Introduction

To date there are two main families of transcription factors involved in mediating Nodal- related signals during early Xenopus development - the Mix and the Fast/FoxHl families. As discussed, selected members of these families interact with Smad2/Smad4 complexes via their Smad-interaction motif in response to Nodal signalling and activate transcription of target genes. The presence of Fast binding sites in a number of Nodal target genes, including Nodal, Lefty2, Mix.2 and goosecoid (Chen et aL, 1996; Labbe et aL, 1998; Norris et aL, 2002; Saijoh et aL, 2000), further supports the role of Fast/FoxHl family members as endogenous mediators of Nodal signalling. There are currently five members of this family of transcription factors: human Fast-1 (FoxHl), mouse Fast-2 (FoxHl), Xenopus Fast-1 (FoxHla) and Fast-3 (FoxHlb), and zebrafish Fast-1 (FoxHl; Chen et aL, 1996; Howell et aL, 2002; Labbe et aL, 1998; Liu et aL,

1999; Pogoda et aL, 2000; Sirotkin et aL, 2000; Zhou et aL, 1998).

In an attempt to better understand how Smads regulate the transcription of target genes, recent work has begun to establish the stoichiometries of active Smad- transcription factor complexes on DNA. This study demonstrated that ARF1/ARF2 is composed of one molecule of XFast-1 or XFast-3, respectively, in complex with a heterotrimer of Smads, containing two molecules of Smad2 and one of Smad4 (Inman and Hill, 2002). This is consistent with studies of the ARE in the Xenopus Mix.2

promoter, which contains one Fast binding site in close proximity to a Smad-binding site, with which Smad4 could interact (Inman and Hill, 2002; Yeo et aL, 1999). In this complex, the Fast interacts via its SIM with the shallow hydrophobic pocket in the Smad2 MH2 domain (Chapter 5; Randall et aL, 2002). However, the Fast molecule only has one SIM and the Smad heterotrimer contains two Smad subunits, leaving open the

Chapter 6____________________________________ A novel Smad2-interactin2 motif

question of how Fast can contact the second Smad2 molecule. Indeed, preliminary experiments conducted by Stéphane Germain (Developmental Signalling Laboratory, Cancer Research UK) in which two point-mutations were made in the XFast-1 SIM in the PPNK core indicated that the ability of this derivative to form ARFl in response to TGF-p was not abolished, as might be expected. This is consistent with previous observations using deletion mutants of the XFast-1 C-terminus which suggested that sequences N-terminal to the SIM are required for formation of active Smad2/Smad4/XFast-l complexes (Chen et aL, 1997a).

Experimental data presented here describe a detailed study of Smad-interaction motifs in Fast/FoxHl family members. I identify a novel Smad-interaction motif, termed the Fast motif (FM), that is present in all known Fast/FoxHl family members, N- terminal to the previously identified SIM. Using extensive site-directed mutagenesis, I have investigated the binding site for this motif on the Smad2/Smad4 heterotrimer. Importantly, this motif is distinct from the SIM in that it recognises only activated Smad complexes, and is able to distinguish between Smad2 and Smad3. Proposed is a model whereby Fasts are able to contact a heterotrimer of Smads by virtue of their two Smad- interaction motifs; the SIM would contact one Smad2 subunit, and the FM would contact the second.

6.2 Results

6.2.1 Identification of the Fast motif

Mix family members that interact with Smad2 share only two regions of amino acid identity; the DNA-binding domain and the SIM (Randall et aL, 2002). In Mixer, mutation of the two prolines in the central ‘PPNK’ of the SIM to alanines completely abolishes interaction with Smad2 and, as a result, TGF-P-induced transcriptional activation mediated by Mixer/Smad2/Smad4 complexes (Germain et aL, 2000). The SIM is also well conserved in all Fast/FoxHl family members, which also interact with Smad2 (Germain et aL, 2000; Randall et aL, 2002). To investigate the importance of the SIM in XFast-1 and XFast-3, ‘PPNK’ was mutated to ‘AAAK’ in the full-length transcription

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Figure 6.1 The FM is required in both XFast-1 and XFast-3 for m ediating TGF-P- induced transcriptional activation through recruitment of active Smad2/Smad4 com plexes

NIH3T3 cells were transfected with the (ARE)3-Luciferase reporter either alone or w ith plasm ids expressing HA-tagged wild-type or mutant derivatives of XFast-1 (A) or XFast-3 (B). Cells were incubated in the presence or absence of TGF-p. Luciferase w as quantitated relative to (3-Gal from the pFFLacZ internal control and the value for TGF-P-induced transcriptional activation of XFast-1 or XFast-3 was set at 100. The data are the mean and standard deviation of three independent experiments. Levels of expression of the mutants are shown in Western blots.

Chapter 6_____________________________________A novel Smad2-interacting motif

factors and their ability to confer TGF-P-induced transcriptional activation on the Mix. 2

ARE was tested. Wild-type XFast-1 and XFast-3 mediated a 29- and 20-fold TGF-P- induced transcriptional activation, respectively, on an ARE-driven luciferase reporter, due to recruitment of endogenous active Smad complexes (Figure 6.1; Germain et aL, 2000). Mutation of the SIM in XFast-1 decreased the induction to 11-fold, but did not eliminate it, and in XFast-3, this mutation had no effect (Figure 6.1). This suggested the presence of an additional motif in the Fasts that could interact with Smad2 and/or Smad4 and thus compensate for the mutated SIM.

Sequence alignment of the 5 known Fast/FoxHl family members revealed a highly conserved region within their C-terminal domains, N-terminal to the SIM. This region, which has been termed the Fast Motif (FM) is characterised by the sequences ‘LPTSY’ and ‘PN(V/A)V(A/M)P(L/P)’, referred to as FMI and FM2, respectively (Figure 6.2). To determine the relative importance of each of these motifs for recruiting active endogenous Smad complexes to DNA, the SIM, FMI and FM2 were mutated individually and in combination, in both XFast-1 and XFast-3 (shown in Figure 6.2). All the derivatives of XFast-1 and XFast-3 were equally well expressed indicating that any effects seen were not the result of different expression levels of protein (Figure 6.1, right panels). In the context of XFast-1, mutation of FMI had a negligible effect on the ability of the transcription factor to recruit active Smad complexes, but mutation of FM2 rendered XFast-1 nearly completely inactive (Figure 6.1 A). Mutation of both FMI and FM2 resulted in a protein that retained only residual activity (4-fold activation), whereas mutation of either FMI or FM2 in combination with a SIM mutation completely abolished activity (Figure 6.1 A). For XFast-3, mutation of FMI had little effect on its ability to mediate TGF-P-induced transcriptional activation, whereas mutation of FMI in combination with the SIM reduced the induction by TGF-p to 7-fold (Figure 6.IB). Mutation of the XFast-3 FM2 completely abolished TGF-p-induced transcription, as did combinations of the FM2 mutation with SIM or FMI mutations (Figure 6. IB).

These results demonstrate that the FM is necessary in both XFast-1 and XFast-3 for mediating TGF-P-induced transcriptional activation via the ARE, and that FM2 is more important than FM I.

m F a s t - 2 3 0 8 WGC z f F a s t - 1 3 5 4 PWE L P IY 1 : SAKYT FM FMI 3LA TL PT--- TSC PQC P S ---SASPAYWSVGTESQGS--- QDLLC PSMRFNGNPFMPLGGIPFYGYGGAHVTTSHLIGHPYWPILPSGPVS--- IQ APPL SMLQS\ IS IySv---WVS ALGSNNQTV DLAAPAPGWLLSWYSM---4 0 1 SPNQYALQNGPSLCKYSL---4 7 2 SIM FM2

Figure 6.2 A lignm ent of the C-terminal regions of the five know n Fast fam ily members

X, Xenopus; h, human; m, mouse; zf, zebrafish. Black dotted underlining indicates the Fast motif (FM), with FMI and FM2 denoted; black underlining indicates the Smad interaction motif (SIM). The dots indicate the residues that were mutated to alanines in the XFast mutants, and also in the Gal4-FM and Gal4-SIM mutants (Figures 6.1, 6.3 and 6.4).