Refining the pH response in A spergillus nidulans: a modulatory triad involving PacX, a novel zinc binuclear cluster protein

Summary The A spergillus nidulans PacC transcription factor mediates gene regulation in response to alkaline ambient pH which, signalled by the Pal pathway, results in the processing of PacC72 to PacC27 via PacC53. Here we investigate two levels at which the pH regulatory system is transcriptionally moderated by pH and identify and characterise a new component of the pH regulatory machinery, PacX. Transcript level analysis and overexpression studies demonstrate that repression of acid‐expressed pal F, specifying the Pal pathway arrestin, probably by PacC27 and/or PacC53, prevents an escalating alkaline pH response. Transcript analyses using a reporter and constitutively expressed pac C  trans‐alleles show that pac C preferential alkaline‐expression results from derepression by depletion of the acid‐prevalent PacC72 form. We additionally show that pac C repression requires PacX. pac X mutations suppress PacC processing recalcitrant mutations, in part, through derepressed PacC levels resulting in traces of PacC27 formed by pH‐independent proteolysis. pac X was cloned by impala transposon mutagenesis. PacX, with homologues within the Leotiomyceta, has an unusual structure with an amino‐terminal coiled‐coil and a carboxy‐terminal zinc binuclear cluster. pacX mutations indicate the importance of these regions. One mutation, an unprecedented finding in A . nidulans genetics, resulted from an insertion of an endogenous Fot1‐like transposon.


Contents
. List of primers used in this work.

Fig. S1 (A). Epifluorescence microscopy of preferentially nuclear localized GFP-tagged PacC 27 reveals that
PacC 27 levels are greatly reduced when co-expressed with PacC 72 in a diploid strain. The pacC c 700 GFP tagged pacC constitutive allele (GFP::PacC5-250) corresponding to PacC 27 is integrated at the pacC resident locus and expressed from the pacC promoter. The pacC -6309 null mutation ] reduced the fluorescence intensity of GFP::PacC5-250 to ~50% (ia or b, iiia and iv), which would be the expected dilution effect. The pacC +/-209 processing recalcitrant mutation (specifying PacC 72 ) had a greater effect resulting in a reduction of fluorescence intensity to less than 25% of that found in the diploid pacC c 700 strain (Ia or b, iia and iv) or a haploid pacC c 700 strain (results not shown). These results are consistent with our proposal that PacC 72 is a repressor of pacC expression. Enhanced images for diploids pacC c 700/pacC +/-209 and pacC c 700/pacC -6309 (iib and iiib, respectively) were obtained by modifying brightness and contrast to improve visualisation of the GFP::PacC5-250 protein to a level comparable to that in the pacC c 700 homozygous diploid (unenhanced, ia and b). Fluorescence images of haploid and diploid pacC c 700 cells (Fig. S1A) were taken using a DMI6000b microscope equipped with a 63x objective 1.4 N.A. and an Orca-ERII camera. Strains were cultivated in WMM [watch minimal medium (Peñalva, 2005)], containing the required supplements and glucose 1% and 5 mM ammonium tartrate as carbon and nitrogen sources, respectively. Cells were grown at 25 o C for 18 hours before epifluorescence analyses. To compare nuclear fluorescence intensities, all images were obtained in the same conditions with an exposure time of 2 seconds. Nuclear fluorescence intensities were estimated using Metamorph 6.3r software by measuring selected areas of 80 pixels into 10 nuclei per strain. The mean of accumulative pixel intensities for areas of 80 pixels are shown in the chart (n=10 nuclei). The diploid strains are pacC c 700/pacC c 700, pabaA1 yA2 glrA1 pacC c 700 / lysA2 pacC c 700 (J2000); pacC c 700/pacC +/-209, lysA2 pacC c 700 / biA1 sB3 pacC +/-209 (J1977) and pacC c 700/pacC -6309, pabaA1 yA2 glrA1 pacC c 700 / pacC -6309 (pacC c 63) pantoB100 (J1999).

Fig. S2
Electrophoretic Mobility Shift Assays (EMSAs) showing the effects of pacX1 on complexes formed by PacC in pacC + (i), pal -(ii), pacC +/-20205 (iii), and pacC c 234 backgrounds. PacC was detected using a 32 Plabelled oligonucleotide containing ipnA2 . The positions of the PacC forms are indicated. Each binding reaction contained 5 g of crude protein. Strains were grown from conidial inocula of 1 -2 x 10 6 ml -1 in 200 ml of appropriately supplemented Aspergillus complete media (Cove, 1966), containing 3% ( w / v ) sucrose, 20 mM MES pH 6.5, at 37C for 16 hours with shaking at 120 rev min -1 . Mycelia were harvested on sterile Miracloth (Calbiochem) and samples of mycelia were cut into small strips of approximately 300 mg and frozen in liquid nitrogen. Cell lysates were prepared essentially as described by Peñas et al. (2007) except that the samples were centrifuged immediately after cell disruption. Double stranded oligonucleotide probes were prepared and binding reactions and electrophoresis were carried out following Perez-Esteban et al. (1993) and Espeso and Peñalva (1994). The conservation scale: 1 2 3 4 5 6 7 8 9

Variable
Average Conserved e -An exposed residue according to the neural-network algorithm. b -A buried residue according to the neural-network algorithm. f -A predicted functional residue (highly conserved and exposed). s -A predicted structural residue (highly conserved and buried). X -Insufficient data -the calculation for this site was performed on less than 10% of the sequences.   and would place the putative C-terminal exon beyond the inverted repeat. In this case a -2 frame shift has resulted in a truncated protein at residue 595, automated intron recognition process "fabricating", in fact, two introns.
It is striking that among the hundreds of loss-of-function mutations obtained by classical genetics and now sequenced, pacX18 is the first and only one due to the insertion of an endogenous transposon.
Attempts to select specifically such events were never successful in A. nidulans. This contrasts with the ready mobility of heterologous Fot1, impala and Minos transposons inserted in the genome of A.
nidulans (Li Destri Nicosia et al., 2001;Carr et al., 2010;Evangelinos et al., 2015) An explanation for the extreme rarity of endogenous transposition in A. nidulans may derive from the absence of active transposase in the cell. Caddick and co-workers have carried out global RNAseq of the A. nidulans transcriptome under 5 different conditions (Sibthorp et al., 2013). These data are accessible on line (http://www.aspgd.org/, JBrowse). A search for each one of the paralogues indicated above showed virtually (and in some cases absolutely) no transcripts. Some transcripts are seen for AN9604, the most divergent of the homologues, which cannot encode an active transposase. In contrast with this data, a signal for a transcript of the correct length was detected in early experiments by conventional Northern blots (Li Destri Nicosia et al., 2001) in culture conditions similar to one of those used by (Sibthorp et al., 2013). The probe used was derived from the only A. nidulans Fot-like element known at the time, whose position within a sequenced cosmid, F2P08, (Kupfer et al., 1997) identifies it unequivocally as AN0975. The high similarity between all Fot1-like paralogues implies that cumulative transcription of all Fot-1 like elements in the genome were detected in this experiment.
The different homologues are spread throughout the genome, are not methylated (Li Destri Nicosia et al., 2001), and active transcription of neighbouring genes show that they are not located in silent regions of the genome. It would be interesting to know if the absence of transcripts derives from very infrequent transcription or from selective degradation of the cognate RNAs though the dicer/argonaute system (Hammond et al., 2008a and b). The strain in which pacX18 was isolated carries an autonomous copy of an impala element from F. oxysporum, belonging to the Tc1/mariner family. While the cognate transposase cannot mobilise a Fot1-like element, the possibility remains that its presence may affect the regulation of its expression. In addition, possible effects of the pacC +/-20205 mutation and the stressful alkaline growth conditions under which the pacX18 mutation was selected may ,have contributed to the activation of the AN 0826 Fot-1-like element Transposons of the Fot1/Pogo family operate through a mechanism of cut and paste, rather than the copy and paste characteristic of retroposons, and among the eukaryotic DNA transposons, of helitrons.
We thus tested the presence of the resident copy of AN0826 in the strain where transposition to pacX had occurred. Fig S7 shows clearly that AN0826 is conserved at its locus in the strain carrying the pacX18 mutation, thus demonstrating a copy/paste mechanism, however, the precise insertion of the inverted repeats and the TA duplication implies that this event was catalysed by the specific transposase.
That copy and paste expansion of eukaryotic type II transposons must occur is indicated by the multiple copies of these elements in genomes where they are present (Daboussi and Capy, 2003;Dufresne et al., 2011, specifically for Fot1 elements), and in the specific case of the Fot1-like transposons of A. nidulans by their polymorphism in different strains of this species (Li Destri Nicosia et al., 2001). A class II "cut and paste" DNA transposon can replicate by transposing from a position behind a replication fork to an un-replicated sequence or by double-stranded gap repair at the donor site by an intact sister chromatid (Rubin and Levy, 1997;Bessereau, 2006;Wicker et al., 2007;Izsvak et al., 2009;Skipper et al., 2013 and refs therein). Both mechanisms imply a temporal coupling of replicative transposition and the S phase of the cell cycle. AN0826 and pacX are both on chromosome VIII at ~180 kb from each other, which is not incompatible with these genes being within the same replicon. This proximity may have facilitated a local transposition event [local hopping, (Tower et al., 1993;Timakov et al., 2002;Carlson and Largaespada, 2005;Muñoz-Lopez and García-Pérez, 2010)] and allowed us to detect this very rare occurrence.

MPR V R VS S SQ N CH EK EG R L L LAV Q AIK K K EI T S I RE A AR R F N VPE ST LRT R L RGT T N R AE SRA -N GH KLT EI EEE V LKQ W Ta nMPP K A SI P SK S QV EQ EG R I L LAI E AIQ K G QI T S I RE A AR V Y D VAR TT LQA R L SGR V F A KN MTN -A RQ KLS NN EEE S LVK W Fo tMPV Y S AD D LE N ----------AI A DFK N G --V S L KT A AK K N G LPP ST LRG R L TGA Q S R QV ARQ -E QL RLT TD QED D LER W Af tMPK S S KI -------N ES Y L L EAC E AAQ A Q KK P N I SK I AR E Y G VPY AT LRD R V KKH V H P RL ANK PV NR ALK GY QEE A LIQ W AN 08 26 80 ILS L D LR G AA P TK AH VR E M A NIL L AKR G S TP I Q T VG Q KW V Y N YTQ RH PE---LES R L S RQ YDC QR AK QEN PK VIQ A WFN T Ta n80 ILS L D KR G AS P RP LD IR D M A NLI I SKR G Y ST V E Q VG I NW A Y S FVK RH ES---LRT R F A RR LNY QR AK MED PE VIK D WFK R Fo t68 ILR Q E KL G HA P TH AQ VR T I V RSV L ARH G D H--A P LG R KW T T R FVE RH PA---LKT K L G RR TDW ER VN AAT PA NIK R LFD V Af t74 IVC M R DR N MP V TP KL LE E Y A NQA L RRA G E S--R Q VS K MW A Y R FEK RL PEH L N LGP A K Q KI KES KR IQ AED AG LLT H WYN Q AN 08 26 57 VRA T I EQ Y GI L PD DI YN F D E TGF A MGL C A HQ K V I TK S E-S C G RRP VL QPG N R -EW V T A IE SIS AS GW AL-PI LIF K GKQ -Ta n57 VQE V I QE Y GI S SD DI YN F D E TGF A MGM I A TY K V V TS S Q-R A G RPS LV QPG N R -EW V T A IE CIR SN GE VLP ST LIF K GKT -Fo t1
1   (Nyyssönen et al., 1996), Aft1 from A. fumigatus (Hey et al., 2008) and Fot1 from Fusarium oxysporum (Daboussi et al., 1992). Alignment carried out with MAFT, G-INS-i, visualised with Box-Shade (http://www.ch.embnet.org/software/BOX_form.html). Nucleotide alignment: inverted repeat (without the TA duplication) of AN0826 at its genomic locus and within the pacX gene in the pacX18 mutation. Inverted repeats detected with einverted:

M M
(http://emboss.bioinformatics.nl/cgibin/emboss/einverted). Right panel, PCR amplification with primers external to the AN0826 inverted repeats at its chromosomal location. M, molecular size markers. pacX + is the parent strain XC34 (yAΔ::Ncpyr4 pabaA1 pacC C 202 pacC +/-20205 pantoB100 niaDΔ::impala::yA + ) for the pacX18 derivative (strain A869). The expected size of the amplified sequence would be 2118 bp in the pacX + strain and 456 bp if the pacX18 mutation resulted from a Cut and Paste mechanism. In the event amplified bands in both strains are of identical size, showing a Copy and Paste mechanism to be operating. Primers used for amplification, AN0826F and AN0826R.