A toolbox for epitope-tagging and genome-wide location analysis in Candida albicans
© Lavoie et al; licensee BioMed Central Ltd. 2008
Received: 05 September 2008
Accepted: 02 December 2008
Published: 02 December 2008
Candida albicans is a diploid pathogenic fungus not yet amenable to routine genetic investigations. Understanding aspects of the regulation of its biological functions and the assembly of its protein complexes would lead to further insight into the biology of this common disease-causing microbial agent.
We have developed a toolbox allowing in vivo protein tagging by PCR-mediated homologous recombination with TAP, HA and MYC tags. The transformation cassettes were designed to accommodate a common set of integration primers. The tagged proteins can be used to perform tandem affinity purification (TAP) or chromatin immunoprecipitation coupled with microarray analysis (ChIP-CHIP). Tandem affinity purification of C. albicans Nop1 revealed the high conservation of the small processome composition in yeasts. Data obtained with in vivo TAP-tagged Tbf1, Cbf1 and Mcm1 recapitulates previously published genome-wide location profiling by ChIP-CHIP. We also designed a new reporter system for in vivo analysis of transcriptional activity of gene loci in C. albicans.
This toolbox provides a basic setup to perform purification of protein complexes and increase the number of annotated transcriptional regulators and genetic circuits in C. albicans.
Candida albicans is an important human fungal pathogen because of its clinical significance as well as its use as an experimental model for scientific investigation . This opportunistic pathogen is a natural component of the human skin, gastrointestinal and genitourinary flora, but it can sporadically cause a variety of infections. Although many Candida infections are not life-threatening (oral thrush and vaginal candidiasis, for example), immunosuppressed patients can be subjected to potentially lethal systemic infections, and therefore Candida infections are a major public health concern [2, 3]. C. albicans can also colonize various biomaterials, and readily forms dense, complex biofilms that are resistant to most antifungal agents. Because of the challenges of drug resistance  and the eukaryotic nature of C. albicans that makes it similar to its human host, extensive efforts are underway to identify new drug targets for therapeutic intervention; many of these take advantage of tools from the genomic era [5–7]. Because of some of its unique biological features, C. albicans is also becoming attractive to studies of more fundamental aspects of genome maintenance, regulatory biology and morphogenesis [8–10].
The diploid nature and the absence of a complete sexual cycle in C. albicans reduces the ease with which genetic manipulations can be achieved . Therefore, biochemical, cell biological and genomic analyses of gene products provide alternate strategies to improve our understanding of this pathogen. In particular, obtaining a better insight into how specific protein complexes assemble in C. albicans would help define targets for drug development. For example, the large-scale definition of protein complexes by tandem affinity purification (TAP), as it was previously done in S. cerevisiae [12, 13], should reveal the architecture of biochemical networks and protein machines specific to C. albicans. This approach would also give insight into the evolution of protein complexes in ascomycetes. As well, the availability of tools allowing comprehensive functional analysis of transcription factors (TFs) on a genome-wide scale could enhance cellular studies. Genome-wide location analysis, or ChIP-CHIP analysis (chromatin immunoprecipitation (ChIP) followed by DNA microarrays (CHIP)) allows the identification of direct targets of a defined TF on a genomic scale . This approach has been comprehensively applied to the model budding yeast S. cerevisiae in order to map its transcriptional regulatory network [15, 16]. While first developed in S. cerevisiae, ChIP-CHIP has since been applied to other organisms including C. albicans [17, 18]. In addition, C. albicans has recently been established as an interesting model organism for the study of the evolution of transcriptional regulatory networks. In fact, it appears that C. albicans differs significantly in its mode of gene regulation from the well-characterized S. cerevisiae transcriptional circuits. It has recently served as a comparative system for studying the evolution of the mating type control circuit and the evolution of ribosomal protein (RP) regulation [19, 20]. Furthermore, changes occuring in the activity of TFs seem to account for part of acquired drug resistance in C. albicans [21, 22]. Moreover, several TFs play a critical role in the C. albicans morphological transitions and in biofilm formation [10, 23]. Therefore, a detailed understanding of the transcriptional regulation mechanisms in C. albicans would be valuable for basic research purposes as well as for improving our understanding of drug resistance and for aiding in the development of new antifungals.
Here we report the construction of a new set of PCR-based epitope-tagging vectors for C. albicans that is successfully applied to perform a biochemical characterization of the small processome subunit via Nop1 tandem affinity purification as well as genome-wide location analysis of the model TFs Tbf1, Cbf1 and Mcm1.
Results and discussion
A set of PCR cassettes for protein tagging
Tandem affinity purification of Nop1
Mass spectrometry results after Nop1-TAP tandem affinity purification
Number of peptides*
Cytosolic large ribosomal subunit
structural constituent of ribosome
cytosolic large ribosomal subunit
structural constituent of ribosome
cytosolic large ribosomal subunit
structural constituent of ribosome
ChIP-CHIP analysis of in vivotagged transcription factors
Mcm1-TAP targets in ChIP-CHIP analysis
Carbamoyl phosphate synthetase
Zinc cluster TF
MADS box TF
ATP-dependent DNA helicase
ATP-dependent DNA helicase
ATP-dependent DNA helicase
ATP-dependent DNA helicase
ATP-dependent DNA helicase
Mcm complex loading
ATPase helicase clamp loader
Origin recognition complex (ORC)
ATPase for DNA binding
However, our data diverges from the other Mcm1 ChIP-CHIP publications at some points. First, our results show Mcm1 binding in the promoters of almost all members of the pre-replication complex (Pre-RC) (Mcm helicases genes MCM2/3/4/5 and 7; pvalue = 1.87E-06; Table 2) while the Tuch et al. (2008) data only uncovered two members of this complex (MCM2 and 7). In addition, we detected reproducible Mcm1 binding at two origin-recognition-complex components (ORC1 and 3) and the loading factor for the Mcm complex, CDC6 (Table 2; see Additional file 1). Second, binding of the promoters of the cyclin genes CLN3, HGC1 and CLB3 by Mcm1 was not detected in our experimental setup (see Additional file 1 and 2). In total, 530 Mcm1 targets are uniquely found in the Tuch et al. gene list (Fig. 3D). These major differences are likely due to variations between the two independently conducted studies. Tuch and collaborators 1) treated their cells with pheromone, 2) used a polyclonal rabbit antiserum raised against an Mcm1 peptide, 3) used signal ratios of cy5 labelled IP versus cy3 labelled whole-cell extract prior to performing the IP and 4) used tiling arrays to determine the promoter targets of Mcm1. Our procedure is somewhat different: we 1) used YPD grown cells, 2) used IgG beads-proteinA interaction to enrich target regions, 3) compared each experimental IP to a mock IP performed in untagged cells and 4) used full genome arrays with smaller coverage (about two probes/intergenic region). All these experimental changes could affect to different degrees the final result of a ChIP-CHIP. First, treating cells with pheromone might dramatically alter Mcm1 binding at some point in the cell cycle and pheromone response elements and deplete it at others (such as the Mcm complex genes). Second, polyclonal anti-Mcm1 antibodies can have cross-specificities that would not be corrected for by using the whole-cell extract as a control instead of a mock IP or an IP performed with a preimmune serum. Third, the different resolutions of the two studies could account for some overlooked targets in our experiments.
We also performed Mcm1-TAP ChIP-CHIP in the yeast to hyphal transition triggered by serum at 37°C. Mcm1 was recruited to a limited set of genes (36) under these conditions (Fig. 3D). Noticeably, it was enriched in the promoters of ALS3, HWP1 and ECE1 after hyphal induction (see Additional files 1 and 2).
Thus, our genome-wide location data showed a significant consistency with previously published results on the three TFs. This suggests that our chromosomally tagged alleles are functional and that the use of a TAP-IgG pull-down protocol is applicable for ChIP-CHIP as previously reported in S. cerevisiae 26 and that this protocol is comparable to a ChIP method based on a anti-HA (for Cbf1 and Tbf1) or rabbit polyclonal (for Mcm1) antibody IP.
In vivobeta-galactosidase reporter assays
Here, we have presented a new set of tools for functional characterization of C. albicans gene products by biochemical methods. We believe that the availability of such tools will greatly help future understanding of the biology of this important human pathogen.
Construction of chromosomal tagging PCR cassettes
Primers used for in this study
CCGGGGAACAGAAGCTTATATCCGAAGAAGACCTCGGAGAGCAAAAGCTCATTTCAGAAGAGGATCTAGGCGAACAGAAACTAATCTCGGAGGAGGACCTCGGTGAACAAAAGCTTATCTCTGAGGAAGATCTTGGCGAGCAGAAGCTCATATCAGAGGAAGACCTAGGG TAG GG
AGAAGAAAGAATTAAACCATTGGAACAATTGACCTTGGAACCTTATGAAAGAGACCATTGTATTGTTGTTGGTAGATACATGAGAAGCGGAATAAAGAAAGGT CGA CGG ATC CCC GGG TT
TAGAGTTGATTAGACCTTATTGTTTTATTTTTCATTTTATTATATATGTCGTATCTTACAGTTCTTTAAATACCAGTGTTTCCAAAATTTTCATTCATTCTCG ATG AAT TCG AGC TCG TT
TGAACAAGCTGTTAGTGAATTGAGTGCTTCAAATGAGAAATTGAAACATGAATTAGAATCAGCTTATCGTGAAATCGAACAATTGAAGAGAGGGAAGAAAGGT CGA CGG ATC CCC GGG TT
TAACATAATTTCAAATACCGAGTAGGAATACACAACCCCAACATCTAACCAGCCATACATTTACATATTTATAATTACATATTAAAACATCGTCAAATTAATCG ATG AAT TCG AGC TCG TT
AACAACAAGAGAAAGAACAACCGGATCAGCAACAACCAGATCAACAACACCCAGATCGACAACAACAAGAGCAGATCCAACAACCAGAAAATCTGGATAAAGGT CGA CGG ATC CCC GGG TT
ATCAACTATTGTGATCCTGCTTAAGTTAGCTTGAACAATTATTCAAATCAATTTACACCTTAAAGATAGATTAATTAACAATACAAATATAATGCTACATGTCG ATG AAT TCG AGC TCG TT
GGT CGA CGG ATC CCC GGG TTA TAC CCA TAC GAT GTT CCT GAC
GGT CGA CGG ATC CCC GGG TTA GAA CAG AAG CTT ATA TCC GAA
TCG ATG AAT TCG AGC TCG TT
The beta-galactosidase reporter was constructed by subcloning a PstI-MluI fragment corresponding to the Streptococcus thermophilus lacZ ORF from plasmid placpoly  between the PstI and AscI sites of plasmid pFA-XFP-URA3 .
PCR reactions were performed in 50-μl volumes containing 1 ng of plasmid template with the Expand Long Template polymerase following manufacturer's instructions (Roche, Germany). PCR parameters were 1 cycle at 94°C, 5 min followed by 35 cycles at 94°C, 30 sec; 58°C, 1 min; 68°C, 3 min.
Construction of C. albicansepitope-tagged strains
Cell growth, transformation, and DNA preparation were carried out using standard procedures [26, 36]. Transformants were selected on either of -Ura, -His or -Arg selective plates. Correct integration was verified by PCR, sequencing and finally Western blotting (Fig. 1A). Rate of correct integration was comparable to a previous study using similar condiitons and was in the range of 40–80% depending on the gene locus considered . We used this strategy to C-terminally fuse Tbf1, Cbf1 and Mcm1 with a TAP tag and to introduce an HA or a MYC tag to the C-terminus of Tbf1 and Cbf1.
Whole cell extracts were obtained by boiling cells at 2 ODs in loading buffer with 100 mM DTT for 10 minutes. Proteins were then separated on a 10% SDS-PAGE gel and transferred to a PVDF membrane (Millipore). Antibodies were prepared in TBS-0.05% Tween20 5% skim milk powder. A rabbit polyclonal antibody directed against the TAP-tag (Open Biosystems) was used at 0.5 μg/ml while monoclonal antibodies anti-HA (12CA5) and anti-Myc (9E10) were used at 5 μg/ml. HRP-conjugated goat anti-rabbit and anti-mouse secondary antibodies (Santa Cruz) were used at 0.04 μg/ml. The HRP signal was revealed with Immobilon™ HRP substrate (Millipore).
TAP purifications and mass spectrometry
Tandem affinity purifications were performed as described http://depts.washington.edu/yeastrc/pages/plasmids.html and then precipitated with Trichloroacetic acid (TCA). For mass spectrometry analysis of the TAP purified proteins, the digestion was performed using Trypsin (Promega) in 50 mM ammonium bicarbonate for 4 hours at 37°C and dried down. One quarter of the TCA precipitate was loaded on a 10% SDS-PAGE gel. The gel was stained with SYPRO Ruby according to manufacturer's instructions (Invitrogen). Excised protein bands were processed as described . Samples were resolubilized in 5% acetonitrile 0.2% formic acid and analyzed on a Eksigent nanoLC system coupled to a Thermo LTQ-Orbitrap MS instrument with a home-made C18 pre-column (5 mm × 300 um) and an analytical column (10 cm × mm × 300 m i.d. Jupiter 3 m C18). Sample injection was 10 ul. The digest was first loaded on the pre-column at a flow rate of 4 ul/min and subsequently eluted onto the analytical column using a gradient from 10% to 60% aqueous acetonitrile (0.2% formic acid) over 56 min at 600 nl/min. Database searches were performed against a non-redundant fungal database using Mascot version 2.1 (Matrix Science).
ChIP-CHIP analysis of Tbf1, Cbf1 and Mcm1by TAP-IgG pull-down in C. albicans
Chromatin immunoprecipitation (ChIP) experiments were performed with chromosomally tagged Tbf1-TAP, Cbf1-TAP and Mcm1-TAP as described . Cells were grown to an optical density at 600 nm of 0.6 in 50 ml of YPD or YPD with 10% FBS for Mcm1-TAP in hyphal state. We followed the ChIP protocol available at http://www.ircm.qc.ca/microsites/francoisrobert/en/317.html with the following exceptions: chromatin was sonicated to an average 300 bp, and 700 μl of whole-cell extract (WCE) were incubated with IgG-Sepharose beads (GE Healthcare). Tagged ChIPs were labeled with Cy5 dye and untagged (mock) ChIPs were labeled with Cy3 dye and were then co-hybridized to our full-genome arrays.
Candida albicansfull-genome arrays, hybridization, scanning and normalization
Our C. albicans full-genome microarrays contain single spots of 5,423 intergenic 70-mer oligonucleotide probes combined with 6,394 intragenic 70-mer oligonucleotide probes already in use in our C. albicans ORF microarray [20, 38]. We designed the 5,423 probes that correspond to the promoter regions of most of the genes in the C. albicans Genome Assembly 21 by using the same algorithm that was successfully applied to the development of our C. albicans ORF oligonucleotide arrays with added weight provided for regions of high homology among Candida species. Our lab has developed full-genome (ORF and intergenic) arrays for use in location profiling experiments and the DNA microarrays were processed and analysed as previously described .
Nucleotide sequence accession numbers
The sequences of plasmids pFA-TAP-URA3, pFA-TAP-HIS1, pFA-TAP-ARG4, pFA-HA-URA3, pFA-HA-HIS1, pFA-HA-ARG4, pFA-MYC-URA3, pFA-MYC-HIS1, pFA-MYC-ARG4 and pFA-LacZ-URA3 have been submitted to GenBank and have been assigned the following accession numbers: FJ160456, FJ160457, FJ160458, FJ160464, FJ160462, FJ160463, FJ160460, FJ160461, FJ160459 and FJ160455, respectively.
Thanks to Eric Bonneil and Pierre Thibeault for expert help in mass spectrometry processing of samples and data analysis. Thanks to E. O'Shea, J. Weissman and J. Wendland for providing plasmid constructs. This work was supported by a grant from Canadian Institute for Health Research (CIHR) to MW and AN (MOP-84341). HL was supported by NCIC grant 17134, AS was supported by a CIHR postdoctoral fellowship and CA by an Alexander Graham Bell CGS-NSERC scholarship. This is NRC manuscript #49576.
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