cell-type-specific or hormone- Notably, retrotransposons are distinguished by long ter- responsive retrotransposon enhancers may determine minal repeats (LTRs} housing the transcriptional regula- the pattern of expression of adjacent cellular genes. For tory domains of the element. In mice, where these pro- example, it has been shown recently that certain mouse virus-like elements were best studied, at least six dis- retrotransposons may impose hormone responsiveness tinct retrotransposon families, each containing from 100 upon an adjacent cellular gene [Stavenhagen and Robins to several thousand members, were discovered {for re- 1988}. view, see Keshet et al. 19901. Transcriptional activation The current study focuses on a particular murine ret- of retrotransposons is a prerequisite and often a rate- rotransposon family designated the VL30 elements limiting step in retrotransposition {Boeke et al. 1985}. (Howk et al. 19781Besmer et al. 19791 Keshet et al. 1980}. GENES & DEVELOPMENT 5:521-532 © 1991 by Cold Spring Harbor Laboratory Press ISSN 0890-9369/91 $3.00 521 Downloaded from genesdev.cshlp.org on October 8, 2021 - Published by Cold Spring Harbor Laboratory Press Schiff et al. Over 100 copies of these potentially transmissible ele- methodology has the advantage that expression confined ments are scattered throughout the mouse genome. The to individual cells within a tissue can be detected by a evolutionary plasticity of retrotransposon LTRs, in gen- screening procedure that covers a large number of tissues eral, and of VL30 LTRs, in particular, has led to a situa- and embryonic organs. Moreover, the effects of physio- tion where different members of the family often possess logic treatments {e.g., the systemic administration of structurally diverse LTRs that display different tran- hormones) on transcription can be monitored without scriptional specificities (Itin and Keshet 1986a). A char- disturbing the natural cellular interactions. acteristic feature of some retrotransposon families is the As a source of a VL30-specific sequences we used a remarkable inducibility of their transcription by a wide 350-bp-long LTR fragment derived from a previously range of agents and physiologic stimuli. For example, characterized genomic clone. We have shown previously VL30 retrotransposons are activated under a variety of that certain VL30 clones are, in fact, mosaic elements experimental conditions. This is reflected in the re- composed of more than one retrotransposon type (kin et peated incidental selection of VL30 cDNA clones in sub- al. 1983}. The use of a defined LTR sequence as a probe structive screening procedures aimed at selecting induc- eliminated the possibility that the probe employed in- ible genes. Thus, VL30 elements were selected as genes cluded sequences that could cross-hybridize with other in which expression is tumed on after epidermal growth genomic elements. VL30 cRNA probes labeled with 3sS factor {EGFI stimulation of quiescent mouse embryo in either antisense or sense orientations were synthe- cells in culture {Foster et al. 1982}; VL30 cDNAs were sized. Embryos and organs were fixed in 4% paraformal- selected as growth-specific elements expressed in SV40- dehyde, embedded, frozen, and sectioned. Frozen sec- transformed, nonconfluent cells but not in the conflu- tions (10 ~m) were processed for in situ hybridization ent, contact-inhibited parental cells {Singh et al. 1985); and were hybridized under stringent conditions, as de- VL30 cDNAs were picked up in a selective procedure scribed in Materials and methods. aimed at identifying genes in lymphoid cells activated by To include as many cell types as possible in each sec- glucocorticoids (Harrigan et al. 1989); VL30 elements tion examined, we initiated our "shotgun" screening by were shown to be activated by chemical carcinogens examining serial sections obtained from whole newborn {Hsieh et al. 1987), whereas other studies have demon- mice or from late embryonic stages [>17 days postcoi- strated a dramatic (500-fold) induction of VL30 expres- turn (p.c.)]. This was followed by focusing on tissues or sion under conditions of anoxia {Anderson et al. 1989). organs that gave the most intense autoradiographic sig- Whereas the patterns of retrotransposon expression nals. have been widely studied in cell culture systems, the activation of mammalian retrotransposons in vivo under Expression of VL30 in the adrenal cortex conditions that allow the correct systemic affects and preserve the authentic cellular interactions has been Figure 1A shows an example of a parasagittal section only poorly studied. Reasoning that in situ hybridization through a 17.5-day-old mouse embryo. Expression of analysis might point at distinct physiologic settings in VL30s could be detected in several tissues, primarily in which resident retrotransposons are transcriptionally ac- the epithelial tissue layers of most internal organs [not tivated, we embarked on a comprehensive in situ hybrid- evident in the low magnification view providedl. How- ization analysis, using specimens derived from mouse ever, it was clearly seen that the predominant site of organs and embryos. Here, we report our observations expression, with highest detectable levels of VL30 RNA, that VL30 retrotransposons are activated in vivo specif- is the embryonic adrenal gland. Histologic details of an ically in steroidogenic cells within the male and female embryonic adrenal gland are better seen in the higher gonads, the adrenal cortex, and the placenta, that is, in magnification view {Fig. 1B). VL30 RNA was mostly de- all four endocrine tissues that specialize in synthesis and tectable in the provisional cortex residing underneath secretion of steroid hormones. Cyclic changes in both the outermost cortical layers {layers that at later devel- levels of expression and types of expressing cells were opmental stages will give rise to the permanent cortex). observed within the female reproductive system in cor- The major