A Comparison of the M and E Complexes Using a Protease Clipping Assay

The treatment o f a protein, or a protein complex, with a dilution series o f a specific protease, will result in a characteristic fi’agmentation profile, called a protease clipping assay. The endoproteases Arg C and Glu C, which attack the peptide bond on the carboxy side o f either arginine or glutamic acid residues respectively, were used to characterise the protein complexes associated with the M and E binding activities. The probes Bio-M and Bio-E were used with the paramagnetic beads to isolate their binding activities, as

described in chapter 6. The protein-DNA complexes were released fi*om the beads by treatment with 20 ^1 o f DTT, after which the eluates were made up to a volume o f 160 }il with the addition o f 0.05 M Tris, pH 8.0. A dilution series of each protease was prepared, by taking 1 |il o f stock (10 ng and successively diluting 1:3, and the proteolytic cleavage was initiated by adding 1 |il o f each dilution to a 10 |il aliquot o f the eluate. The reaction proceeded at room temperature for 15 min., after which the samples were

resolved on a 6 % (w/v) PAG (figure 7,5). The Arg C degradation o f the M complex was almost identical to that o f the E complex: the main binding activity was reduced in

intensity as the protease concentration increased, while simultaneously, a slightly smaller activity appeared (figure 7.5a). The Glu C profiles were similar to those o f Arg C in that, with the increase in protease concentration, the main activity became weaker to be

replaced with a smaller activity (figure 7.5b). At the highest concentration o f Glu C there was a further decrease in the mobility o f the protein-DNA complex.

Figure 7.5 Protease Clipping Assays.

The degradative profiles after treatment with (a) Arg C, and (b) Glu C. (See text for details).

Kev: - = Minus extract.

Chapter 7 - Biochemical Characterisation of the Xsna Promoter Binding Activities 168

(a) Bio-M Bio-E

+ — + ■■I t / t i ' i I * ' uTij Binding Activities [ • • • • Free Probe - (b) Bio-E + Binding Activities ^ Free Probe - = Glu C

The most striking aspect of the fragmentation patterns was that for each enzyme the pattern associated with the two probes was almost identical. This suggests that the organisation o f the proteins within the M and E protein complexes is very similar.

7.6 Discussion

The high apparent molecular weight of the P complex on gel filtration (-370 kDa) relative to the size o f the proteins identified by UV crosslinking (-90 kDa), suggest that the

binding complexes are multimeric. This may explain the sensitivity o f the binding activities to freezing and to salt elution. It may be possible using a protein-protein chemical

crosslinking agent such as bis[sulphosuccinimdyl] suberate in conjunction with UV crosslinking o f the protein to the DNA, to demonstrate contact between polypeptides within the complex. Samples that have been treated in this manner would contain larger components than those observed after UV crosslinking alone, from which the size o f individual components could be calculated.

The apparent similarity in protease fragmentation products o f the M and E complexes using two different proteases is quite surprising. It would be expected that these two endoproteases would produce distinctly different patterns (for example, Snape et al., 1991; Roux et al., 1989). However, other data suggest there may be a relationship between the binding activities (see section 5.5).

7.7 Summary

1) A large multi-subunit complex o f -370 kDa is responsible for the binding activities observed using EMSA.

Chapter 7 - Biochemical Characterisation of the Xsna Promoter Binding Activities 170

of about 97 kDa: the mesodermal peptide is the smaller o f the two. 3) In stage 14 embryos, these peptides are apparently 90 kDa.

4) The protease clipping profiles for the M complex are almost identical to that of the E complex, possibly indicating a surprising similarity in organisation.

The Regulation of Xsna

8.1 Introduction 172

8.2 Xsna Induction is Regulated by PKCa 174

8.3 Discussion 174

Chapter 8 - The Regulation of Xsna 172

8.1 Introduction

The ectodermal expression pattern of Xsna that appears at stage 11 - 11.5 is the first indication that the prospective neural crest is distinguishable fi'om the prospective neural plate (Essex et al, 1993). This expression, which begins as a low arc on the dorsal aspect, outlines the neural plate during subsequent extension of the latter (termed the neural plate border; Essex et al., 1993). By the open neural plate stage, Xsna is down-regulated in the anterior fold prior to its incorporation into the forebrain, but remains strongly expressed on the lateral edges which later become the neural folds (Essex et al., 1993).

For some time it has been suspected that the patterning of the nervous system in

Xenopus involves signals emanating from the underlying mesoderm and through the plane

between the organiser and the ectoderm (Spemann and Mangold, 1924), and several molecular candidates for the inductive signals have emerged (Ferreiro and Harris, 1994). More recently it has been suggested that interactions between the dorsal and ventral ectodermal domains (chapter 1), lead to induction of the prospective neural crest (Mayor et al., 1995; Mancilla and Mayor, 1996)).

Using the specific neural crest marker (Mayor et al., 1995), a paralogue of

Xsna, an expression cloning study was conducted in which it was discovered that the

translation initiation factor eIF4AI and eIF4AII could specifically induce the genes of the neural plate border (Morgan and Sargent, submitted). Over-expression o f eIF4AI, or the murine homologues mmeIF4AI and mmeIF4AII, up-regulated the neural crest markers Xslu, Xsna and Pax-3 (but not twist), the anterior fold markers XANF and hairy 2A , and the cement gland marker XGCl. There was no up-regulation of the neural plate markers NCAM, Tubulin-p, Hox-B9 and Xif-6, or the mesodermal markers Xbra, Xhox-3 and myo-D.

RNAse protection analysis showed that eIF4AI was expressed in all tissues at all embryonic stages and that eIF4AII mRNA increased sharply after stage 11, with more than 90 % being confined to the dorsal ectoderm (Morgan and Sargent, submitted). eIF4AII was up-regulated by a variety of neural induction regimes but the strength o f the signal determined whether neural plate or neural plate border genes were expressed. The

minimum amount o f each inducer which up-regulated eIF4AII, also up-regulated Xslu and suppressed epidermal markers, but as the amount of inducer was increased, NCAM was expressed and Xslu repressed (Morgan and Sargent, submitted).

The expression o f the Xenopus protein kinase C a (PKCa), which has been implicated in the acquisition of competence for neural induction (Otte et al., 1988, 1990,

1991; Otte and Moon, 1992), is transcriptionally up-regulated by mmeEF4AII. Ectopic expression o f either eIF4AII or PKCa mRNA sensitised the ectoderm to neural induction by noggin: micro-injection of PKCa reduced the amount of noggin needed to induce NCAM and suppress Xslu to 1 % (Morgan and Sargent, submitted). The

bis-indolylmaleimide GF109203X is a specific inhibitor of the calcium dependent isoforms of PKC (a, ft and y; Toullec et al., 1991). Ectopic expression of eIF4AII in the presence o f GFl 09203X resulted in the suppression oïX slu, but not E F la and the inhibitor had no effect on axial mesoderm formation. These data suggest that induction o f Xslu is

dependent on PKC activity, and that eIF4AII and PKCa are intermediaries in neural crest induction. Since Xsna expression is upregulated by overexpression o f eIF4AII and PKCa,

and is inhibited by GFl 09203X, it seems likely that the activity of the ectodermal element of the Xsna promoter is dependent directly or indirectly on phosphorylation.

Another aspect o f the role o f phosphorylation came to light in experiments

Chapter 8 - The Regulation of Xsna 174

Xsna gene in animal cap assays. This autoregulatory loop was enhanced in the presence of GF109203X.