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The Three Clades of the Telomere-Associated TLO Gene Family of Candida albicans Have Different Splicing, Localization, and Expression Features

ABSTRACT Candida albicans grows within a wide range of host niches, and this adaptability enhances its success as a commensal and as a pathogen. The telomere-associated TLO gene family underwent a recent expansion from one or two copies in other CUG clade members to 14 expressed copies in C. albicans. This correlates with increased virulence and clinical prevalence relative to those of other Candida clade species. The 14 expressed TLO gene family members have a conserved Med2 domain at the N terminus, suggesting a role in general transcription. The C-terminal half is more divergent, distinguishing three clades: clade α and clade β have no introns and encode proteins that localize primarily to the nucleus; clade γ sometimes undergoes splicing, and the gene products localize within the mitochondria as well as the nuclei. Additionally, TLOα genes are generally expressed at much higher levels than are TLOγ genes. We propose that expansion of the TLO gene family and the predicted role of Tlo proteins in transcription regulation provide C. albicans with the ability to adapt rapidly to the broad range of different environmental niches within the human host.

T he ability to respond to stress is critical for survival, especially in organisms that reside in a dynamic environment such as the varied niches within a host organism. Candida species are the most prevalent fungal pathogens of humans, causing mucosal infections of the mouth, genitourinary tract, and skin, as well as life-threatening bloodstream infections. Candida albicans resides as a harmless commensal in the human gastrointestinal tract, yet it causes Ͼ50% of all systemic fungal infections. A number of traits, including the ability to switch to hyphal growth and to undergo phenotypic switches, likely contribute to the higher virulence of C. albicans than of other Candida species (35).
Genetic responses to growth in new, stressful environments include changes in gene copy number, which provide a rapid mechanism to adapt available genetic material to cope with altered conditions (7,10,15,33,42). Telomeric regions of the genome exhibit the most variation, and variation accumulates rapidly in these regions (7,9,12). For example, in Saccharomyces cerevisiae the SUC, MAL, and MEL families have expanded to different extents in strains bred to ferment different carbon sources (sucrose, maltose, and melibiose, respectively) (3,7,10,43); the subtelomeric family of FLO genes, which encode the ability to adhere to different cellular and abiotic surfaces, have expanded in some fermentation and clinical isolates (22,43,45).
The telomere-associated (TLO) gene family in C. albicans is a remarkable example of gene family expansion near the telomeres. The TLO gene family is the gene family that has expanded most in C. albicans relative to the less pathogenic Candida species (6). C. albicans has 14 annotated TLO genes, compared to two TLO genes in the closely related oral pathogen Candida dubliniensis and a single TLO gene in most other Candida species (6,44).
All but one of the TLO genes are located within 12 kb of a telomere and are often the most terminal predicted open reading frame (ORF) of each chromosome arm. A single TLO is found at an internal locus on chromosome 1 (Chr1), although whether this TLO is expressed is not known (44). In S. cerevisiae, the most terminal ORFs contain the subtelomeric gene families Y= and X, which encode RNA helicases and transcriptional silencers, respectively (26,27), and are actively transcribed (47).
The first TLO gene to be identified was named CTA2 and was isolated in an S. cerevisiae one-hybrid screen for C. albicans transactivating proteins (24). This implies that Tlo proteins bind (directly or indirectly) to DNA and have the potential to regulate transcription. Indeed, the predicted Tlo proteins all include a domain with high similarity to Med2, a component of the Mediator complex, which regulates the transcription of class II genes by bridging general transcriptional activators and RNA polymerase II (PolII) (20,24,34). A recent study (48) revealed that some Tlo proteins function as Med2-like components of the Mediator complex.
Here, we characterize the structure and expression patterns of the C. albicans TLO gene family. Phylogenetic analysis indicates that there are three clades of expressed TLO genes, ␣, ␤, and ␥, all of which include a predicted Med2 domain. They differ primarily by the presence of long terminal repeat (LTR) insertions that alter the coding sequences. In addition, we identified a 15th TLO gene, organized in a head-to-tail arrangement with a TLO pseudogene copy that lacks the Med2 domain. We found that members of the TLO␥ clade produce both spliced and unspliced transcripts and that the splice junctions are different in different TLO␥ genes. Tlo proteins encoded by all three clades are detected in the nucleus, and the Tlo␥ proteins also localize to mitochondria. TLO␣ genes are expressed at the highest levels, with TLO␥ clade transcripts and proteins expressed at much lower levels under a range of physiologically relevant growth conditions. This broad range of Tlo expression levels and different localization patterns is predicted to result in a similarly broad range of Mediator complex subunit compositions, perhaps facilitating adaptation to the broad range of host niches that C. albicans occupies.

MATERIALS AND METHODS
Growth conditions used. Standard growth conditions were rich medium (YPAD at 30°C) (39). Assays were performed by inoculating cells in YPAD and growing them at 30°C overnight. Cultures were then diluted 1:100 in fresh YPAD and grown at 30°C for 4 h.
Assignment of TLO genes to chromosome arms. SC5314 DNA was collected as previously described (17). PCR was performed using armspecific primers together with a pan-TLO primer, which are all listed in Table S1 in the supplemental material. The arm-specific primers were designed against the region of unique sequence closest to the TLO.
Construction of the right arm of Chr1 was performed by amplifying an ϳ4-kb fragment using the pan-TLO primer and the Chr1 right-arm-specific primer listed in Table S1 in the supplemental material. Sequential sequencing of the amplicons was used to construct the full sequence. The start codon of TLO␥4 was confirmed using primers 5= ATGCCAGAAAA CCTCCAAAC 3= and 5= CACACATCAGGTGATGACAG 3=, which correspond to the well-conserved TLO start codon and a TLO␥4-specific primer, respectively.
Strain construction. Strains are listed in Table S2 in the supplemental material. Transformations were performed using lithium acetate (36). Integration of the construct at the expected locus was confirmed by PCR and sequencing. C-terminal green fluorescent protein (GFP)-tagged TLO strains were constructed by PCR amplification from plasmid p1602 (13), which contains GFP and URA3, using primers with at least 70 bp of homology to the target TLO (see Table S3 in the supplemental material). The resulting tagged TLO was identified by amplifying a fragment with a GFP-specific primer and the pan-TLO primer and subsequently sequenced. To detect the chromosome into which the construct integrated, chromosomes were separated on contourclamped homogeneous electric field (CHEF) karyotype gels and Southern blotting (38) was used to detect the URA3 insertion. The URA3 probe was amplified by PCR using digoxigenin (DIG)-11-dUTP nucleotides (Roche, Mannheim, Germany) with primers 5= AGACCT ATAGTGAGAGAGCA 3= and 5= CAAACAATCCTCTACCAACA 3= according to the manufacturer's instructions. We used only strains in which insertion was detected on only one chromosome and with one, unambiguous PCR fragment from a single chromosome arm.
Fluorescence microscopy. Strains were inoculated in YPAD, grown at 30°C overnight, diluted 1:100 in fresh synthetic complete dropout (SDC) medium, and grown at 30°C for 3 to 4 h. 4=,6-Diamidino-2-phenylindole (DAPI) (Sigma, St. Louis, MO) diluted 1:1,000 and Mito-Tracker (Invitrogen, Carlsbad, CA), which labels mitochondria by reacting with accessible thiol groups found in the mitochondrial matrix and inner membrane, diluted 1:1,000 were added, and cultures were incubated for 25 min. Cells were washed twice in fresh SDC medium and imaged using differential interference contrast (DIC) and epifluorescence microscopy with a Nikon Eclipse E600 photomicroscope (Chroma Technology Corp., Brattleboro, VT). Digital images were collected using a CoolSnap HQ camera (Photometrics, Tucson, AZ) and MetaMorph software, version 6.2r5 (Universal Imaging Corp., Downingtown, PA). A total of 12 fields with 8 to 15 fluorescent images along the z axis, in 1-m increments, were collected for each cell to ensure that any signal present was captured throughout the diameter of the cell body. The z series stack was then collapsed into a single image by using the stack arithmetic/maximum function of MetaMorph for analysis and image construction for presentation.
Quantitative reverse transcription-PCR (qRT-PCR) of TLO transcription. RNA was collected using the Masterpure yeast RNA extraction kit (Epicentre, Madison, WI), and cDNA was synthesized using the Protoscript M-MuLV first-strand cDNA synthesis kit according to the manufacturer's instructions using d(T) 23 VN (New England Biotech, Ipswich, MA). PCR of cDNA using intron-spanning primers to RPL6 confirmed a lack of contaminating genomic DNA. Transcript abundance was quantified by SYBR green incorporation using a Lightcycler 480 II qPCR machine (Roche, Mannheim, Germany) and analyzed with the Lightcycler 480 software package v1.5.0 (Syni Corporation, Los Angeles, CA). Absolute quantification of SYBR fluorescence using the second derivative maximum value was used to calculate the threshold cycle (⌬CT) value for each TLO gene by using ACT1 and TEF1 as controls. Briefly, the ⌬CT value of each TLO gene was calculated as the difference in cycle threshold between TEF1 and each TLO gene (the ratio of TEF1/ACT1 was consistent across experiments). Results represent at least three independent experiments with standard deviations.
Oligonucleotide sequences are provided in Table S4 in the supplemental material. Due to the high levels of DNA sequence homology between TLO family members, TLO-specific primer sets were designed such that one oligonucleotide hybridized to a limited number of TLO sequences (1 to 4 instead of all 14 TLO sequences) and the second oligonucleotide was specific to a single TLO transcript by including unique single nucleotide polymorphism (SNPs). TLO-specific PCR amplicons were verified by DNA sequencing of cDNA.
Splicing abundance assay. SC5314 cDNA was used to PCR amplify both spliced and unspliced TLO␥16 transcripts using the primers described to identify both RNA isoforms above. Amplified products were run by gel electrophoresis on the same 1% gel, and band intensities were quantified using Fiji/ImageJ v1.46 (NIH, Bethesda, MD). Three independent experiments were run for each strain tested.
Western blotting assay of Tlo abundance. Protein lysates were collected as previously described (14). Western blot assays were performed with mouse anti-GFP (Roche, Penzberg, Germany) and goat anti-Cdc28 (Santa Cruz Biotechnology, Santa Cruz, CA).

RESULTS
Identification and mapping of TLO clades. Fourteen telomereassociated (TLO) genes were previously defined and mapped to the assembled C. albicans genome sequence, primarily by using bioinformatics criteria (44). To compare the relative similarities of members of this gene family, we aligned the nucleotide sequences using MUSCLE (11). Based on sequence similarity, the TLO genes are organized into three major regions: the 5= region (nucleotides [nt] 1 to 315), which has homology to the Med2 domain (Fig. 1A, green regions), as discussed below and in reference 48; a middle region of variable length (up to 140 nt) that contains indels with gene-specific adenosine-rich stretches; and the 3= half, which is organized in a clade-specific manner (Fig. 1A) interspersed with clade-specific unique sequences (Fig. 1A, yellow and magenta). To distinguish these differences, we renamed the TLO genes by adding the clade letter (␣, ␤, or ␥) between "TLO" and the notation of van het Hoog et al. (44), which numbers the TLO genes based upon their chromosomal arm positions, e.g., TLO␤2.
The TLO␣ clade has 6 members, each contained by a single exon. TLO␤2, the single member of the TLO␤ clade, contains two unique sequences in the 3= half of the ORF, resulting in a single exon that is 240 bp longer than in TLO␣ genes (Fig. 1A). The most 5= TLO␤-specific sequence encodes a predicted EF-hand calciumbinding domain (P Ͻ 1EϪ5). Each of the 7 TLO␥ clade members has been predicted to encode transcripts with two possible RNA isoforms, either a single exon or a spliced transcript (44) that would produce proteins differing in length by 8 to 43 amino acids. Unspliced TLO␥ clade transcripts are similar to TLO␣ clade members but contain several short deletions through the clade-specific region. We detected full-length (unspliced) transcripts for TLO␥5, TLO␥7, TLO␥13, and TLO␥16 (Fig. 1A). Furthermore, TLO-specific primers for either TLO␥5 or TLO␥13 detected both unspliced and spliced products (Fig. 1A).
Unexpectedly, the splice junctions previously predicted for TLO␥ clade members (44) could not be detected using primers bridging the splice junction at nt 243 (data not shown). Instead, we identified a putative splice junction at nt 333 of TLO␥5 and TLO␥13, immediately upstream of the adenosine-rich, gene-specific region, using GeneScan to analyze the TLO␥ clade sequences (5). GeneScan predicted a 314-nt intron, which includes a 5= splice site at nt 333, a lariat branch point at nt 531 (UUCUUAC), and a 3= splice junction at nt 647. The second 372-nt exon spans nt 647 to 1019 and carries 102 predicted codons. A PCR primer designed to span the 5= splice junction at nt 333 (Fig. 1A) produced a product whose sequence confirmed that the splice junction conformed to the GeneScan prediction. The TLO␥5 and TLO␥13 spliced tran-scripts were sequenced, and their ORFs predict proteins with a Med2 domain most similar to the TLO␥ clade and genes with a 3= region similar to TLO␣ coding sequences.
Because the predicted splice donor sequence in TLO␥5 and TLO␥13 was not detected in the other 5 TLO␥ clade members, it was unclear if these genes are spliced in the same manner. Indeed, analysis of TLO␥16 by 3= rapid amplification of cDNA ends (RACE) identified two transcripts: the predicted single exon (528 nt) and a spliced form with 5= and 3= junction sites very different from those in TLO␥5 and TLO␥13. The 5= splice site at nt 498 is only 30 nt upstream of the stop codon in the single-exon form of the gene. The second exon is short, only 45 bp long, and carries a TLO␣-like sequence (Fig. 1A). TLO␥16 is likely to be unique in having this particular spliced isoform, because no other TLO sequence contains the necessary GT splice donor site at this position. The proportions of spliced-to-unspliced TLO␥16 mRNA were roughly equivalent (Fig. 1B). Interestingly, multiple TLO␥ transcripts (e.g., TLO␥16 and TLO␥13) ended immediately following the translational termination codon followed by a poly(A) tail, suggesting that some TLO transcripts completely lack a 3= untranslated region (UTR) (data not shown). Transcripts lacking a 3= UTR have not been detected for other C. albicans genes and are likely to affect transcript stability and translation efficiency (23).
High sequence similarity between TLO gene family members and complex sequence structure in the subtelomeres could have complicated the bioinformatic assignment of specific TLO genes to chromosome arms. To experimentally test the genome position of each TLO sequence, we amplified the TLO from each chromosome arm by PCR with a single arm-specific primer anchored in a the TLO gene family members in the SC5314 genome and distinctions between the ␣, ␤, and ␥ clades. The TLO DNA sequence is roughly divided into three regions: a conserved region with the Med2 domain, highlighted in green; a gene-specific region consisting of highly variable adenosine-rich sequences; and a clade-specific region, which in the single-exon form is dissimilar between clades and highly conserved among genes within clades. A predicted nuclear localization signal (NLS) is indicated by a red line. A series of indels differentiates the TLO␣/␤ and single-exon TLO␥ sequences. Insertions of clade-specific sequences are connected by lines to indicate their positions relative to one another and color coded as indicated. Two differentially spliced transcripts were detected for TLO␥5 and TLO␥13 using a primer spanning the splicing event and against the poly(A) tail, indicated as half arrows. Sequencing confirmed splicing at nt 310 in TLO␥8 and at nt 333 in TLO␥13, in both cases joined to a second exon of ϳ372 bp starting at nt 647 and 639, respectively. unique sequence, together with a single pan-TLO primer with homology to the highly conserved Med2 domain ( Fig. 2A). In addition to the expected TLO amplicons in strain SC5314 (Fig. 2C; Table 1), the sequenced amplicons identified two tandem TLO ORFs on the right arm of Chr1 (Fig. 2B). The centromere-proximal one of these two TLO genes, previously named TLO4 (44), contains only a partial TLO sequence lacking the Med2 domain and the conserved TLO promoter sequence. qRT-PCR and 3= RACE studies did not detect a TLO4 transcript (data not shown). Thus, we named this gene TLO4, to indicate that it is a pseudogene. The second TLO ORF on Chr1R was telomere proximal to TLO4 (at coordinates Chr1:3187887 to Chr1:3187464). This locus, now named TLO␥4, carries a TLO␥ clade ORF that is disrupted by an LTR in the 3= half of the gene. TLO␥4 is expressed and is transcribed toward the centromere, like all other TLO genes. The TLO␥4 transcript terminates within the LTR sequence, producing a protein that contains the Med2 domain and a short (36-amino-acid [aa]) C-terminal tail that is homologous to TLO␥ clade members. Thus, SC5314 contains 14 transcribed TLO genes and one pseudogene.
Localization of Tlo proteins. The presence of a Mediator complex subunit 2 (Med2) domain (P ϭ 7.3EϪ24) at the 5= end of predicted TLO ORFs suggests that the TLO genes are C. albicans Med2 homologs. The Mediator complex is a large, multisubunit transcriptional coactivator for polymerase II (PolII)-transcribed genes (8) expected to localize to the nucleus, and the identification of TLO genes as having DNA binding activity (21) bolsters the prediction. Furthermore, nuclear localization signals (NLSs) are predicted in TLO␣ and TLO␤ sequences just 3= of the Med2 domain (Fig. 1A). Thus, we performed fluorescence microscopy on TLO gene products that were tagged with green fluorescent protein (GFP) at the C terminus of their single-exon forms. Sequence containing the highest level of polymorphism within 150 bp 3= of the TLO coding sequence provided the basis for targeting GFP to specific TLO genes. To identify the TLO gene tagged with GFP, we sequenced an amplified fragment from the GFP tag into the TLO Med2 domain. To ensure that this gene was on the expected chromosome end, we also analyzed chromosomes separated on a CHEF gel by Southern blotting with a probe against URA3, the selectable marker used to introduce the GFP tag. Tlo␣9-GFP, The end of the transcript terminated in the gamma LTR sequence shortly after the Med2 domain. This cDNA was cloned three times independently, and no alternate RNA isoforms were identified. (C) TLO assignment to chromosome arms in strain SC5314 is depicted and reflects PCR and sequencing information obtained as in panel A. TLO genes are color coded by clade as indicated. (D) The ancestral C. albicans TLO was most similar to the TLO␣ clade. A rho LTR disrupted an ancestral TLO, producing a novel 3= end. The disrupted TLO could splice out the LTR sequence and recreate the TLO␣-like sequence. This disruption is the progenitor of the TLO␥ clade. Further evolution has occurred on Chr1 where a second LTR insertion has disrupted the TLO␥ clade gene and produced TLO␥4, which is truncated, terminating in the more recent LTR insertion, compared to other TLO␥ clade members. The numbers indicate the position of the TLO gene on chromosome 1.
Tlo␣12-GFP, and Tlo␤2-GFP colocalized with DAPI staining of nuclear DNA in a single large focus per cell in Ͼ80% of cells imaged (Fig. 3A and D). In contrast, Tlo␥16-GFP and Tlo␥13-GFP exhibited a more complex pattern: some of the GFP signal colocalized with DAPI in the nucleus and some colocalized with DAPI to regions outside the nucleus at the cell periphery ( Fig. 3B and C). This peripheral Tlo␥16-GFP and Tlo␥13-GFP colocalized with Mito-Tracker stain, which detects mitochondrial inner membrane and matrix proteins. Tlo␥16 and Tlo␥13 localized to mitochondria and the nucleus in ϳ60% of cells, to mitochondria in only ϳ38% of cells, and exclusively to nuclei in only ϳ2% of cells imaged (Fig. 3D). Spliced Tlo␥5/Tlo␥13 proteins had primarily nuclear localization like that seen for Tlo␣ and Tlo␤ proteins, likely due to the NLS encoded in the second exon (data not shown). Thus, unlike Tlo␣ and Tlo␤ proteins, the Tlo␥ proteins produced from unspliced transcripts localized both to the mitochondria and to the nucleus.
TLO gene transcription levels. Individual members of expanded subtelomeric gene families are often expressed at different levels (25,40,47). To ask if all TLO genes are transcribed, we analyzed deep sequencing (RNA-Seq) data reported for C. albicans strain SC5314 by Bruno et al. (4). The abundances of most TLO transcripts appeared to be similar under standard growth conditions, suggesting that TLO genes from all clades were expressed at similar levels (Fig. 4A).
A concern with RNA-Seq analysis is ambiguity in the mapping of sequencing reads to specific genes that have high levels of homology to each other. For the C. albicans RNA-Seq data, each ambiguous read was resolved independently by uniform assignment to one, and only one, of the matching sequences. Given two differentially expressed genes with similar coding sequences, ambiguous mappings would be expected to make the transcript levels appear more similar than the biological reality.
To measure expression from individual TLO genes, we used quantitative reverse transcription-PCR (qRT-PCR) (31) together with gene-specific primers designed to align with single-exon sequences that were most distinctive for each TLO gene (described in detail in Materials and Methods). All assayed TLO family members, including the internal TLO␣34, produced detectable transcripts, with the exception of pseudogene TLO4. However, in stark contrast to the predictions from RNA-Seq, steady-state levels of TLO␣ and TLO␤ transcripts were 100-to 1,000-fold more abundant than the steady-state levels of TLO␥ transcripts ( Fig. 4B; note log scale on y axis). Consistent with this, much higher levels of the corresponding proteins were detected by Western blotting (Fig. 4C) (48). Therefore, we conclude that TLO␣ and TLO␤ expression is significantly higher than TLO␥ expression.

DISCUSSION
Telomere-associated genes tend to expand when they provide an adaptive advantage for growth under a new environmental condition (7,37). The C. albicans TLO gene family has expanded dramatically since the divergence of C. albicans from C. dubliniensis (20 million years ago [MYA], 2 copies) and the other CUG clade members (50 to 200 MYA, 1 copy) (30). While the mechanism by which it provides an advantage, presumably to life as a commensal and/or pathogen within the human host, is not yet clear, here we begin to characterize the nature of the expansion. We found that TLO genes have diversified into three clades from a single ancestral gene (19), that the TLO␥ clade is regulated differently at the level of mRNA splicing, and that the proteins encoded by the different clades also localize, to some degree, to different cellular compartments.
The 14 TLO genes (plus one TLO pseudogene) of SC5314 likely arose from a single ancestral TLO␣ member that expanded and produced the current TLO diversity (19). The close association of LTRs with TLO ORFs, as well as with the tandem TLO gene and pseudogene on Chr1R, suggests that LTR retrotransposition likely drove TLO diversification (Fig. 2D) (29). We speculate that an initial LTR retrotransposition event disrupted a TLO␣-like ancestral gene, producing the TLO␥ clade, with a different 3= end in the single-exon form. Splicing of the resulting TLO␥ gene, as in the case of TLO␥13 and TLO␥5, would excise the LTR and produce a transcript with a 3= end that resembles that of TLO␣ clade members and that might retain a closely related function. Interestingly, the localization of the single-exon Tlo␥ proteins to the mitochondria portends a function that may differ from that of Tlo␣ and Tlo␤ proteins (49). Distinguishing the function of mitochondrial Tlo proteins will require biochemical and further genetic analysis.
The mechanism that facilitated TLO expansion is unclear, but clues exist in the structure of the subtelomeres. A kappa LTR sequence resides telomere proximal to all TLO genes, and the Tlo␥ proteins produced from unspliced mRNAs are predicted to include C-terminal sequences translated from the rho LTR (44). A subsequent LTR disruption of TLO␥4 on Chr1R suggests that these elements have continued to play a role in TLO diversification. Interestingly, the one internal TLO gene, TLO␣34, also is flanked by kappa LTR and rho LTR sequences, indicating that TLO genes associate with LTRs irrespective of their chromosome location and LTRs may have facilitated TLO expansion and clade differentiation (29,46). Additionally, long tracts of highly homologous sequence that reside centromere proximal to the TLO genes may have facilitated recombination and TLO expansion. The conserved N-terminal Mediator 2 (MED2) homology of all transcribed TLO genes strongly suggests that they encode functional components of the Mediator complex, which participates in transcription regulation (16,24), an idea recently confirmed biochemically (48). The ratio of nonsynonymous to synonymous evolutionary changes (dN/dS ratio) of the Med2 domain across all TLO family members (0.625) suggests purifying selection for the Med2 sequence, similar to what is seen for MED2 homologs from other yeast species (2). The C termini of Med2 proteins are highly variable and species specific, presumably conferring specificity for different transcription factors in different species (32).
It is quite unusual that multiple genes encode a single Mediator subunit, but this is not completely unprecedented: plants and some other yeast species encode multiple paralogs of a single Me-diator subunit (28,41). The large number of Med2 homologs encoded by the TLO genes and the splicing variants of TLO␥ clade members suggest that Mediator is not monolithic. Rather, Mediator is likely to be a related set of different variant complexes that, in C. albicans, differ by the Med2 component (18). In metazoans, Mediator complexes differ by interchangeable use of duplicated kinase module subunits and the incorporation of additional novel subunits in the core modules (2). It is tempting to speculate that different Mediator variants have different affinities for a range of transcription factors and that they ultimately impact the expression of different sets of target genes (1). Thus, the TLO gene family expansion may have greatly increased the repertoire of possible transcriptional responses by generating a broad variety of options for transcriptional outputs. The fraction of cells with Tlo-GFP localized to various cellular compartments across 10 fields is displayed alongside each clade. Two Tlo␣ and Tlo␥ clade members were localized to ensure clade reproducibility. Tlo␣ and Tlo␤ clade members localized primarily to the nucleus, and Tlo␥ clade members localized to the mitochondria or mitochondria and nucleus.