Genomic organization, chromosomal assignment, and expression
analysis of the mouse Suppressor of fused gene (Sufu) coding a Gli
* Claudie Lamour-Isnard,
Unite´deGe´ne´tique des Mammife`res, Institut Pasteur, 25 rue du Dr Roux, 75724 Paris Cedex 15, France
Institut Jacques Monod, Laboratoire “Ge´ne´tique du De´veloppement et Evolution,” Universite´s Pierre et Marie Curie et Denis Diderot, 2 place Jussieu,
75251 Paris Cedex 05, France
Received: 14 January 2000 / Accepted: 31 March 2000
Abstract. Suppressor of fused (Sufu) is a negative regulator of the
Hedgehog pathway both in Drosophila and vertebrates. Here, we
report the genomic organization of the mouse Sufu gene (mSufu).
This gene comprises 11 exons spanning more than 30 kb and
encodes a protein with a putative PEST sequence. DNA-consensus
sequences recognized by basic helix-loop-helix (bHLH) proteins,
referred to as E-box motifs, are found in the 5Ј flanking region.
Analysis by single-strand conformation polymorphism and radia-
tion hybrid positioned the Sufu locus to the distal end of mouse Chr
19 between D19Mit102 and D19Mit9, near the Fgf8 and dactylin
genes. Mouse Sufu is expressed in various tissues, particularly in
the nervous system, ectoderm, and limbs, throughout the develop-
ing embryo. Sufu binds with all three Gli proteins, with different
affinities. This report, in conjunction with recent studies, points out
the importance of Sufu in mouse embryonic development.
The Suppressor of fused gene (Sufu) has been characterized in
Drosophila as a genetic suppressor of the fused (fu) mutant phe-
notype (Preat 1992). Genetic analyses have identified the Ser/Thr
kinase Fu and the Sufu protein as respectively activator and re-
pressor of Hedgehog (Hh) signaling (Preat et al. 1993; Pham et al.
In Drosophila, loss-of-function Sufu mutants are viable, fertile
(Preat 1992), and display only very weak developmental defects in
wings (Ohlmeyer and Kalderon 1998). These Sufu mutations fully
suppress all embryonic, wing, and ovarian defects associated with
loss of function fused alleles, showing that Fused activity is no
longer required in absence of Sufu (Preat et al. 1993). The Dro-
sophila Sufu gene encodes a novel PEST protein (Pham et al.
1995), which can bind directly to Fu and to the transcriptional
regulator Cubitus interruptus (Ci) (Monnier et al. 1998), a Gli
family member (Alexandre et al. 1996; Dominguez et al. 1996).
Sufu should thus be included in the large cytoplasmic complex
containing Ci, Fu, and the kinesin-like protein Costal-2 (Cos-2)
(Robbins et al. 1997; Sisson et al. 1997). This complex has been
involved in the control of Ci activity (see review in Ingham 1998).
In the absence of Hh, Ci is cleaved, presumably through the
targeting of PKA-phosphorylated Ci (Chen et al. 1998) to the
ubiquitin-proteasome pathway by the F-box/WD40-repeat protein
Slimb (Jiang and Struhl 1998), to give a 75-kDa product which
acts as a repressor of Hh target genes (Aza-Blanc et al. 1997). In
cells receiving Hh, the cleavage of Ci is blocked, resulting in the
accumulation of a full-length 155-kDa product, which is turned
into a transcriptional activator (Ingham 1998). Three negative
regulators of the Hh pathway are now identified: Cos-2, cyclic
AMP-dependent protein kinase (PKA), and Sufu; they seem act
through distinct mechanisms to inhibit the activity of the full-
length Ci (Chen et al. 1999; Wang et al. 1999).
All the components of the Hh pathway, except the PKA, were
first identified in Drosophila. Most of them were recovered in
vertebrates with similar roles (see review in Ingham 1998). We
have recently characterized the mouse Sufu gene, which encodes a
484-amino acid protein (MW: 53.5 kDa) with 38% amino acid
identity and 56% similarity with Drosophila Sufu (Delattre et al.
1999). In the course of our analysis in mouse, other groups have
shown that the vertebrate Sufu protein is a conserved negative
regulator of the Shh signaling pathway (Pearse et al. 1999; Ko-
german et al. 1999; Ding et al. 1999). Present in both nuclear and
cytoplasmic compartments, Sufu can sequester Gli1 in the cyto-
plasm (Kogerman et al. 1999; Ding et al. 1999), and also regulate
the nuclear activity of Gli1 by enhancing Gli1’s ability to bind
DNA (Pearse et al. 1999) thereby inhibiting Gli1’s transcriptional
activity (Kogerman et al. 1999; Ding et al. 1999).
In this paper, we report the complete structure of the mouse
Sufu gene and its localization on Chr 19 near the Fgf8 and dactylin
genes (Sidow et al. 1999). We show that the Sufu 5ЈUTR contains
E-boxes that are found in the control regions of numerous tissue-
specific genes. We have also analyzed the expression of Sufu
during embryogenesis, and in the yeast two-hybrid method and an
in vitro assay we show that some differences could exist in pro-
tein:protein interactions between Sufu and the three Gli proteins.
Materials and methods
SSCP and radiation hybrid.
For SSCP analysis, PCR reactions were
performed on 100 ng of genomic DNA with Taq polymerase (Promega)
of each primer. Primers P1: 5Ј-CAAGGTGTCAACATAAGC-
3Ј and P2:5Ј-GCTCATCTCTACAGAGAC-3Ј were used to amplify a 137-
bp fragment of Sufu intron 1. Amplification was done in a PTC200 MJ
Research multicycler with an initial denaturation at 94°C for 2 min, fol-
lowed by 30 cycles of 94°C for 30 s, 55°C for 45 s, and completed by a
final extension step at 72°C for 3 min. PCR products, previously denatur-
Correspondence to: M.-F. Blanchet-Tournier; E-mail: blanchet@
The nucleotide sequence data reported in this paper have been submitted to
GenBank and have assigned the accession number AJ131692.
* These authors made equal contributions to this work
© Springer-Verlag New York Inc. 2000Mammalian Genome 11, 614–621 (2000).