Amh (anti-Müllerian hormone) is a single copy gene which is expressed strongly in Sertoli cells in the foetal testis and participates in the onset of sexual differentiation. Its promoter driving the expression of a reporter gene (d2EGFP) has been used to analyse the role of certain defined putative elements and a downstream enhancer element in gene expression. These experiments were carried out in vitro using a line of pre-pubertal mouse Sertoli cells, transienly transfected with circular DNA constructs with variously mutated promoter elements. A downstream enhancer element, situated immediately 3’ of the polyadenylation (PA) signal for Amh, has been inserted in an equivalent position in the d2EGFP construct. When the Amh promoter is unmodified, the downstream enhancer (DE) is positively associated with a large increase in EGFP expression. This is at least partly the consequence of an increased rate of expression by individual cells. Experiments using variously truncated Amh promoters indicate that an upstream region (-214 to -336) may play a minor role in facilitating enhancement. However mutation of the Wilms tumour factor-1 element, situated between the tata box and the start of translation, results in an almost complete suppression of enhancement.
Amh, a member of the TGFbeta (BMP) family of transforming growth factor genes, plays a key role in early sexual differentiation in male mammals [1] and possibly a more subtle and protracted role in females [2]. Understanding the way in which gene expression is controlled in pre-Sertoli cells in the foetal testis, may help in decyphering the differentiation cascade triggered by the initial expression of Sry [3]. In the experiments to be described here the Amh promoter has been used to drive expression of a reporter gene (d2EGFP) in vitro, thus allowing a simple and quick way to investigate the role of constituent elements of the Amh promoter [4]. This approach largely confirms results obtained in vivo with an Amh promoter driving the expression of AMH [5-9]. It therefore seems likely that the in vitro system affords a convenient and economical approach to understanding other aspects of the control of gene expression by the Amh promoter.
Previously it was suggested that 1 - 3 kb of DNA immediately downstream of the Amhpolyadenylation (PA) signal, might play a role as a modulator of expression [10]. Further analysis of this region now shows that the 3’ part of this sequence is in the open reading frame (ORF) of yet another gene in the cluster around Amh. This gene is transcribed in an anti-sense direction and codes for JP45 (Jsrp1—junctional sarcoplasmic reticulum protein [11]. JP45 runs from 15260 to 11973 in mouse genomic sequence X83733—the Amh enhancer sequence (DE) runs from 11106 to 11195.
The results described in this paper now confirm that 89nt immediately downstream of the Amh PA signal is effective as an enhancer of Amh promotion. The enhancement role of this stretch of DNA and its dependence on the Wilms Tumour factor-1 element (Wt) upstream in the Amh promoter, has been established but leaves as a matter of conjecture the exact molecular mechanism of enhancement.
2. MATERIALS AND METHODS
SMAT cells (pre-pubertal Sertoli), TM4 cells (post-pubertal Sertoli) [12] and 3T3 (fibroblast) cells, are all mouse cell lines which adhere to tissue culture plasticware. They were grown in DMEM-F12 medium with glutamax and with 10% foetal calf serum plus penicillin and streptomycin. Assays were based on quadruplicate cultures in Costar 24-well plates (~2 × 105 cells/well). Transfections with LipofectAmine 2000 were carried out in antibiotic free medium. Transfections for SMAT and 3T3 cells were made with 800 ng DNA and 2 µl LipofectAmine and TM4 cells with 1200 ng DNA and 3 µl Lipofect-Amine per well.
Details of the flow cytometric analysis of d2EGFP reporter gene expression and of the maintenance of the cell lines, have been described previously [4,10]. An index of EGFP (green) fluorescence was measured using aflowcytometer monitoring red (Iexas red) and green (fluorescein) channnels. This enables autofluorescence to be defined accurately to allow a window for cells expressing EGFP specific fluorescence to be measured accurately. The index of expression is the product of the number of cells in the green window expressed as percent of total live cells (%) and the geometric mean brightness (Gm) of these cells (I = % × Gm).
Free DE and control DNA was prepared by synthesizing complimentary 90 nt oligo-nucleotides which were annealed by mixing them in equal molar proportions, heating them to 98˚ and allowing the mixture to cool very slowly overnight in a Dewar flask.
3. RESULTS
Previous results [4] hinted that the increased response index seen when an Amh promoter with a mutated proxGata element was used to drive expression of the reporter gene (d2EGFP), was explicable in terms of an increase in Gm. This implies that the mean expression per cell is increased. Figure 1 illustrates this point but also indicates that the same conclusion cannot be used to explain fully a decreased index with a mutated Se1 element.
Figure 2 illustrates the effectiveness of a downstream enhancer (DE) on responses driven by three different truncations of the Amh promoter in pre-pubertal Sertoli cells. Truncation X (see figure 3) has virtually no effect while truncations Y and Z lead to a highly significant reduction in the level of EGFP expression. The presence of DE immediately 3’ of the Amh PA signal, leads to a very large increase in the response driven by the intact (control) Amh promoter. The efficacy of DE on the responses driven by all three of the truncations is very much less. This result hints that something in the
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