The spineless-aristapedia and tango bHLH-PAS proteins interact to control antennal and tarsal development in Drosophila
Richard B. Emmons
1
Dianne Duncan
1
Patricia A. Estes
0
Paula Kiefel
1
Jack T. Mosher
0
Margaret Sonnenfeld
0
Mary P. Ward
0
Ian Duncan
1
Stephen T. Crews
)
0
0
Department of Biochemistry and Biophysics, the University of North Carolina at Chapel Hill
,
Chapel Hill, NC 27599-7260
,
USA
1
Department of Biology, Washington University
,
St. Louis, MO
,
USA
SUMMARY
The Drosophila spineless (ss) gene encodes a
basic-helixloop-helix-PAS transcription factor that is required
for proper specification of distal antennal identity,
establishment of the tarsal regions of the legs, and normal
bristle growth. ss is the closest known homolog of the
mammalian aryl hydrocarbon receptor (Ahr), also known
as the dioxin receptor. Dioxin and other aryl hydrocarbons
bind to the PAS domain of Ahr, causing Ahr to translocate
to the nucleus, where it dimerizes with another
bHLHPAS protein, the aryl hydrocarbon receptor nuclear
translocator (Arnt). Ahr:Arnt heterodimers then activate
transcription of target genes that encode enzymes involved
in metabolizing aryl hydrocarbons. In this report, we
present evidence that Ss functions as a heterodimer with
the Drosophila ortholog of Arnt, Tango (Tgo). We show that
the ss and tgo genes have a close functional relationship:
loss-of-function alleles of tgo were recovered as dominant
enhancers of a ss mutation, and tgo-mutant somatic clones
show antennal, leg, and bristle defects almost identical to
those caused by ss- mutations. The results of yeast
twohybrid assays indicate that the Ss and Tgo proteins
interact directly, presumably by forming heterodimers.
Loss-of-function mutations in the Drosophila spineless (ss)
gene cause three distinct phenotypes: transformation of the
distal antenna to distal second leg, deletion of most of the tarsal
region in each leg, and reduction in the size of almost all
bristles (Struhl, 1982; Lindsley and Zimm, 1992; Duncan et
al., 1998). Consistent with these phenotypes, ss is expressed in
the distal portion of the antennal imaginal disc, in the tarsal
regions of the leg discs, and in bristle precursor cells (Duncan
et al., 1998). ss is also expressed in the antennal segment,
gnathal segments, limb primordia, and peripheral nervous
system of the embryo. ss appears to be a primary determinant
of distal antennal identity, as ectopic expression causes
transformation of the distal leg, maxillary palp, and rostral
membrane to antenna. In the legs, ss appears to function in the
establishment of the tarsal primordia, as it is expressed only
Coexpression of Ss and Tgo in Drosophila SL2 cells
causes transcriptional activation of reporters containing
mammalian Ahr:Arnt response elements, indicating that
Ss:Tgo heterodimers are very similar to Ahr:Arnt
heterodimers in DNA-binding specificity and
transcriptional activation ability. During embryogenesis,
Tgo is localized to the nucleus at sites of ss expression. This
localization is lost in a ss null mutant, suggesting that Tgo
requires heterodimerization for translocation to the
nucleus. Ectopic expression of ss causes coincident ectopic
nuclear localization of Tgo, independent of cell type or
developmental stage. This suggests that the interaction of
Ss and Tgo does not require additional signals, unlike the
ligand-dependent interaction of Ahr and Arnt. Despite the
very different biological roles of Ahr and Arnt in insects
and mammals, the molecular mechanisms by which these
proteins function appear to be largely conserved.
early in the tarsal region, and is required for later expression
of the tarsal gene bric brac (Duncan et al., 1998).
The protein encoded by ss is a member of the
basic-helixloop-helix-PAS domain (bHLH-PAS) family of transcription
factors. PAS is named for the founding members of the family,
Period, Aryl hydrocarbon receptor (Ahr) and Single-minded
(Sim) (for review see Crews, 1998). PAS domains can function
in dimerization (Huang et al., 1993), dimerization specificity
(Pongratz et al., 1998), binding of aryl hydrocarbons (Dolwick
et al., 1993) and interaction with non-PAS proteins
(Coumailleau et al., 1995; Gekakis et al., 1995). Recently, it
has become clear that several bHLH-PAS proteins share a
common bHLH-PAS dimerization partner. In mammals, this is
the so-called Aryl hydrocarbon receptor nuclear translocator,
or Arnt. Arnt serves as the dimerization partner for Ahr and
probably at least five other mammalian bHLH-PAS proteins
(Crews 1998). These include the small family of proteins
related to Hypoxia-inducible factor-1a (HIF-1a ) that function
in the physiological and developmental aspects of oxygen
homeostasis (Semenza, 1998), and Sim1 and Sim2, the
mammalian orthologs of Drosophila Sim. The Sim1 gene
controls the formation of several hypothalamic nuclei
(Michaud et al., 1998) and Sim2 has been implicated in Down
Syndrome (Michaud and Fan, 1997). In Drosophila, a homolog
of Arnt, called Tango (Tgo), has been shown to dimerize in
vivo with the bHLH-PAS proteins Sim and Trachealess (Trh)
(So (...truncated)