Transcriptional regulation of the Drosophila melanogaster muscle myosin heavy-chain gene

Abstract

We show that a 2.6kb fragment of the muscle myosin heavy-chain gene (Mhc) of Drosophila melanogaster (containing 458 base pairs of upstream sequence, the first exon, the first intron and the beginning of the second exon) drives expression in all muscles. Comparison of the minimal promoter to Mhc genes of 10 Drosophila species identified putative regulatory elements in the upstream region and in the first intron. The first intron is required for expression in four small cells of the tergal depressor of the trochanter (jump) muscle and in the indirect flight muscle. The 3'-end of this intron is important for Mhc transcription in embryonic body wall muscle and contains AT-rich elements that are protected from DNase I digestion by nuclear proteins of Drosophila embryos. Sequences responsible for expression in embryonic, adult body wall and adult head muscles are present both within and outside the intron. Elements important for expression in leg muscles and in the large cells of the jump muscle flank the intron. We conclude that multiple transcriptional regulatory elements are responsible for Mhc expression in specific sets of Drosophila muscles.

Figure 1. Structure of MHC-lacZ fusion and reporter gene constructs

Constructs A, B, C and G were made by fusing the lacZ gene of pOX3 (Raghaven et al., 1986) in frame to the Mhc coding region. Constructs D, E and F were derived from pDM30 (Mismer and Rubin, 1987), which contains the Drosophila hsp70 minimal promoter linked to a lacZ reporter. The parental lacZ plasmids also provide a polyadenylation signal. All constructs were inserted into a P element plasmid vector prior to germline transformation. Details are given in Experimental Procedures.

Figure 2. Staining of organisms in X-gal solution reveals the relative level and tissue-specificity of MHC-lacZ fusion gene expression

(A) Dissected adult containing pπMHC-lacZ 1 and stained in X-gal solution. Indirect flight muscles (IFM), proboscis muscles (P), abdominal body wall muscles (A) and gut muscles (G) are clearly stained. (B) Cryosection of an adult lacking a fusion gene. Only minor staining of abdominal structures is seen. (C) Parasagittal section of adult containing pπMHC-lacZ 1 shows ß-galactosidase activity in various muscle tissues including indirect flight muscles (IFM), proboscis muscles (P), abdominal body wall muscles (A) and leg muscles (L). (D) Horizontal section of adult carrying pπMHC-lacZ 1. Dorsal-lateral IFMs (DLM) and dorsal-ventral IFMs (DVM) are clearly stained, as is the tergal depressor of the trochanter muscle (TDT). (E) Whole mount of embryo carrying pπMHC-lacZ 1. All body wall muscles are stained. (F) Parasagittal section of fly transformed with pπMHC-lacZ Δi1, which deletes most of intron 1, reveals intense staining in the TDT. (G) Horizontal section of adult thorax expressing pπMHC-lacZ Δi1. The TDT is well stained, the direct flight muscles (DFM) exhibit clear staining, but the IFM shows extremely low levels of staining. (H) Higher magnification of (G) showing that the four smaller anterior cells (S) are poorly stained compared to the other cells of the TDT. (I) Parasagittal section of fly transformed with pMHC-1.5-hsplacZ, which contains intron 1, shows staining in the indirect flight muscles (IFM), proboscis muscles (P) and abdominal body wall muscles (A). The TDT barely stains. (J) Horizontal section of fly transformed with pMHC-1.5-hsplacZ shows staining in the indirect flight muscles (IFM) and proboscis muscles (P), but little or none in the TDT or direct flight muscles (DFM).

Figure 3. Staining of embryos in X-gal solution reveals the level and tissue-specificity of MHC-lacZ transgene expression

Lateral (left panels) and dorsal (right panels) views are shown. (A, B) Embryos containing pπMHC-lacZ Δi1, which lacks most of intron 1, express ß-galactosidase in body wall and pharyngeal muscles (Ph) in a similar manner to those expressing the minimal myosin promoter pπMHC-lacZ 2 (Fig. 2E). (C, D) Some pπMHC-lacZ Δi1 embryos show differential muscle staining. (E, F) Embryos expressing pMHC1.5-hsplacZ, which contains the intron 1 enhancer, show body wall and pharyngeal muscle staining similar to those expressing pπMHC-lacZ 2. (G, H) Deletion of the 330 terminal bp of pMHC1.5-hsplacZ to yield pMHC1.2-hsplacZ results in greatly reduced body wall muscle expression. (I, J) Expression of pπMHC-lacZ ΔAT, which was produced by deletion of the 330 bp intron fragment from pπMHC-lacZ 2, allows high levels of embryonic ß-galactosidase expression.

Figure 4. DNAse footprint reaction of proteins from 10-16 hour old Drosophila embryos

Sephacryl S400 HR column fractions of nuclear extracts were reacted with an end-labeled Mhc gene intron sequence (+1355 to +1685). Following DNAse digestion and gel electrophoresis, AT-rich regions were shown to be protected (brackets) by column fractions 74, 76 and 78, compared to column fraction 60 or a no-protein sample (-).