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Cement gland-specific activation of the Xag1 promoter is regulated by co-operation of putative Ets and ATF/CREB transcription factors

Fiona C. Wardle^*, Daniel H. Wainstock and Hazel L. Sive

Whitehead Institute for Biomedical Research, and Massachusetts Institute of Technology, Nine Cambridge Center, Cambridge, MA 02142, USA
^* Present address: Wellcome Trust/Cancer Research UK Institute, Tennis Court Road, Cambridge CB2 1QR, UK

View larger version (25K): [in a new window]   Fig. 1. (A) Two major classes of Xag1 transcripts are detected by 5' RACE. One class initiates downstream of a TATA box (+1; black arrow). The second class consists of several transcripts that initiate 150-100 bp upstream of the TATA box (gray arrows). (B) Northern blot analysis of RNA isolated from blastula through tailbud (stage 9-30) embryos shows the first class of transcript is the most abundant in the embryo. Both probes recognize in vitro transcribed Xag1 upstream sequence (not shown). (C) Sequence of the Xag1 genomic region used in this study. 3 Ets-like binding sites (EBS; dark-gray boxes), an ATF/CREB-like binding site (CRE, light-gray box) and the TATA box (unshaded box) are indicated. The black arrow indicates the initiation site of the major transcript, the gray arrow indicates the 5' limit of the rarer transcripts that were isolated. Also shown are the 14 bp regions (1-7) and the 4 bp region 8 that were replaced in the linkerscan analysis (see Fig. 3).

View larger version (38K): [in a new window]   Fig. 2. Upstream genomic Xag1 sequence drives cement gland-specific expression. In situ hybridization for endogenous Xag1 (A,B) or gfp transcripts (C,E-J) in early neurula (A,C,E,G) or tailbud (B,F,H-J) embryos. (D) GFP fluorescence in a transgenic tailbud embryo. Endogenous Xag1 is expressed at the anterior of the embryo from the end of gastrulation (A) in the cement gland (black arrowhead) and hatching gland primordia (black arrow), and in the cement gland (white arrowhead) and hatching gland (white arrow) in the tailbud embryo (B). This expression pattern is replicated by 8 kb of Xag1 genomic sequence driving a GFP reporter gene (C,D). (E-H) Cement gland-specific gfp expression is driven from early neurula stages by –275 bp (E,F) and –102 bp (G,H), but not 73 bp of upstream sequence (I) or the Xag1 TATA box (J) (arrowheads indicate location of cement gland). Expression is weaker with the –102 bp construct (G,H) than with the longer –275 bp construct (E,H), especially at neurula stages (arrowheads). Po, posterior; np, neural plate.

View larger version (19K): [in a new window]   Fig. 3. Structure of Xag1 upstream region and reporter constructs. (A) Potential transcription factor-binding sites and the start site of transcription (black arrow) are indicated. Cement gland-specific expression is driven at tailbud stages by –102 bp of upstream sequence. For the 8 kb construct, hatching gland expression is also seen. (B) Linkerscan analysis of this short promoter shows linkerscan (LS) regions 1-3 and 5-6 are most important for this expression. (C) In the context of the larger –275 bp promoter, mutation of these regions has no effect on gfp expression, although the distal promoter alone drives expression only very rarely. A large part of the distal compensation can be attributed to the two EBS found in this region, because when these are mutated the frequency of expression in the cement gland falls significantly. Cement gland expression of gfp in more than 25% of embryos is indicated by +++ (18-25%), ++ (9-17%), + (2-8%), +/– (no change) and – (<2%) (see Table 1; Materials and Methods).

View larger version (26K): [in a new window]   Fig. 4. The EBS and CRE are sufficient to drive cement gland expression in the tailbud embryo and cooperate to increase expression. (A) Multimerized 14 bp regions containing the proximal EBS (region 2) or CRE (region 3) are sufficient to drive cement gland-specific expression of GFP. When the EBS or CRE are mutated (3 bp substitution in core binding site; see Materials and Methods), their ability to drive this expression is abolished. (B) In front of a heterologous promoter (adenovirus major late promoter; MLP) the 5xEBS and 5xCRE are extremely inefficient at driving gfp expression, but when multimerized together (3EBS/2CRE) the sites cooperate to drive robust expression in the cement gland. Cement gland expression of gfp in more than 25% of embryos is indicated by +++ (18-25%), ++ (9-17%), + (2-8%), +/– (no change) and – (<2%) (see Table 2; Materials and Methods).

View larger version (26K): [in a new window]   Fig. 5. (A) Gel shift analysis shows a complex formed on the Xag1 promoter at neurula stages. Cell extracts were made from cement gland primordia dissected out of the embryo at stages 15-17 (mid-neurula) and subjected to electromobility shift analysis with labeled Xag1 probe in the presence or absence of cold competitors. Lane1, probe only; lane 2, probe plus extract; lane 3, probe plus extract and Xag1 competitor; lane 4, probe plus extract and EBS competitor; lane 5, probe plus extract and mutated EBS competitor; lane 6, Xag1 probe plus extract and CRE competitor; lane 7, probe plus extract and mutated CRE competitor; lane 8, probe plus extract and OCTA competitor. (B) Sequence of Xag1 probe and cold competitors used.

View larger version (45K): [in a new window]   Fig. 6. The CRE responds to Otx2 activity, while the EBS is independent of Otx2. (A) Embryos were injected at the 1- to 2-cell stage with 150 pg otx2 or globin mRNA plus 50 pg of either 5xEBS, 5xEBSmut, 5xCRE, 5xCREmut, 3EBS/2CRE or TATA-only nGFP constructs. Promoter constructs contained the Xag1 TATA box (see Fig. 4). Animal caps were cut at stage 9, cultured until stage 16 and expression of Xcg, Xag1, gfp and odc analyzed by RT-PCR. (B) RT-PCR of ectodermal explants dissected from embryos injected with otx2 or globin mRNA plus the indicated reporter construct. Expression of cement gland markers (Xcg and Xag1) and induced gfp were examined using odc expression as a loading control. Lane 1, uninjected animal caps; lane 2, globin mRNA plus TATA-only nGFP DNA; lane 3, globin mRNA plus 5xEBS.nGFP DNA; lane 4, globin mRNA plus 5xCRE.nGFP DNA; lane 5, globin mRNA plus 5xEBSmut.nGFP DNA; lane 6, globin mRNA plus 5xCREmut.nGFP DNA; lane 7, globin mRNA plus 3EBS/2CRE.nGFP DNA; lane 8, otx2 mRNA plus TATA-only nGFP DNA; lane 9, otx2 mRNA plus 5xEBS.nGFP DNA; lane 10, otx2 mRNA plus 5xCRE.nGFP DNA; lane 11, otx2 mRNA plus 5xEBSmut.nGFP DNA; lane 12, otx2 mRNA plus 5xCREmut.nGFP DNA; lane 13, otx2 mRNA plus 3EBS/2CRE.nGFP DNA.

View larger version (22K): [in a new window]   Fig. 7. Model to show how transcription factor activity is integrated at the Xag1 promoter to bring about cement gland-specific expression. Markers of differentiation, such as Xag1, are activated at the end of gastrulation in the cement gland primordium (CG), which is defined by the overlap of up to three larger domains: anterodorsal (AD), ventrolateral (VL) and ectodermal outer layer (EO). Surface views of early neurula stage embryos are shown for the AD and VL panels, and sagittal section schematics for the EO and cement gland panels. The AD domain is defined by otx2 expression, the VL domain by BMP4 or some downstream readout, such as activated Smad1, while an unknown factor (XXX) defines the outer ectodermal layer. Xag1 expression in the cement gland primordium requires an ATF/CREB factor, which binds to the CRE and lies downstream of otx2. This acts in cooperation with an Ets factor that binds to the EBS and may lie downstream of BMP4 and/or XXX. Other inputs may influence Xag1 expression, but are not sufficient on their own to drive expression. CG, cement gland; ar, archenteron; Bc, blastocoel; A, anterior; P, posterior; D, dorsal; V, ventral.