Zachau, 1984; Mason et al., 1985; Parslow et al., 1987;. Boulet et al., 1986). ...... Atchison,M. and Perry,R.P. (1987) Cell, 48, 121-128. Banerji,J., Rusconi,S. and ...
The EMBO Journal vol.7 no.1 pp.177- 188, 1988
A cis-acting element and associated binding factor required for CNS expression of the Drosophila melanogaster dopa decarboxylase gene
S.J.Bray, W.A.Johnson, J.Hirsh, U.Heberlein' and R.Tjian1 Department of Biological Chemistry and Molecular Pharmacology, Harvard Medical School, Boston, MA 02115 and 'Department of Biochemistry and Molecular Biology, University of California Berkeley, Berkeley, CA 94720, USA Communicated by J.Tooze
A cis-acting sequence from the Drosophila melanogaster dopa decarboxylase (Ddc) gene is selectively required for Ddc expression in the central nervous system. We analyze several parameters influencing the function of the sequence element and describe a factor which interacts with it and mediates CNS expression of Ddc. The element, element I, can function in vivo when included on a synthetic oligonucleotide inserted near its normal location, or closer to the RNA startpoint. It displays partial activity when inverted. Two different 2-bp mutations in element I abolish its ability to stimulate neuronal Ddc expression in the CNS. A factor present in embryonic nuclear extracts specifically protects element I in DNase I footprinting assays. The binding affinity of this factor is reduced by each alteration of element I that inhibits neuronal expression, indicating a role in mediating CNS expression of Ddc. Element I alone has no detectable activity when placed adjacent to a heterologous promoter, although 2.2 kb of 5' Ddc sequences direct correct cell-specific expression of a heterologous promoter. Key words: Drosophilaldopa decarboxylase/cis-acting element/gene expression
Introduction Many eukaryotic genes contain regulatory domains which receive inputs from multiple discrete regulatory pathways. For inducible promoters such as those from metallothionein (Stuart et al., 1984; Searle et al., 1985; Stuart et al., 1985), heat shock (Pelham, 1982; Bienz and Pelham, 1986), glucocorticoid (see Yamamoto, 1985) and TPA inducible genes (Angel et al., 1987; Lee et al., 1987) single short sequence elements are sufficient to confer induction, although often more than one copy of the sequence is required for full induction. These elements can co-exist with each other and with other more complex regulatory elements. Similar short cis-acting sequences also appear to play a role in the regulation of tissue and cell specific expression (Falkner and Zachau, 1984; Mason et al., 1985; Parslow et al., 1987; Boulet et al., 1986). However, the requirements for tissue specific regulation may be more complex than for inducible promoters, involving multiple, often partially redundant, components (Grosschedl and Baltimore, 1985; Edlund et al., 1985; Lenardo et al., 1987; Hammer et al., 1987a; Schirm et al., 1987). ©IRL Press Limited, Oxford, England
An analysis of the components involved in regulating the expression of a gene in specific cells and tissues requires an in vivo assay for their activity. It is not possible to reproduce in cell culture the conditions that participate in the specification of gene expression within a subset of cells in a complex tissue such as the central nervous system (CNS). Also, many eukaryotic genes are expressed several times during development in a variety of tissues. The fruit fly, Drosophila melanogaster, provides a system in which to study those aspects of tissue- and cell-specific gene expression that are difficult to approach in cell culture systems. In vitro altered genes can readily be integrated into the germline via P element vectors (Rubin and Spradling, 1982), and usually the site of integration has little effect on the expression of the integrated gene (Scholnick et al., 1983; Spradling and Rubin, 1983; Goldberg et al., 1983; Hazelrigg et al., 1984). Using this system, we have begun to dissect the regulatory mechanisms governing the expression of the gene encoding the enzyme dopa decarboxylase (Ddc) (Hirsh et al., 1986; Scholnick et al., 1986; Morgan et al., 1986). Ddc is expressed at high levels in the hypoderm and in a small number of cells in the CNS (Wright et al., 1976; Hirsh, 1986; Beall and Hirsh, 1987; Konrad and Marsh, 1987). Ddc is required in the hypoderm for the synthesis of dopamine which is metabolized to compounds used in cuticle hardening and pigmentation (Lunan and Mitchell, 1969; Wright et al., 1976) and in the CNS for the synthesis of serotonin and dopamine (Livingstone and Tempel, 1983; Morgan et al., submitted). Expression of Ddc in each tissue is regulated differently during development (Kraminsky et al., 1980; Marsh and Wright, 1980; Hirsh, 1986) and this regulation is mediated by both transcriptional and posttranscriptional mechanisms. The Ddc primary transcript is alternately spliced in the CNS and hypoderm, leading to gene products that differ by inclusion of a 33-35 amino acid amino terminus contained in a CNS-specific exon (Morgan et al., 1986). Cis-acting elements regulating the transcription of Ddc have also been defined. A comparison of the Ddc genes from the diverged species D.melanogaster and D. virilis identified several 8-16 base pair (bp) conserved sequence elements within 120 bp of the RNA startpoint (Bray and Hirsh, 1986). The functional importance of these elements was demonstrated by analyzing the expression in vivo of Ddc genes with deletion end-points in this region (Scholnick et al., 1986). Several regions are required for normal hypodermal Ddc expression and one, localized between -59 and -83, is required for CNS Ddc expression. Within this region there is only one element defined by evolutionary sequence conservation, element I, a 16 bp sequence perfectly conserved between the diverged Ddc genes. A more detailed analysis, using a Ddc antiserum to study the precise spatial pattern of Ddc expression, has shown that element I alone is insufficient to confer correct expression of Ddc within the CNS (Beall and Hirsh, 1987). The wild type Ddc gene is expressed in - 130 neuronal cell bodies 177
S.J.Bray et al.
Fig. 1. Element I oligonucleotides and structure of Ddc genes used for analysis. (A) The wild-type location of element I with respect to the Ddc 'TATA' box and RNA start-point is diagrammed. The extent of the Ddc promoter deletions used in this study are also shown. These mutant Ddc genes contained 2.2 kb of 5' flanking sequences and 1 kb of 3' flanking sequences as shown in (B). The Ddc exons are drawn as shaded boxes (Morgan et al., 1986). The vertical black lines indicate the XbaI overhanging ends bounding deletions between -208 and either -60 or -38 (Scholnick et al., 1986), to which the double-stranded element I oligonucleotides were ligated. The oligonucleotides containing either wild-type or mutant element I sequences are shown at the bottom. These oligonucleotides contain element I and 5 additional base pairs normally found adjacent to element I, plus the nucleotides which form XbaI overhanging ends to facilitate insertion of the oligonucleotide into Ddc. When the oligonucleotides are inserted at -60, element I is 9 bp farther from the RNA startpoint than in the wild-type gene, at -38 it is 13 bp closer. A short palindrome within the element I sequence is indicated by arrows and the bases changed in the mutant oligonucleotides are boldfaced. The bases selected for mutation in the ml oligonucleotide were chosen because they interrupt the palindrome, those in m2 are adjacent to, not part of, the conserved palindrome. The minimal extent of DNase protection by the element I binding factor described in this manuscript is indicated. The altered Ddc genes shown were inserted into a P-element vector containing alcohol dehydrogenase (Adh) as a selectable marker (Scholnick et al., 1986; Bray and Hirsh, 1986).
and in a subset of cortical glial cells. A second element, located between 800 and 2200 bp from the RNA startpoint, is required in conjunction with element I to generate the normal neuronal pattern of Ddc expression. Deletion of the distal element results in near-normal levels of CNS expression, but most Ddc product is located within the glial cells, where it is expressed to much higher levels than in wild type. Deletion of element I results in a loss of expression in both cell types. Studies of the interactions between these two elements will provide insights on how a complex pattern of cell-specific gene expression can be generated. In this communication we study further the role of element I in CNS expression of Ddc. We show that it can function with a degree of positional flexibility and that its function can be inactivated by mutation. Furthermore, we have detected a binding factor whose binding affinity is significantly reduced by alterations to element I that inhibit in vivo function. 178
Results Characterization of a cis-acting sequence required for CNS expression of Ddc To characterize the activity of the cis-acting sequence, element I, we tested the ability of an oligonucleotide containing element I to rescue CNS expression when inserted into the 5' flanking region of a Ddc gene lacking the element. The host gene contains a promoter deletion, [A(-208, -60), Figure IA] and shows essentially no expression in the CNS (Scholnick et al., 1986; BeaU and Hirsh, 1987; Figure 2D). Insertion of the element I oligonucleotide at -60 specifically restores CNS Ddc expression. The levels of Ddc enzyme activity in the larval CNS and in white prepupae were assayed in homogenates from the transformant strains. Ddc enzyme activity in whole white prepupae is largely a reflection of hypodermal expression since > 90 % of enzyme activity is in the hypodermal fraction (Scholnick et al., 1983;
Dopa decarboxylase gene expression
I Fig. 2. A wild type element I oligonucleotide rescues CNS expression of Ddc in an orientation independent manner. Isolated larval CNS were incubated with an anti-Ddc serum and detected in whole mount preparations with a FITC-labeled secondary antiserum (Beall and Hirsh, 1987).
Staining of a CNS from (A) Canton S wild type, shows a regular pattern of neuronal cell bodies in the segmented ventral ganglion (lower left), and also cell bodies in the brain lobes (top). The meshwork of staining in the ganglion and near the medial regions of the brain lobes is a network of glial cells (Beall and Hirsh, 1987). Similar staining is observed in transformant strains containing the element I oligonucleotide in (B) normal orientation, strain I(A-208,-60)b, or (C) inverted orientation, strain Iinv(A-208,-60)a. Neuronal cell body staining is somewhat weaker than normal in both strains. Inset in (C) is of the abdominal ganglion of a Iinv(A-208,-60)a larval CNS that shows more clearly neuronal cell body staining. The glia are not in the plane of focus in (B). Panel (D) shows a control strain, A(-208,-60)a, which lacks element I and shows no detectable neuronal or glial staining.
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S.J.Bray et al. Table I. Ddc expression in element I mutant strains Strain
Ddc enzyme activity Summary from figures % Canton S wild type Neuronal Relative Larval CNS White prepupa expression element I (whole animal) binding
Canton S
100
100
+
1
zA(-208,-60) a b
6 4
36 32
-
NA
I(A -208,-60) a b c
72 68 26
40 34 36
+
1
Iinv(A -208, -60) a 34 90 A(-208,-38) 2
55 36
+/-
1
4
-
I(A-208,-38) a
130 160
24 23
+
1.2
3 b 10 c nd
12 6 12
-
