Differential transcription of the repetitive R-element in various mouse cell types

105

Gene, 53 (1987) 105-i 11 Elsevier GEN 01955

Short communication Differential transcription of the repetitive R-element in various mouse cell types (R~ornb~~t DNA; Northern hyb~dization; genomic library ‘; clones; plasmid vectors ,; in situ hyb~dization; regulated gene expression)

T. Weber*, H.P. Schmitt and A. Alonso

Received 17 December 1986 Revised 14 January 1987 Accepted 15 January 1987

SUMMARY

Clones that contain R-elements separated from the rest of the Ll-repeat have been isolated from a mouse genomic DNA library. Spot hybridization of DNA from various species (from mammals to plants) with one representative and well characterized mouse DNA clone shows that at least this sequence hybridizes only with mouse DNA. In addition, we demonstrate that the R-element repeat is transcribed differentially in various tissues or cell types. Furthermore, the amount of R-transcripts is regulated at varying rates in the different cell types of a tissue as shown by in situ hyb~d~ation.

INTRODUCTION

About 20% of the rodent genomic DNA is made up of sequence repeated more than 100 times

Correspondence to: Dr. A. Alonso, German Cancer Research Center, Institute of Experimental Pathology, Im Neuenheimer Feld 280, 6900 Heidelberg (F.R.G.) Tel. 49-6221-4841. * Present address: Institute of Zoology I, University of Wiirzburg, Rontgenring 10, 8700 Wtlrzburg (F.R.G.) Tel. 49-931-31625. Abbreviations: ds, double stranded; hnRNA, heterogeneous nuclear RNA; kb, kilobase pair(s); Ll-repeat, the long interspersed repeat family of the mouse DNA; nt, nucleotide(s); R-element, the 5’ end region of the Ll repeat; SSC, 0.15 M NaCl, 0.015 M Na, . citrate pH 7.6.

(B&ten and Kohne, 1968; Singer, 1982) that are usually scattered throughout the genome. These sequences are normally organized into families (Jelinek and S&mid, 1982; Rogers, 1985) whose , members show slight variations from a common consensus sequence (Singer, 1982; Rogers, 1985). In the mouse genome two main families have been described. One of them, the Bi/B2 group is composed of short fragments that are repeated up to several hundred thousand times (Georgiev et al., 1985; Singer, 1982). The second family is the Ll-repeat, whose members are about 7 kb in length and are repeated up to 100000 times (Loeb et al., 1986; Voliva et al., 1984; Bennett and Hastie, 1984). The 3’ end of the Ll repeats has been identified as the former R-repeat already described (Gebhard

0378-I119/87/$03.50Q 1987EIsevier Science Publishers B.V.(BiomedicalDivision)

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et al., 1982; Gebhard and Zachau, 1983). Although part of the complete Ll fragment, the R-repeat element has also been found separated from the rest of the repeat and distributed in a random manner throughout the rodent genome. The biological role of the repeated sequences is not known but some results

point to their possible role as control elements in the expression of unique genes (Davidson and B&ten, 1979; Singer and Skowronski, 1985). It is now well established, that a large proportion of the repetitive elements are transcribed in vivo and that this transcription may be dependent on the cellular stage

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Fig. 1. Restriction map and sequence of the genomic Fragment containing the isolated R-element. (A) A mouse genomic DNA library cloned in the EcoRI site of phage 1 Charon4A (mean length of the insert is 14 kb) was screened with a radioactive probe from dsRNA prepared as described by Schmitt et al. (1986). Alter two cycles of dilution and plating, DNA was prepared from isolated phages. Several clones were further characterized. Most of them contained two or more repetitive segments. This panel shows a restriction map of one of the clones used. The 14-kb insert was rescued by EcoRI digestion and the resulting fragments were cloned into plasmid pSPT18 (Pharmacia, Uppsala). The leftmost L&RI fragment was shown to contain a B2 repetitive sequence. In the lower (expanded) restriction map, the thick line gives the position of the R-repeat. N, NcoI; B, BamHI; X, XbuI; S, SstI; Sa, SalI; H, HindIII; E, EcoRI. (B) Computer comparison between the cloned fragment and the consensus sequence of the R-element (Gebhard and Zachau, 1983). A 92% homology was found between the nt 669 and 1138 from the 1.4-kb fragment (upper sequence) and the consensus sequence (lower sequence) of the R-repeat. Asterisks between nucleotides denote identity in the nucleotide sequence. Y and R represent pyrimidine or purine transitions, respectively. Hyphens between nucleotides represent deletions, made by the computer to maximize homology. The plasmid used in these studies contains the complete R-sequence shown above plus 120 nt upstream. These 120 nt were unique as shown by Southern hybridization to total genomic DNA. Downstream from the R-element, the sequence continues for several nucleotides, to be homologous with the Barn-5 repeat (Voliva et al., 1984), changing thereafter abruptly to unique sequences as demonstrated by Southern hybridizations.

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(D’Ambrosio et al., 1986; Jackson et al., 1985; Bennett et al., 1984). In this communication, we describe the characterization of Ll non-linked R-elements and the transcriptional properties of this repeated sequence in different mouse tissues. We also localize their distribution in different cell types by using in situ hybridization to histological slices.

EXPERIMENTAL

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AND DISCUSSION

(a) Identification of R-repeats We have isolated several clones hybridizing strongly with dsRNA from a mouse genomic DNA library. Several of these clones hybridized with the R-repeat fragment of the Ll-repeat. One of these clones was recognized to contain one B2 repeat and one R-repeat (Fig. 1A). The position of both repeats was located in the genomic fragment and the corresponding subfragments were subcloned into plasmid vectors. The R-repeat was found to be 92% homologous to the R-consensus sequence using a computer program (Fig. 1B) (Gebhard and Zachau, 1983). Both the 3’ and the 5’ flanking regions were unique as demonstrated by Southern hybridization to genomic DNA blots. To analyse the distribution of this sequence in different species, DNA was isolated and spotted onto nitrocellulose filters and then hybridized to radioactively labelled cloned DNA. The cloned fragment used for these studies, named pAW1, was demonstrated to contain solely the R-repeat together with a few unique base pairs at their 5’ end (see legend to Fig. 1). A significant hybridization signal was obtained with mouse DNA but not with other mammalian species or plants (Fig. 2). Only Ratfus rattus gave a very weak signal after long exposure times. (b) Transcription of the R-elements in different mouse tissues Although the function of the repeated sequences is not understood, a differential expression of the R-elements in different cell types would support the possibility that these sequences may be involved in some way in controlling gene expression. To test this

Fig. 2. Species-specificity of the R-repeat. DNA (1 pg) of the species indicated below was spotted onto nitrocellulose filters and hybridized to nick-translated plasmid pAW1 (Maniatis et al., 1982). The filters were hybridized in 50% formamidex SSC-50 mM phosphate buffer-100 g/ml denatured salmon sperm DNA-5 x Denhardt reagent at 42°C for 24 h. Alter hybridization, they were washed in 2 x SSC at room temperature for 60 min and twice in 0.1 x SSC at 55°C for 60 min. The filters were autoradiographed on Kodak X-Omat films. The species used for this experiment were: (1) Mus musculus (liver); (2) Mus musculus (F9 cells); (3) Mus musculus (3T3 cells); (4) Mus mu.scu1u.r(liver, strain 129/J); (5) Homo sapiens (placenta); (6) Rufh*s rattus (liver); (7) Bos taurus (liver); (8) Paracenrrorus lividus; (9) Drosophila melanogaster; (10) Zea mays; (11) Vicia faba; (12) Physarum polycephalum.

possibility RNA from different tissues or cell lines was spotted onto nitrocellulose filters and hybridized to radioactive pAW1 DNA. Fig. 3A shows the results of this experiment. Contrarily to the results shown by Jackson et al. (1985) we were able to find differences in the transcriptional properties of the R-repeats. Whereas pancreas, stomach and liver contain only small quantities of complementary RNA, spleen contains about 20 times more and the RNA isolated from retinoic-acid-treated F9 cells contains at least 3-4 times more complementary RNA. These results prompted us to analyse the size distribution of the specific RNA in different tissues.

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Fig. 3. Analysis of R-transcripts in different tissues. (A) RNA spot hybridization: liver (1), stomach (2), spleen (3), retinoic-acid treated F9 cells (Strickland and Mahdavi, 1978) (4), and pancreas (5) was spotted onto nitrocellulose filters as described by Maniatis et al. (1982). The filters were hybridizided to nicktranslated plasmid [32P]DNA (see Fig. 1) under the same stringent conditions as described in Fig. 2. (B) Northern hybridizations. RNAs (10 pg) isolated using the guanidinium isothyocyanate method (Maniatis et al., 1982) from F9 stem cells (a), brain (b), or spleen (c) were denatured with formaldehydeformamide and separated by 0.8% agarose gel electrophoresis, as described by Schmitt et al. (1986). The RNA was transferred to nylon membranes and hybridizided with plasmid pAW1 and plasmid pMg-actin ( a clone containing the 3’ non-coding region of the mouse &actin mRNA) 32P-labelled by nick translation or random priming (Feinberg and Vogelstein, 1983). Hybridization conditions were as described in Fig. 2. Washing was at room temperature for 60 min in 2 x SSC and twice in 0.1 x SSC at 50°C for 60 min. The filters were exposed to x-ray film for 3 h. The band labelled by the arrow is the b-actin mRNA used in these experiments as an internal marker. Lane d shows the hybridization pattern of F9 RNA hybridized only to the B-actin DNA. Arrowheads denote the position of 16s and 23s rRNA.

RNA from F9 cells, brain or spleen was separated by denaturing agarose gel electrophoresis, blotted onto nitrocellulose and hybridized to a radioactive mix of clone pAWl and a clone containing the mouse ,&actin gene. Fig. 3B shows the results of this experiment. A smear pattern was obtained in all three RNAs used, whereas the actin RNA band could be readilly recognized as a band of about 2 100 nt. These results demonstrate that the R-element is present in all size classes in similar molar amounts. In the case of spleen RNA, a stronger hybridizing signal was observed as shown previously with the dot hybridization. This stronger signal was probably not due to DNA rearrangements in the spleen genome, as shown by Southern hybridizations to DNA restricted with different enzymes (results not shown). That this hybridization pattern was exclusively due to transcripts of the R-element was further reinforced by the fact that the flanking regions to the R-repeat did not show smear hybridization at all, according also to their unique nature (see legend to Fig. 1). The presence of a smear pattern in the Northern hybridizations can be interpreted as transcription of the R-elements present in introns of different genes, or R-elements located at the 3’-end and 5’-end non-coding regions of mRNAs (Jackson et al., 1985). In fact we have isolated a clone from a &tlO cDNA library of F9 cells which contains an R-repeat at their 3 ‘-end non-coding region (A.A. and T.W., unpublished results). Taken together, it seems probable that most of the R-elements are transcribed as parts of larger transcription units (independently of the Ll transcription units; Jackson et al., 1985), similar to the Bl-B2 repeats. In contrast to these other repeats, however, we could never demonstrate the presence of discrete RNA bands hybridizing with the R-repeat (Georgiev et al., 1985). (c) In situ hybridization The results presented in Fig. 3 were obtained with RNAs isolated from tissues with a heterogeneous cell population. It was thus impossible to ascertain whether all cell types transcribe the repeat at the same intensity or not.

Fig. 4. In situ hybridizations. Histological slices were prepared and hybridized with DNA from plasmid pAW1 labelled with [35S]dATP and [35S]dCTP to a specific activity of4-5 x 10’ cpm/pg. Hybridization was in 50% formamide, 600 mM NaCl, 1 mM EDTA, 1 mg/ml serum albumin, 1 x Denhardt reagent, 50 pg/ml of denatured salmon DNA, 10% dextran sulfate, 10 mM Tris, pH 7.4 (Rentrop et al.,

1986). For control were observed

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(results not shown). Autoradiography

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We therefore performed an in situ hybridization with clone pAW1. Fig. 4 shows the results with three different tissues. In mouse liver, most of the silver grains are localized in the nucleus, indicating that most of the R-transcripts are probably located in hnRNA (treatment with RNase totally abolished the hybridization signal). From the number of silver grains, it can be deduced that the different hepatocytes transcribe the repeat at similar ratios. Contrary to the findings in liver, spleen shows the grains localized mainly in the white pulp, whereas the red pulp contains only minute amounts of silver grains. Interestingly, the grains in the spleen are localized mainly in the cytoplasm. Finally, in situ hybridization with skin shows that the silver grains are mainly localized in the basal cells, whereas only a scarce labelling is found in the stratum comeum. Furthermore, it is worthwhile to note the reduced amount of silver grains in the dermal cells, in which the radioactivity is located in both the cytoplasmic and the nuclear compartments. These results demonstrate that the different cell types in the spleen and skin transcribe the R-repeat to different extents. These findings support the possibility that repetitive sequences may be involved in the regulation of gene expression. Nevertheless, the fact that most, if not all, of the R-transcripts are part of large transcriptional units makes their possible involvement in the regulation of gene expression at least questionable. One possibility that should be kept in mind, however, is that the intronic R-transcripts may in some way be involved in the processing or splicing of the hnRNA to mRNA, although no supporting experimental evidence is available. A final possibility is the existence of R-transcripts with their own promoter, separate from large transcriptional units, which are independently regulated. Such small RNA transcripts have not been described.

ACKNOWLEDGEMENTS

This work was supported by the Deutsche Forschungsgemeinschaft (Al 158/3). We thank M. Rentrop for his help with the autoradiography.

REFERENCES Bennett, K.L. and Hastie, N.D.: Looking for relationship between the most repeated dispersed DNA sequences in the mouse: small R elements are found associated consistently with long MIF repeats. EMBO J. 3 (1984) 467-472. Bennett, K.L., Hill, R.E., Pietras, D.F., Woodworth-Gutai, M., Kane-Haas, C., Houston, J.M., Heath, J.K. and Hastie, N.D.: Most highly repeated dispersed DNA families in the mouse genome. Mol. Cell. Biol. 4 (1984) 1561-1571. Britten, R.J. and Kohne, D.E.: Repeated sequences in DNA. Science 161 (1968) 529-540. Davidson, E.H. and Britten, R.J.: Regulation ofgene expression: Possible role of repetitive sequences. Science 204 (1979) 1052-1059. D’Ambrosio, E., Waitzkin, S.D., Witney, F.R., Salemme, A. and Furano, A.V.: Structure of the highly repeated, long interspersed DNA family (LINE or LlRn) of the rat. Mol. Cell. Biol. 6 (1986) 41 l-424. Feinberg, A.P. and Vogelstein, B.: A technique for radiolabeling DNA restriction endonuclease fragments to high specific activity. Anal. Biochem. 132 (1983) 6-13. Gebhard, W. and Zachau, H.G.: Organization of the R family and other interspersed repetitive DNA sequences in the mouse genome. J. Mol. Biol. 170 (1983) 255-270. Gebhard, W., Meitinger, T., Hbchtl, J. and Zachau, H.G.: A new family of interspersed repetitive DNA sequences in the mouse genome. J. Mol. Biol. 157 (1982) 453-471. Georgiev, G.P., Kramerov, D.A., Ryskov, A.P., Skryabin, K.G. and Lukanidin, E.M.: Dispersed repetitive sequences in eukaryotic genomes and their possible biological significance. Cold Spring Harbor Symp. Quant. Biol. 49 (1985) 1109-l 121. Jackson, M., Heller, D. and Leinwand, L.: Transcriptional measurements of mouse repeated DNA sequences. Nucl. Acids Res. 13 (1985) 3389-3403. Jelinek, W.R. and S&mid, C.W.: Repetitive sequences in eukaryotic DNA and their expression. Annu. Rev. Biochem. 51 (1982) 813-44. Loeb, D.D., Padgett, R.W., Hardies, SC., Shehee, W.R., Comer, M.B., Edgell, M.H. and Hutchison III, C.A.: The sequence of a large LlMd element reveals a tandemly repeated 5’ end and several features found in retrotransposons. Mol. Cell. Biol. 6 (1986) 168-182. Rentrop, M., Knapp, B., Winter, H. and Schweizer, J.: Aminoalkylsilane-treated glass slides as support for in situ hybridization of keratin cDNAs to frozen tissue sections under varying fixation and pretreatment conditions. Histochem. J. 18 (1986) 271-276. Rogers, J.H.: The origin and evolution of retroposons. Int. Rev. Cytol. 93 (1985) 187-279. Singer,M.F. : Highly repeated sequences in mammalian genomes. Int. Rev. Cytol. 76 (1982) 67-l 12. Singer, M.F. and Skowronski: Making sense out of LINES: long interspersed repeat sequences in mammalian genomes. Trends Biochem. Sci. 10 (1985) 119-122.

111 Schmitt, H.P., Ktlhn, B. and Alonso, A.: Characterization of cloned sequences complementary to F9 cell double-stranded RNA and their expression during differentiation. Differentiation 30 (1986) 205-210. Strickland, S. and Mahdavi, V.: The induction of differentiation in teratocarcinoma stem cells by retinoic acid. Cell 15 (1978) 393-403.

Voliva, Ch.F., Martin, S.L., Hutchinson III, CA. and Edgell, M.H.: Dispersal process associated with the Ll family of interspersed repetitive DNA sequences. J. Mol. Biol. 178 (1984) 795-813. Communicated by H.G. Zachau.