Download The tightly regulated promoter of the xanA gene of

Molecular Microbiology (2007) 63(6), 1577–1587
doi:10.1111/j.1365-2958.2007.05609.x
The tightly regulated promoter of the xanA gene of
Aspergillus nidulans is included in a helitron
Antonietta Cultrone,1†§ Yazmid Reyes Domínguez,1‡§
Christine Drevet,1 Claudio Scazzocchio1,2* and
Rafael Fernández-Martín1,3
1
Institut de Génétique et de Microbiologie, Université
Paris-Sud, Bâtiment 409, UMR 8621 CNRS, 91405
Orsay Cedex, France.
2
Institut Universitaire de France.
3
Laboratorio de Biotecnología Animal, Departamento de
Producción Animal, Facultad de Agronomía, Universidad
de Buenos Aires, Avenida San Martin 4453, 1417
Buenos Aires, Argentina.
Summary
In Aspergillus nidulans the xanA gene codes for a
xanthine a-ketoglutarate-dependent dioxygenase, an
enzyme only present in the fungal kingdom. The 5⬘
region of this gene, including its putative promoter
and the first 54 codons of the open reading frame,
together with the first intron is duplicated in the
genome. This duplication corresponds to a helitron, a
eukaryotic element proposed to transpose replicatively by the rolling circle mechanism. We show that
the regulation of xanA conforms to that of other
genes of the purine degradation pathway, necessitating the specific UaY transcription factor and the AreA
GATA factor. The promoter of the duplicated region is
active ectopically and the difficulty in detecting an
mRNA from the duplicated region is at least partially
due to nonsense-mediated decay. Comparative
genomic data are only consistent with the hypothesis
that the 5⬘ region of xanA pre-existed the helitron
insertion, and that a ‘secondary helitron’ was generated from an insertion 5⬘ to it and a pre-existing 3⬘
consensus sequence within the open reading frame.
It is possible to propose a role of helitrons in promoter shuffling and thus in recruiting new genes into
specific regulatory circuits.
Accepted 10 January, 2007. *For correspondence. E-mail
[email protected];
Tel.
(+33) 1 69155706;
Fax
(+33) 1 69157808. Present addresses: †C.R.A. Istituto Sperimentale
per l’Agrumicoltura, Corso Savoia 190, 95024 Acireale (CT), Italy;
‡
Fungal Genomics Unit, Austrian Research Centers and BOKU
Vienna, Muthgasse 18, 1190 Vienna, Austria. §These two contributed
equally.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd
Introduction
In Aspergillus nidulans genes encoding enzymes involved
in nitrogen source utilization are, as in most saprophytic
organisms, subject to a dual control mechanism (Arst
and Scazzocchio, 1985; Marzluf, 1993). These genes are
induced by a metabolite specific for each pathway and
repressed by favoured nitrogen sources such as ammonium and glutamine (nitrogen metabolite repression).
Among the pathways involved in nitrogen utilization,
the purine degradation has been thoroughly studied
(Darlington et al., 1965; Scazzocchio and Darlington,
1968; Scazzocchio et al., 1982; Scazzocchio, 1994;
Cecchetto et al., 2004). The specific inducer of the transporters and enzymes of this pathway is uric acid (Scazzocchio and Darlington, 1968; Scazzocchio, 1973; 1994;
Sealy Lewis et al., 1978; Scazzocchio et al., 1982; Cecchetto et al., 2004). Induction is mediated by UaY (Scazzocchio et al., 1982; Suarez et al., 1991a,b; Oestreicher
and Scazzocchio, 1995; Oestreicher et al., 1997), a
protein belonging to the 6-cysteine zinc binuclear cluster
family of transcriptional activators. UaY binds to inverted
repeat sites of the form 5′-CGG-X6-CCG-3′ (Suarez
et al., 1995).
Nitrogen metabolite repression is mediated by the
GATA factor AreA (Arst and Cove, 1973; Kudla et al.,
1990). AreA is a, positive acting, transcription factor necessary for efficient transcription of most genes involved in
the utilization of nitrogen sources (Arst and Cove, 1973;
Kudla et al., 1990). Ammonium and glutamine repress
transcription by counteracting AreA activity at a number of
levels including by promoting the degradation of its
cognate mRNA (Morozov et al., 2001).
Xanthine is hydroxylated in A. nidulans, as in other
organisms, by a classical molybdenum-cofactorcontaining xanthine dehydrogenase (Lewis et al., 1978;
Glatigny and Scazzocchio, 1995; xanthine dehydrogenases reviewed by Hille, 1996 and Hille and Nishino, 1995),
which is co-induced with other enzymes of the purine
degradation pathway (Scazzocchio et al., 1982; Glatigny
and Scazzocchio, 1995). Recently, we have established
that in A. nidulans and some other fungi, xanthine can also
be hydroxylated by a uniquely fungal enzyme, a xanthine
a-ketoglutarate-dependent dioxygenase (enzymes of this
group reviewed by Hausinger, 2004). The physical and
kinetic properties of the enzyme have been studied in detail
1578 A. Cultrone et al.
Results
into the sequences corresponding to the first exon at
position +88. These sequences are present at 21 and 40
different sites in A. nidulans genome respectively. The
xanA gene and the psxA sequences show a 97% identity
over a length of 739 bp.
A search in the repeated sequences database Repbase Update (http://www.girinst.org) revealed that the
duplicated sequence corresponds exactly to a nonautonomous helitron element of A. nidulans as described
by Kapitonov and Jurka (Galagan et al., 2005, Supplementary material S6; Kapitonov and Jurka, 2003c; where both
sequences are called Helitron-N1_AN). Both duplicated
sequences are flanked by a 5′ A and a 3′ T, they show 5′ TT
and a 3′ CTTG terminus, considered to be the diagnostic
mark of the A. nidulans helitrons (Galagan et al., 2005,
supplementary material S6). Upstream of both xanA and
psxA, following the 5′ TT putative helitron terminus, we
found 98 bp identical to the 5′ sequence of the putative
autonomous helitron element (Helitron-1_AN) described
by Kapitonov and Jurka (2003d). The transposon character
of the 5′ sequence of the xanA gene, including its promoter,
posits the problem of whether it is subject to the same
regulation pattern as other genes of the purine utilization
pathway as preliminary evidence seemed to indicate
(Sealy Lewis et al., 1978; Cultrone et al., 2005). The
sequence of the putative promoter region suggests this to
be the case. Four putative GATA and two putative canonical UaY binding sites are present, in both the genuine xanA
upstream region and the duplicated sequence. The promoter region of psxA shows seven nucleotide changes but
none affect the putative AreA or the UaY binding sites
(Fig. 1). The distance between the UaY binding sequences
and the 5′ transcription start point is 70 bp (from the
downstream binding site, counting from the last G of the
CCG 3′ sequence) and 85 bp from the upstream binding
site. These distances are quite similar to those observed
for hxA, uaZ and uapA (70, 80 and 79 bp respectively)
(Suarez et al., 1995). We thus proceed to investigate the
regulation of the xanA promoter.
The 5⬘ sequence of xanA is a duplicated helitron
Patterns of xanA expression
This is shown in Fig. 1. The duplication was first detected
in Southern blots of a xanA-deleted open reading frame
(ORF) (Cultrone et al., 2005) and then located between
the positions 73914 and 72729 of contig 1.134, supercontig 11, in chromosome II. The duplication, to be called
psxA (pseudo-xanA), includes the putative promoter
sequence and 144 nucleotides from the coding region
including the first intron of the xanA gene. psxA is interrupted by two defective retroposon sequences already
identified by Kapitonov and Jurka (2003a,b). Copia-2LTR_AN is located 5′ to the sequences corresponding to
the xanA putative promoter (see below) and is 131 nucleotides long; LTR-1_AN is an insertion of 300 nucleotides
The transcript of xanA is about 1.4 kb (Cultrone et al.,
2005). Only this transcript is revealed with a xanA probe
(‘common probe’ see Experimental procedures) which
should reveal also any transcript originating from the psxA
region as it includes sequences common to xanA and
psxA. We investigated the presence of a psxA transcript in
a xanAD (CF067) strain, which carries a deletion of the
entire xanA ORF. A Northern blot of RNA obtained from
mycelia grown under induced conditions was hybridized
with the ‘common probe’. No mRNA hybridizing with this
probe is seen in the deletion strain (not shown, see
below). Thus the work reported in the following two sections concerns exclusively the xanA transcript.
and will be published separately (G. Montero-Morán et al.,
unpublished; M. Li et al., unpublished).
In A. nidulans this novel enzyme is coded by the xanA
gene (Sealy Lewis et al., 1978; Cultrone et al., 2005). We
have shown that this enzyme is responsible for the utilization of xanthine as nitrogen source in Schizosaccharomyces pombe, which lacks xanthine dehydrogenase, and
homologues are present in a number of fungi, including
many yeasts able to utilize oxidized purines as sole nitrogen sources. Many fungi contain homologues of xanA and
we have shown that the one from Neurospora crassa fully
complements a xanA deletion (Cultrone et al., 2005). In
this article we investigate whether this gene is subject to
the same regulatory signals as all other enzymes of the
purine degradation pathway. We observed that the promoter element of xanA is comprised in a helitron. Helitrons
are a recently described group of eukaryotic transposons,
which are supposed to undergo replicative transposition,
based on a rolling-circle mechanism (Kapitonov and Jurka,
2001). Since their first description, based entirely on an in
silico analysis, they have been found in a number of
organisms including the fungi Phaenerochaete chrysosporium (Poulter et al., 2003), Microbotrium violaceum (Hood,
2005) and A. nidulans, but not in the related A. oryzae and
A. fumigatus (Galagan et al., 2005, Supplementary material S6). A number of recent articles describe putative roles
of helitrons in generating gene diversity in maize, including
the generation of pseudogenes and the shuffling of exons
(Buckler et al., 2006 for review). We have reported previously that the 5′ region of the xanA gene on chromosome
VIII is duplicated in chromosome II (Cultrone et al., 2005).
Inspection of recent accessions to the databases (http://
www.girinst.org) classifies both the 5′ region of xanA and
the duplicated sequence as non-autonomous helitron elements. As the duplicated sequences include the entire
putative xanA promoter, it is doubly interesting to investigate the transcriptional regulation of the xanA gene.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587
A fungal helitron includes a regulated promoter 1579
Fig. 1. Alignment of xanA with the psxA
duplication. This figure shows the alignment
of the 5′-terminus of the xanA gene with the
psxA region. The putative termini of the
helitron (Helitron-N1_AN, Kapitonov and
Jurka, 2003c) are in black with white letters.
The A and T at the putative insertion point are
in dark grey. The two insertions corresponding
to retroposon sequences (Kapitonov and
Jurka, 2003a,b) are indicated by triangles.
The corresponding name and the length of
each insertion as found in Repbase Update
(http://www.girinst.org) is indicated. The
common part between the autonomous
Helitron-1_AN (Kapitonov and Jurka, 2003d)
and the two copies of the non-autonomous
helitrons Helitron-N1_AN shown here are
over-lined with broken lines. Putative UaY
binding sites are boxed. Putative AreA binding
sites are underlined. The starts of
transcription and translation of xanA are
indicated by bold letters. In the xanA gene the
exons are overlaid in light grey.
In Fig. 2 we show that hypoxanthine, xanthine and uric
acid induce the expression of xanA in the wild type. In this
and other figures the steady-state levels of xanA are
compared with that of another gene belonging to the purine
utilization pathway, uapA, encoding the specific xanthineurate permease (Gorfinkiel et al., 1993; Oestreicher and
Scazzocchio, 1995; Ravagnani et al., 1997; Cecchetto
et al., 2004). In Fig. 2 we compare induction by the three
oxy-purines in a wild-type strain and in a strain carrying
loss-of-function mutations in both the hxB and the xanA
genes and thus totally unable to metabolize other purines
to uric acid. Only uric acid acts as an inducer in the double
mutant, as shown previously for other genes involved in
this pathway (Scazzocchio and Darlington, 1968) including
hxA (Scazzocchio, 1973; Sealy-Lewis et al., 1978) and
recently for all the purine transporters (Cecchetto et al.,
2004), thus uric acid is presumably the effector of the UaY
transcriptional activator. Figure 3 (first three lanes of both
panels A and B, showing independent experiments) shows
that as uapA and all other genes of this pathway, xanA is
subject to both induction by uric acid and repression by
ammonium.
xanA expression depends on the UaY transcription
factor
Figure 3 shows the expression of xanA in uaY mutants.
The uaY2 mutation is a complete loss-of-function mutation
that results in a chain termination codon at residue 392 of
the protein (Suarez et al., 1995). The uaY2 mutation completely abolishes the inducibility of xanA (Fig. 3A). The
uaYc462 allele, selected as a constitutive mutation (Suarez
et al., 1991a), affects differentially the basal uninduced
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587
1580 A. Cultrone et al.
A
B
Fig. 2. Uric acid is the physiological inducer of the xanA gene. In A we compare the induction by xanthine and uric acid of xanA, of a
wild-type (hxB+xanA+) strain with a double mutant strain (hxB20 xanA1) blocked in the conversion of both hypoxanthine and xanthine to uric
acid. In B we carry out the comparison of inducibility by hypoxanthine and uric acid. The panels represent independent experiments. Below
the Northern blots we show the phosphoimager quantification. U, urea (non-induced conditions); Xa, xanthine; UA, uric acid; Hx,
hypoxanthine. These indicate the metabolites tested for their ability to induce xanA (see in the Experimental procedures for details). The blots
were hybridized with the xanA and the uapA probes, the latter is an independent control of induction and ammonium repression (Gorfinkiel
et al., 1993; Cecchetto et al., 2004). The membranes were then hybridized with a acnA (actin) probe as control of RNA loading.
Quantifications of the xanA and uapA transcripts were corrected according to the corresponding acnA transcript intensity. All values are
expressed in relation to the wild type induced with uric acid, given the arbitrary value of 100 and corresponding to the experiment shown; nd,
not detectable.
level, the induced level and the ammonium repressed level
of different genes under UaY control. This mutation is a
one-base change in codon 222, resulting in a serine to
leucine change (Oestreicher and Scazzocchio, 1995).
xanA expression is constitutive, hyperinducible and partially derepressed in this context (Fig. 3B).
xanA expression depends on the AreA GATA factor
We studied the expression of xanA in areA600 and
areA102 strains grown under different physiological conditions (see Experimental procedures). areA600 is a chain
termination mutation at codon 646 (Kudla et al., 1990)
A
B
resulting in a null phenotype; the latter is a specificity
mutant (Leu683Val) in the DNA binding motif (Kudla et al.,
1990). This mutation results in overexpression of some
genes such as amdS and nil expression of others such as
uapA (Arst and Scazzocchio, 1975; Hynes, 1975; Gorfinkiel et al., 1993; Diallinas et al., 1995; Ravagnani et al.,
1997) and still virtually unchanged expression of others
such as azgA (Cecchetto et al., 2004). Figure 4A shows
that the mutation areA600 strongly impairs but does not
abolish the expression of xanA. We studied the expression
of xanA using both hypoxanthine and uric acid as inducers,
as an areA102 mutant is drastically impaired in the uptake
of uric acid but not of hypoxanthine (Arst and Scazzocchio,
Fig. 3. xanA gene induction depends on uaY
activator.
A. uaY+: wild-type allele, uaY2:
loss-of-function allele.
B. uaY+: wild-type allele; uaYc/d462:
constitutive derepressed allele.
The strains were grown as described in
Experimental procedures. U, urea; UA, uric
acid; UA/NH4+, uric acid and ammonium L (+)
tartrate. Growth conditions: see Experimental
procedures for details, probes and
quantification of xanA and uapA transcripts as
in Fig. 2; nd, not detectable.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587
A fungal helitron includes a regulated promoter 1581
A
B
1975). Figure 4B shows that xanA transcription is equally
induced by uric acid or hypoxanthine in an areA102 background. Nevertheless the level of the induction is almost
half that observed in the areA+ strain. Figure 5 shows the
expression of xanA in an extreme derepressed areA allele,
called for historical reasons xprD1 (Arst and Cove, 1973;
Cohen, 1973). This is a pericentric inversion in chromosome III which results in a deletion of the carboxy-terminus
of the AreA protein and of the 3′ UTR which is responsible
for the areA mRNA instability in the presence of ammonium
as well as a carboxy-terminal domain of the protein which
Fig. 5. Derepression of the xanA transcript. areA+: wild type; xprD1
(areAd): derepressed allele of areA. Growth conditions, probes and
quantification of xanA and uapA transcripts as in Fig. 2.
Fig. 4. xanA transcription depends on the
GATA factor AreA.
A. areA+: wild-type; areA600: null allele;
B. areA+: wild-type; areA102: mutation
conferring altered specificity, see text.
U, urea; UA, uric acid; UA/NH4+, uric acid +
ammonium L (+) tartrate; Hx, hypoxanthine;
Hx/NH4+, hypoxanthine and ammonium L (+)
tartrate, for details, probes and quantification
of xanA and uapA transcripts as in Fig. 2; nd,
not detectable.
is post-translationally responsive to nitrogen metabolite
repression (Arst, 1982; Kudla et al., 1990; Morozov et al.,
2001). For both xanA and uapA, xprD1 results in derepression, but also in an elevated basal level of message in both
the presence and absence of inducer.
Is the promoter of the psxA region active?
As reported above, no transcript corresponding to psxA
could be detected. We investigated whether the psxA
promoter was itself active as expected by its high similarity with the xanA promoter. We thus assembled by double
joint PCR (DJ-PCR Yu et al., 2004) the molecule
psxA5⬘::CDSriboB:: psxA3⬘ (see Experimental procedures and primers list in Table S1, for all the details of the
construction) where the riboB gene is driven by the psxA
promoter. The psxA5⬘::CDSriboB:: psxA3⬘ molecule was
used to transform the riboB2 strain CS2752. We selected
transformants in the absence of riboflavin and in the presence of uric acid. We obtained 35 transformants. Of these
14 were purified and re-tested, 13 are conditional
prototrophs, being able to grow fully in the absence of
riboflavin if uric acid is present in the medium. One transformant is a riboflavin protoproph and arose presumably
by gene conversion at the riboB locus. This shows that not
only the psxA promoter is active, but that is regulated, at
least qualitatively, in the same pattern as the xanA
promoter. This is shown in Fig. 6.
The conditional activity of the psxA promoter could be
explained if psxA was located in a silent region of the
A. nidulans genome. We thus inserted a reporter gene in
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587
1582 A. Cultrone et al.
Fig. 6. The psxA promoter is active and responds to uric acid induction. Strains were grown on minimal media with 5 mM urea as nitrogen
source and appropriate supplements, riboflavine (Rib) or uric acid (UA) are added where indicated. ‘riboB+’ is a pabaA1 strain used as the wild
type throughout this article, CS2752 is a riboB2 strain used as a recipient for the riboB+ gene driven by the psxA promoter. T1 and T2 are two
transformed strains obtained with this construction as described in the text and in Experimental procedures.
the psxA region. We assembled by DJ-PCR (Yu et al.,
2004) the molecule psxA5⬘::riboB:: psxA3⬘::yA (see
Experimental procedures and primer list for all details).
This construction contains 2 kb of upstream and downstream from psxA. At the ATG position of psxA we inserted
the riboB gene with its own putative promoter. At the 3′
extreme of the psxA 2 kb downstream sequence, we
inserted the yA gene, complete with its promoter
sequences (Aramayo and Timberlake, 1990). We used
psxA5⬘::ribo:: psxA3⬘::yA to transform the CS2752 strain
(yAD niaD303 pabaA1 riboB2 argB2). Insertions of riboB
in the psxA region by homologous recombination will not
include the yA+ gene, and thus these events will be recognized as transformants which are yA– and riboB+,
whereas a yA+ phenotype indicates ectopic integration.
These transformants will be obtained only if the riboB
gene is expressed when inserted in the psxA region, while
we expect no transformants showing this phenotype if
riboB is silenced when inserted in psxA. In about 200
transformants, 75 riboB+ colonies, showing complete
complementation of the riboB2 phenotype were also yA–.
Fifteen of these transformants were characterized by
Southern blots (not shown). Three transformants (YR3,
YR9 and YR8) containing a single integration of the riboB
gene into the predicted site of the psxA locus were
identified. Thus the riboB locus is normally expressed
when inserted in the psxA region. However, an alternative
explanation is possible. It could be that in the three transformants analysed a double event had occurred, a riboB
insertion at the psxA locus which may or not be expressed
plus a gene conversion at the riboB resident locus. To
exclude this possibility we amplified the resident riboB
gene from these transformants by PCR using the ribupsf
and ribdowr primers (Table S1) and we use the amplification product to transform a yAD niaDD353 pabaA1 riboB2
argB2 strain. No transformants were obtained on a selective media without riboflavine. In a control experiment,
carried out in parallel, in which a riboB+ allele was amplified by PCR with the ribupsf and ribdowr primers using as
a template a wild-type DNA, 170 transformants per mg
were obtained. This confirms that YR3, YR9 and YR9 still
carry the riboB2 mutation. Transformant YR3 was crossed
to a wild-type strain and among 24 progeny four riboB–
strains were recovered, excluding the possibility that a
gene conversion in the riboB locus could have occurred.
We then investigated the expression of psxA placed in
other regions of the A. nidulans genome. We amplified the
psxA region by PCR using the primers PSXA2160F and
PSXA7280R (Table S1), this region includes psxA, Copia2-LTR_AN, LTR-1_AN sequences, plus 2 kb upstream and
downstream of psxA (Fig. 1). We co-transformed a xanAD,
yA2, biA1, pantoB100 strain together with the panB
plasmid, carrying a pantoB+ gene (see Experimental procedures). About 200 pantoB+ transformants were obtained.
Southern blots on 15 of them showed that four transformants carried ectopically intact copies of the psxA region,
with a number of copies ranging from one to seven. No
psxA transcript was detected using the ‘common probe’
under conditions of induction in any of these transformants
(not shown). The above results suggest that the psxA
promoter is active, but that the mRNA transcribed from the
psxA region is too unstable to be detected. In order to test
this possibility, we constructed a strain carrying both xanAD
and the recently described nmdA1 mutation (NmdA being
the A. nidulans orthologue of the Nmd2/Upf2 proteins),
which is defective in the nonsense decay process
(Morozov et al., 2006). The results are shown in Fig. 7. An
inducible transcript of about 600 nucleotides is present in
the xanAD nmdA1 strain, which suggests that nonsensemediated decay is one of the mechanisms resulting in the
absence or near absence of the psxA transcript.
Discussion
We have shown that transcription of xanA is subject to the
same regulatory signals as all other genes involved in the
purine degradation pathway that have been investigated,
induction by uric acid mediated by UaY, together with a
requirement for an active AreA protein which accounts for
its sensitivity to ammonium repression. Thus xanA shows
the same pattern of regulation as hxA, encoding xanthine
dehydrogenase (Glatigny and Scazzocchio, 1995), uaZ,
encoding uricase (Oestreicher and Scazzocchio, 1993),
uapA, uapC and azgA, encoding three different purine
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587
A fungal helitron includes a regulated promoter 1583
Fig. 7. Nonsense-mediated decay of the psxA transcript.
nmdA+xanA+: wild type; xanAD nmdA+: a strain carrying the xanA
deletion described by Cultrone et al. (2005); xanAD nmdA1: a strain
carrying both a deletion of the xanA gene and a mutation in a gene
necessary for nonsense-mediated decay (Morozov et al., 2006).
The latter two strains are isogenic pabaA1 auxotrophs arising from
the same cross. One other strain with identical (xanAD nmdA1)
relevant genotype but carrying different auxotrophies gave identical
results. Experimental procedures as in the previous figures, except
that the probe used to detect both the xanA and psxA transcript
was a 551 nucleotide probe, amplified from the psxA region and
overlapping 166 nucleotides with xanA.
transporters (Gorfinkiel et al., 1993; Diallinas et al., 1995;
Ravagnani et al., 1997; Cecchetto et al., 2004) and the
UaY-mediated regulation of hxB, which encodes the sul-
phurase necessary for both xanthine dehydrogenase and
nicotinate hydroxylase (Amrani et al., 2000) and responds
additively to the regulatory signals specific to the purine
and the nicotinate utlization pathways (Amrani et al.,
1999).
The nil or highly reduced expression of xanA in both the
uaY and areA loss-of-function mutant tested, could be
due either to a direct interaction of these transcription
factors with the region upstream the xanA ORF and/or to
the exclusion of the inducer, as UaY and AreA also control
the transporters which incorporate purines into the cell
(Gorfinkiel et al., 1993; Diallinas et al., 1995; Cecchetto
et al., 2004). The organization of the UaY and AreA
canonical binding sites in the xanA promoter region
strongly suggest that both transcription factors act directly
eliciting xanA transcription (Figs 1 and 8).
For UaY, the results with the gain-of-function mutant
uaYc469 can only be interpreted as a direct effect on the
xanA promoter, as increased transcription is seen in the
absence of any inducer, similarly, the results for AreA
demonstrate a direct action of the transcription factor in
xanA transcription. The results with xprD1, including an
elevated base level of expression, overinducibility and
derepression of xanA are only consistent with a direct
action of AreA on the xanA (and uapA, which was shown
independently, Ravagnani et al., 1997) promoter. The
specificity mutant areA102 results in strongly decreased
Fig. 8. Origin of the xanA promoter non-autonomous helitron element.
Top diagram. Hypothetical structure of the 5′ region of the xanA gene before the insertion of the helitron element, showing exon 1, intron 1
and part of exon 2. Grey double arrows indicate GATA sites, black double arrows indicate UaY binding sites. xanA promoter structures similar
to this are found in all sequenced Aspergillus genomes with the exception of A. fumigatus; where the xanA gene is absent (see text).
Middle diagram. An autonomous helitron element inserts 434 bp upstream the ATG, leaving the promoter intact. Helitron sequences not to
scale.
Bottom diagram. A deletion of most to the helitron sequences occur leaving as a fossil the presence of the helitron 98 bp identical to the 5′ of
the autonomous helitron element. The TT sequence at the 5′ of the fossil sequence and the serendipitous presence of a CTTG sequence
within exon 2 generate a non-autonomous helitron element, which would be the origin of psxA.
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587
1584 A. Cultrone et al.
binding to A/CGATAR sites and increased binding to
TGATAR sites. In an areA102 there is no transcriptional
induction of either the uapA or uapC genes which contain
only A/CGATAR sites (Gorfinkiel et al., 1993 Ravagnani
et al., 1997; Cecchetto et al., 2004), and the utilization of
uric acid but not of hypoxanthine is strongly impaired (Arst
and Scazzocchio, 1975). The results with the areA102
allele are surprising, but fully consistent with a direct role
of AreA on the xanA promoter. All the AreA binding sites in
the xanA promoter are A/CGATAR sites, and nevertheless
xanA is expressed in an areA102 background. There are
two possible reasons for this result. AreA sites function
cooperatively (Ravagnani et al., 1997; Muro-Pastor et al.,
1999), and in contrast to the well-studied uapA promoter,
where only two adjacent sites are extant, four sites are
present in the xanA promoter. Mutations in either the
uapA and uapC promoters, resulting in the duplication of
the upstream regions containing both AreA and UaY
binding sites suppresses specifically the transcriptional
impairment seen in areA102-carrying strains (Gorfinkiel
et al., 1993; D. Gómez et al., unpubl. results). But it is
more relevant that we have shown that the introduction of
a second UaY binding site, 10 nucleotides upstream from
the one present in the uapC promoter, suppressed the
effect of the areA102 mutation for this promoter (D.
Gomez et al., unpubl. results). This is the natural situation
in the xanA promoter, where two nearby UaY canonical
binding sites, flanked by AreA binding sites are extant.
The above result is consistent with an interaction of AreA
and UaY at the level of the promoter. The second surprising result is that, in an areA102 background, the induction
of xanA is identical when hypoxanthine or uric acid are
used as inducers. That implies that in spite of the nil
induction of uapA and the low basal expression of uapC
(Gorfinkiel et al., 1993; Ravagnani et al., 1997; Cecchetto
et al., 2004 and checked in Fig. 3 of this article for uapA),
resulting in the inability of areA102 strains to use uric acid
as nitrogen source (Arst and Scazzocchio, 1975; Hynes,
1975), enough uric acid enters the cell to induce transcription of xanA.
The sequence that we have called psxA lies in a region
of about 2 kb between autocalled genes AN7871.2 and
AN7872.2. The latest release of the A. nidulans genome
records a short ORF of 144 bp (Autocalled gene
AN11581.3) starting at the ATG of the duplicated psxA
sequence corresponding to the first exon of xanA and
extending a few base pairs in the LTR-N1_AN insertion. A
sequence, nearly identical to the xanA promoter, with the
conservation of all putative UaY and AreA binding sites is
comprised in psxA (see Fig. 1). This sequence is presumably responsible for the inducible transcription of the
600 nucleotide mRNA which we detected in the nmdA1
strain (Fig. 7) and can also drive the transcription of a
riboB gene, resulting in full complementation of riboB2.
The complementation depends on the presence of the
inducer, uric acid, which strongly suggests that the promoter is both active and tightly regulated.
As the psxA region includes a helitron and two degenerated retroposons, we investigated if this region is
heterochromatic. At the qualitative level the answer is
negative, a riboB gene inserted in the psxA region affords
complete complementation of a riboB2 mutation.
In the solo LTR which interrupts the first exon of psxA a
termination codon is present at 55 nucleotides from its 5′,
this would result in translational termination followed by
nonsense-mediated decay (Morozov et al., 2006). The
results in Fig. 7 suggest that nonsense-mediated decay is
at least partly responsible for the absence of a psxA
mRNA and moreover show that the cognate short
message is inducible by uric acid. All these data taken
together argue against the heterochromatin status of the
region surrounding psxA.
Helitron elements have only be detected in silico. They
are proposed to mediate DNA replicative transposition via
a rolling circle mechanism (Kapitonov and Jurka, 2001).
The almost total identity of the 5′ of xanA with psxA is
consistent with a very recent duplication. We have not
found a psxA sequence in any of the Aspergillus databases
(with the obvious exception of A. nidulans), while a xanA
gene is present in all the sequenced Aspergillus species
with the exception of A. fumigatus. The fact that an intron is
perfectly conserved in psxA argues that the duplication did
not involve a cDNA intermediate, which is consistent with
the operation of a helitron element. The position of the
xanA first intron, included in the helitron, is perfectly
conserved in A. oryzae (AO090001000152, http://
www.bio.nite.go.jp/dogan/MicroTop?GENOME_ID=ao),
A. terreus (positions 131452–132659, supercontig 1.7,
http://www.broad.mit.edu/annotation/genome/aspergillus_
terreus/Home.html), A. niger (e_gw1.1. 1797.1, http://
genome.jgi-psf.org/Aspni1/Aspni1.home.html) and A.
flavus (AFST000501, http://www.aspergillusflavus.org)
and outside the Aspergilli in N. crassa (NCU09738.2,
http://www.broad.mit.edu/annotation/genome/neurospora/
Home.htm, other fungal species not checked). The portion
of the xanA ORF which is contained in the helitron is also
clearly conserved. Moreover, in the putative promoters
of the A. oryzae, A. terreus, A. niger and A. flavus
xanA genes, we find within 200 bp upstream of the ATG
that the two UaY binding sites are strictly conserved,
together with a multiplicity of GATA sites (from 3 to 5 with
the position of two sites conserved vis-à-vis the UaY
binding sites).
No helitron elements have been detected in the
A. oryzae genome (Galagan et al., 2005; S6) and no
sequences corresponding to the autonomous element
helitron described by Kapitonov and Jurka (2003c) have
been found by us in the genomes of A. terreus, A. niger or
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587
A fungal helitron includes a regulated promoter 1585
A. flavus. These data strongly argue that the xanA gene is
not a chimeric gene where the 5′ originated from a helitron
inserted upstream of the non-repeated section of the ORF,
but that the 5′ of the xanA gene, including its promoter,
pre-existed the helitron insertion. The extreme 5′ of the
duplicated sequences contains the 5′ border consensus of
a helitron element (A)TT and 98 nucleotides of the 5′ of the
autonomous Helitron-1_AN helitron element defined by
Kapitonov and Jurka (2003c). We propose that the helitron
insertion occurred in the intergenic region upstream of the
xanA promoter, that this was followed by a deletion of most
of the helitron sequences and that a secondary nonautonomous element was generated comprising the original 5′ TT sequences, the first 98 bp of the original helitron
and additional sequences up to a CTTG(T) sequence
pre-existing in the xanA ORF. This sequence of events is
illustrated in Fig. 8. This non-autonomous helitron would
then have replicated and transposed to chromosome II,
driven by the helicase motif-containing helitron ORF of the
autonomous element. As stated before, psxA has suffered
two additional insertions, its promoter does not drive an
active gene, and it is thus an evolutionary dead end. But the
fact that the 5′ of xanA can be subject to replicative
transposition illustrates the potential of helitrons to generate new regulatory patterns. A transposition of the xanA
helitron element in phase with an ORF of an active gene
will result in assimilating this gene into the regulation
pattern of the purine utilization pathway, including its
control by the UaY and AreA transcriptional activators. A
number of recent studies have dealt with the role of
helitrons in the evolution of domesticated plants, essentially maize (reviewed by Buckler et al., 2006). The duplication of the xanA promoter in A. nidulans suggests that
this role may also be extant in the fungal kingdom.
Experimental procedures
Media and growth conditions
The standard media and growth conditions for A. nidulans
were used (Cove, 1966; Scazzocchio et al., 1982). Nitrogen
sources were used at the following concentration: 5 mM urea;
5 mM ammonium L (+) tartrate; hypoxanthine, xanthine and
uric acid: 0.1 g l-1 (around 700 mM). Strains were cultured for
8 h in urea at 37°C and shaken at 150 rpm. Mycelia were
then filtered and cultured for a further 2 h in urea (neutral
conditions), urea + uric acid or xanthine or hypoxanthine
(inducing conditions), urea + uric acid or hypoxanthine +
ammonium (inducing/repressing conditions). Alternatively, filtration was not carried out and the metabolites indicated
above were added after 8 h of growth and cultures continued
for further 2 h.
Strains
The following A. nidulans strains were used in northern
experiments: pabaA1 (CS2498) was used as the wild type;
uaYc/d462, pantoB100 (CS2459); uaY2, pantoB100, yA2,
fpaD43 (CS0836); areA600, pabaA1, biA1, sb43 (CS1318);
areA102, pyroA4, fwA1 (CS1094); hxB20, xanA1, yA2
(CS2473); xprD, yA2, pabaA1; xanAD, pantoB100, pabaA1
(CF067); nmdA1, xanAD, pabaA1.
The following A. nidulans strains were used in transformation experiments: yAD::pyr4, niaDD353, pabaA1, riboB2,
argB2 (CS2752); xanAD, pantoB100 and xanAD, yA2, biA1,
pantoB100. Strain pabaA1 xanAD nmdA1 was constructed by
conventional crossing of a pyrG89 hxA18 xanAD pantoB100
strain with the strain pabaA1 nmdA1 kindly provided by Herb
Arst.
DNA constructions
All the DNA molecules were assembled by DJ-PCR as
described (Yu et al., 2004). All the primers are listed in
Table S1. The psxA5⬘::CDSriboB:: psxA3⬘ molecule was constructed as following: PSXA2001F and PSXA4762R primers
were used to amplify the psxA5⬘ fragment; primers PSXARIBOATG and RIBOSXA4809R were used to amplify the fragment CDSriboB, and primers PSXA4763F and PSXA7280R
were used to amplify psxA3⬘ fragment. Nested primers
PSXA2160F and PSXA7200R were used to amplify the complete assembled molecule.
psxA5⬘::riboB:: psxA3⬘::yA molecule was constructed as
follows: psxA5⬘ and psxA3⬘ were amplified as described previously, the riboB fragment was amplified from the pPL5
plasmid (Oakley et al., 1987), using primers RIBOSXA4809R
and RIBOSXA4712F, primers YASXA7230F and YA3546R
were used to amplify the yA gene from genomic wild-type
DNA. Nested primers PSXA2160F and YA3375 were used to
amplify the complete assembled molecule.
RNA manipulations
RNA was isolated from A. nidulans mycelia with RNA-PLUS
following the supplier’s instruction (Q-BIOgene) and separated on glyoxal agarose gels as described by Sambrook
and Russell (2001). The hybridization technique is
described by Church and Gilbert (1984). [32P]-dCTP-labelled
DNA molecules used as gene-specific probes were prepared using Megaprime DNA labelling system kit following
the supplier’s instructions (Amersham, Little Chalfort,
England). A 1.4 kb fragment including most of the xanA
ORF (from position +43 downstream from the ATG to position 205 downstream from the stop codon) was amplified by
PCR from plasmid pAC001 using XANA1F and XANA4R
(Cultrone et al., 2005) and used to detect the xanA messenger. A 2.7 kb XbaI fragment from plasmid pAN503 (Gorfinkiel et al., 1993) was used to detect the uapA messenger.
A 2.5 kb BamHI-KpnI fragment of plasmid pSF5 (Fidel et al.,
1988) was used to detect the actin messenger as a control
of mRNA loading. The 995 bp long common probe was
amplified from the xanA region with specific primers 5xan1F
and 5xan2R so as to detect also psxA transcription. It overlaps 339 bp with the xanA transcript. The 551 bp long ‘psxA
probe’ (Fig. 7) was amplified with oligonucleotides psxfw
and psxrv. It overlaps all transcribed or putatively transcribed homologous regions of xanA and psxA, respectively, from a position at the start of transcription of the xanA
© 2007 The Authors
Journal compilation © 2007 Blackwell Publishing Ltd, Molecular Microbiology, 63, 1577–1587
1586 A. Cultrone et al.
gene to the 3′ end of the Helitron-N1_AN sequences and
covers the LTR1_AN element. The intensities of RNA bands
were quantified with a 400 A PhosphoImager (Molecular
Dynamics, Sunnyvale, USA). Data were analysed with
ImageQuant.
Acknowledgements
We thank John Clutterbuck and Vladimir Kapitonov for helpful
discussion. This work was supported by the CNRS, the Université Paris-Sud, the Institut Universitaire de France and EU
Contract HPRN-CT-1999-00084 (XONET) which also provided a studentship to A.C. and a fellowship to R.F.-M., who
was later supported by a Marie Curie Fellowship (MCFI2001-01084). Y.R.-D. was supported by a predoctoral studentship of the Ministère de l’Education Supérieure et de la
Recherche and the CONACYT (México).
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Supplementary material
The following supplementary material is available for this
article online:
Table S1. Oligonucleotides used in this work.
This material is available as part of the online article from
http://www.blackwell-synergy.com
Please note: Blackwell Publishing is not responsible for the
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