Various Adh gene regions shown in. Figure 1 were cloned into plasmid vectors at the corresponding restriction sites: pSal 8.4, pRI-S 3.5, pRI-H 2.9 in pBR 327; ...
Volume 15 Number 19 1987
Nucleic Acids Research
Roles of cis-acting elements and chromatin structure in Drosophila alcohol dehydrogenase gene
expression Cheeptip Benyajati*, Stephen Ayer, Jeffrey McKeon, Amy Ewel and Jin Huang
Department of Biology, University of Rochester, Rochester, NY 14627, USA Received May 6, 1987; Revised and Accepted August 30, 1987 ABSTRACT
The alcohol dehydrogenase (Adh) gene of D. welanogaster is transcribed from two different promoters during fly development: the distal (adult) and the proximal (embryonic-larval). Certain aspects of Adh gene regulation are represented in
Drosophila continuous cell lines. We have used Drosophila tissue culture cells in an in vivo transient expression assay to delimit cis-acting sequences affecting &fr expression, and to investigate the role of chromatin structure in Adh gene regulation. These studies show that positive cis-acting elements of the distal promoter can exist in at least 2 alternative chromatin configurations. There is a close correlation between specific transcriptional activity of the Adh distal promoter and a defined, localized chromatin structural change that indicates altered DNA - protein interactions. Thus, chromatin structure appears to play a role in regulating the accessibility of defined positive cis-acting regulatory sequences of Adh to transcription factors and the transcription machinery. INTRODUCTION
The alcohol dehydrogenase (Adh) locus of Drosophila melanogaster displays regulated patterns of gene expression during development. ADH activity per organism is high in larvae and adult flies and low in embryos and pupae. Enzyme activity is also restricted to certain tissues (1). A single Adh gene encodes two mature ADH transcripts, which differ only in part of their 5'-untranslated regions; the protein-coding and 3'-untranslated sequences are identical (2). These two ADH mRNAs are transcribed from two different promoter regions, each containing a TATA box, located about 700 bp apart in the genome. The transcript of the distal promoter is found primarily in adult flies, while the transcript of the proximal promoter is found in larvae (2). However, a recent detailed study indicates a more complex pattern of alternative promoter usage (3). The wadult", distal promoter is also active transiently in 9- to 14-hour embryos, and mid-third-instar larvae. A low level of the "larval", proximal transcript also accumulates throughout adult life. Furthermore, both transcripts are maternally inherited by the zygote (O to 2-hour embryos). Several approaches have been taken to study the molecular mechanisms © I R L Press Limited, Oxford, England.
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Nucleic Acids Research regulating Drosophila Adh gene expression during development. Using P-elementmediated germ line transformation, cis-acting sequences necessary for correct developmental expression of Adh have been shown to reside in an 11.8 kb genomic DNA fragment containing the gene (4). Similar analysis using specific deletion mutants generated from the cloned gene indicates that correct temporal- and tissue-specific expression from the distal promoter requires the sequences within 663 bp of the distal RNA start site (5). Sequences within 770 bp of the proximal RNA start site confer the tissue-specific activity of the proximal promoter in larvae following germ line transformation (5), as well as in the in vivo transient tissue-specific gene expression assay (6). Another region, which lies more than 2 kb upstream of the proximal promoter is also required for normal levels of Adh transcription in larvae (5). In vitro studies have identified some DNA binding factors that interact with specific sequences in the Adh promoter regions. Both distal and proximal promoters can be transcribed accurately in an in vitro transcription system derived from nuclear extract of an ADH-negative (ADH) Drosophila cell line (79). A promoter-specific transcription factor, Adf-1, has been partially purified from the nuclear extract and characterized (9). Adf-1 is required for maximum in vitro transcription of the Adh distal promoter. In footprinting experiments, Adf-1 protects the sequences -86 to -46 of the distal RNA start, as well as -150 to -105 of the proximal start site. However, the binding of Adf-I is not required for proximal promoter transcription in vitro.The proximal promoter also contains two additional specific binding regions, suggesting multiple components are involved in accurate initiation of transcription. In this study, we have used Drosophila tissue culture cells to characterize the cis-acting elements required for Adh gene expression, and to identify potential regions where trans-acting factors may bind the Adh region in vivo. We have shown previously that both the proximal and distal promoters of Adh are functional in the in vivo transient expression assay using ADH Drosophila Schneider line 2 (SL2) cells (10). These results indicate that SL2 cells contain all the trans-acting factors required for Adh transcription initiation at both promoters. We report here the analysis of cis-acting regulatory elements of Adh by assaying the expression of deletion mutants transfected into SL2 cells. However, SL2 and several other ADH continuous Drosophila cell lines appear to possess intact endogenous genes (see below). These observations then raise further question of why endogenous Adh genes are not expressed in SL2 cells when exogenous genes are. One possible explanation is that the inactive endogenous genes may be in a chromatin configuration that does not permit
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Nucleic Acids Research recognition of promoter sequences by transcription factors. To investigate the chromatin structure at the Adh locus and its role in Adh gene regulation, we have taken advantage of the availibility of several new continuous Drosophila cell lines (11), some of which transcribe Adh specifically from the distal promoter (ADHA) MATERIALS AND METHODS Cells. Schneider line 2 (SL2) cultured Drosophila cells were grown in Schneider's Drosophila medium (GIBCO) containing 10% fetal calf serum at 230C. Other Drosophila continuous cell lines were kindly provided by A.A. Simcox, and some have been described (11). These cells were maintained at 230C in M3 medium containing 10% fetal calf serum (12). Transfection of Drosophila Cells. The calcium phosphate-DNA transfection of the SL2 cells was performed as described (10).SL2 cells were seeded at 2x107 cells per T-75 flask in 10 ml of Schneider's medium containing 10% fetal calf serum 24 hr before transfection. Supercoiled plasmid DNA (10 ug) precipitated with calcium phosphate (1 ml) was added. Cells were harvested at 48 hr and processed for RNA, protein and ADH enzyme activity determinations as described (10). Recombinant plasmids. Supercoiled plasmid DNA for transfection was isolated by the cleared lysate method and was twice purified by CsCl/ethidium bromide equilibrium centrifigation. More than one preparation of individual plasmids was used in the transfection experiments. Various Adh gene regions shown in Figure 1 were cloned into plasmid vectors at the corresponding restriction sites: pSal 8.4, pRI-S 3.5, pRI-H 2.9 in pBR 327; pSst 11.8 and pRI 4.8 in pUC 12 in the same orientation; pXba 3.2 and pXba 2.8 in pUC 13 in the same orientation. pXba 3.2 was a gift from J. Posakony. RNA analysis. Cytoplasmic RNA was isolated as described (13), treated with 1-3 units of RNase-free DNAase (Promega Biotec) for 30 min at 370C to eliminate any DNA that may have contaminated the RNA preparation, and extracted with phenol/chloroform. Relative concentration and integrity of RNA were determined on denaturing formaldehyde-agarose gels (13). RNA samples were analyzed by the riboprobe-RHase mapping technique (14). Quantitations of the protected, labeled RNA bands were performed by densitometric tracing of autoradiograms. Nuclei isolation and DNAase I digestion. Nuclei were prepared from each Drosophila continuous cell line, digested with DNAase I (DPFF grade, 1735 units/mg; Cooper Biomedical), and DNA purified as described (15). High molecular weight, protein-free DNA was prepared from nuclei and digested with DNAase I as for nuclei, but with 50 - 100 fold lower concentrations of DNAaseI.
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Nucleic Acids Research Gel electro0horesis and hybridization. DNA samples (10 ug) were digested with restriction enzymes and separated on 25 cm long agarose gels. To accurately position DNAase I hypersensitive sites, the internal size standards were used on each gel: *X174 DNA cleaved with HaeIII, A DNA cleaved with HindIII, and purified Drosophila genomic DNA or cloned Adh plasmids cleaved with appropriate restriction enzymes. After blotting, the filters were probed with 32P-labeled, nick-translated DNA (Specific activity > 1 x 109 cpm / ug DNA). RESULTS Transient expression of recombinant Adh plasmids in Drosovhila SL2 cells. Supercoiled plasmids containing wild-type Adh can be transcribed correctly from their own regulatory sequences when introduced into ADH SL2 cells by the calcium phosphate-DNA transfection technique (10). This results in functional larval- and adult-type ADH transcripts that have the correct 5' ends, and are properly spliced. The levels of ADH protein and enzyme activity in these transfected cells 48 hr after transfection correlate directly with the levels of ADH transcripts (10). In the present study to identify cis-acting elements, we analyzed the expression in SL2 cells of several Adh constructs containing various 5' and 3' flanking sequences of Adh. The relative expression of different constructs is summarized in Figure 1. Maximum transient expression of the cloned Adh gene in SL2 cells requires the 3.2 kb sequences between the two XbaI sites flanking the gene (pXba 3.2). The absence of sequences upstream of the 5' XbaI and downstream of the 3' XbaI appears not to affect the level of Adh expression. We have localized the cisacting element in the 5' flanking region of Adh that is necessary for efficient expression from the distal promoter to a region between -663 and -63 of the distal RNA start (A). When this region is not present (compare the expression of pXba 3.2 to pRI-S 3.5), the level of the distal but not the proximal transcript is reduced five fold; both RNA initiation sites remain unaffected. An internal deletion that removes the -63 (A) to +327 (A) sequence (compare pXba 3.2 to pXba 2.8), which include the distal TATA box (between -26 (A) and -32 (A)), the distal RNA start site (position +1) and part of the distal RNA intron, completely abolishes distal RNA transcription but does not affect transcription from the proximal promoter. Thus, this region is required only for correct initiation of the distal ADH transcript. Analysis of the cis-acting element(s) of the proximal promoter is more complicated. The proximal promoter, which is 700 bp downstream of the distal promoter, appears to be less active than the distal one in the transient expression assay: only about 20 - 30 % of the ADH transcripts are of the 7906
Nucleic Acids Research AATAAA TATAAATA > JX"G TA~~pA TATTTAA
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Fig.l. The Drosophila uelanogaster Adh gene, recombinant plasmids containing various Adh regions, and their relative expression in the SL2 cells. TATTTAA and TATAAATA (TATA boxes) represent the distal (adult) and the proximal (larval) promoter regions, located at 25 and 24 bp upstream of the distal (A) and the proximal (L) RNA start sites respectively. ATG and TAA indicate the initiation and termination codons; AATAAA and pA refer to the poly(A)-addition signal and the mature 3' end of both the larval- and the adult-type ADH RNAs. For plasmid constructs containing the various Drosophila genomic Adh sequences, the distance in bp from the proximal RNA start (L) and the distal RNA start (A) are shown for the 5' flanking, and from the proximal RNA start for the 3' flanking. The table of relative expression represents the compilation of data from several transfection experiments using the same set of Adh plasmids, in which both the ADH transcripts and the ADH protein were quantitated. The lowest detectable level of expression (from the proximal promoter of pRI-H 2.9) was assigned the relative expression of 1; it was then used as a basis to compare levels of expression of other Adh constructs in the same experiments. The relative expression was reproducible within each experiment; variations between experiments were no more than 25%. Quantitation of plasmid DNAs in Hirt supernates(22) of transfected cells by Southern blot hybridization indicated equal amounts of plasmid DNA in the cells at the time of harvest.
proximal type. Removal of the sequence between -770 and -214 of the proximal start site (L, compare pRI-S 3.5 to pRI-H 2.9) results in a three-fold reduction of proximal transcript level; the RNA start site remains unchanged. Thus, this region, which contains the distal promoter and the distal RNA start site, has a positive influence on quantitative transcription from the proximal promoter. To dissect this cis-acting element further, we analyzed an Adh plasmid construct with an internal deletion (pXba 2.8) that removes the sequence between -770 (L) and -383 (L) encompassing the distal promoter and distal start site. The level of the proximal transcript was not affected (compare pXba 2.8 to pXba 3.2 and pRI-S 3.5). This result suggests that the 7907
Nucleic Acids Research sequence between -383 (L) and -214 (L), which is present in pXba 2.8 but not pRI-H 2.9, exerts a positive effect on the quantitative transcription from the proximal promoter. However, an upstream region between -1370 (L) and -770 (L), has also been brought closer to the proximal promoter in this mutant. It is possible that changing the spatial relationship of this upstream sequence to the proximal promoter could affect its transcription in this assay. It is also possible that novel regulatory element(s) were generated in this construct. Further experiments are necessary to distinguish amongst these possibilities. Other studies, using different experimental approaches, indicate that the cisacting elements of the proximal promoter are more complex than that of the distal promoter (5,6,9; see also DISCUSSION). Role of chromatin structure in the reoulation of the Drosovhila Adh Gene. To determine whether chromatin structure plays a role in regulating AO and to map the DNA regions where non-histone protein factors may interact, we have compared chromatin structure of Adh in several Drosophila cell lines, and correlated the results with specific transcription of Adh in these cell lines. Analysis of ADH transcripts in Drosophila tissue culture cells. During the past few years, a large numbers of new Drosophila melanogaster continuous cell lines have been established and maintained (11). All available Drosophila cell lines in continuous culture were derived from embryos, usually between 4 and 18 hr old (17,18). Although the tissue origins of cell lines that have survived the culturing conditions are unknown and most lines have not yet been cloned, each line has a distinct, easily recognizable, and stable morphology. Since these cell lines can be obtained in large quantities, they provide convenient and relatively homogenous starting materials for chromatin and biochemical studies, in contrast to intact animals and tissues. And as shown below, aspects of Adh gene regulation in vivo are mimicked in these cell
lines. To investigate whether any cell lines express ADH, we screened extracts of 60 Drosophila cell lines for ADH by protein immunoblotting and by assaying for ADH enzyme activity. ADH was detected in 10 cell lines at varying levels approximately 20 to 50-fold lower than that in mature adult flies. We next analyzed ADH transcripts by quantitative riboprobe-RNase mapping. Results for some of the cell lines are shown in Figure 2. In agreement with the protein study, 3 cell lines, 1006-2, OX736-3 and UC13-1, representing ADH-positive (ADH+) cells, possess ADH transcripts at the level at least 10-fold higher than SL2 cells (Figure 2) and other ADH cell lines (data not shown). Of interest, in these lines as well as in other characterized ADH+ cell lines ADH RNA
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Nucleic Acids Research 1
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