Cognate Site Identifier analysis reveals novel binding properties of the Sex Inducer homeodomain proteins of Cryptococcus neoformans

Abstract

Homeodomain proteins function in fungi to specify cell types and control sexual development. In the meningoencephalitis-causing fungal pathogen Cryptococcus neoformans, sexual development leads to the production of spores (suspected infectious particles). Sexual development is controlled by the homeodomain transcription factors Sxi1alpha and Sxi2a, but the mechanism by which they act is unknown. To understand how the Sxi proteins regulate development, we characterized their binding properties in vitro, showing that Sxi2a does not require a partner to bind DNA with high affinity. We then utilized a novel approach, Cognate Site Identifier (CSI) arrays, to define a comprehensive DNA-binding profile for Sxi2a, revealing a consensus sequence distinct from those of other fungal homeodomain proteins. Finally, we show that the homeodomains of both Sxi proteins are required for sexual development, a departure from related fungi. Our findings support a model in which Sxi1alpha and Sxi2a control sexual development in a homeodomain-dependent manner by binding to DNA sequences that differ from those defined in previously established fungal paradigms.

Fig. 1

The Sxi proteins are sequence-specific DNA-binding proteins. A. Schematic representation of the Sxi proteins. Illustrations show the full-length and 6x-histidine-tagged protein truncations of Sxi1α and Sxi2a with the homeodomain indicated as a dotted box, and the 6x-histidine tag shown in black. B. The Sxi proteins bind to a 19 bp site of the CLP1 upstream region. Lanes 1–4 contain 0, 8, 16 and 32 nM, respectively, of purified Sxi1α-N protein. Lane 5 contains 16 nM of purified Sxi1α-N and an anti-6x-histidine antibody. Lanes 6–8 contain 5, 10 and 20 nM, respectively, of purified Sxi2a-HD protein. Lane 9 contains 10 nM Sxi2a-HD and an anti-6x-histidine antibody. C. Sxi1α-N binds DNA with specificity. Lanes 1 and 2 contains the 177 bp fragment of the CLP1 upstream region, which includes the 19 bp binding site (clp19) as probe. Lanes 3 and 4 contain the CLP1 upstream region in which the clp19 site has been removed. Lanes 1 and 3 contain probe only. Lanes 2 and 4 include 16 nM of purified Sxi1α-N. D. Sxi2a-HD binds DNA with specificity. Lane 1: probe only. Lanes 2–11 each contain 50 nM Sxi2a-HD and 75 nM clp19 probe. Lanes 3–6 also contain increasing concentrations of unlabelled clp19 (specific competitor); lanes 8–11 contain increasing concentrations of unlabelled random 19-mer (non-specific competitor) (5-, 10-, 50- and 75-fold molar excesses for both competitors). E. Sxi2a-HD has an apparent K_d of 25 nM. Lanes 1–7 contain a 177 bp of the CLP1 upstream region as probe. Lanes 2–7 contain 5, 10, 25, 50, 100 and 200 nM Sxi2a-HD.

Fig. 2

The Sxi2a-HD binds clp19 in vivo. A. Schematic representation of the reporter plasmid pLGΔ-178 + clp19. The CYC1 promoter drives lacZ expression. B. The Sxi2a-HD activates lacZ expression in the clp19 reporter. CPRG assays were performed in triplicate for an n = 6 for each sample. Average values and standard error are reported and an asterisk indicates P < 0.0001. An increase of 40% and 36% in lacZ expression is observed in the presence of Sxi2a-HD alone and in combination with Sxi1α-FL respectively.

Fig. 3

CSI arrays and MEME define a core motif bound by Sxi2a. A. The binding profile of Sxi2a-HD is determined using CSI arrays. Purified Sxi2a-HD is labelled with a Cy5-conjugated antibody that recognizes the 6x-histidine tag of Sxi2a-HD and applied to an array that contains hairpins comprised of a 10-mer sequence (N₁–N₁₀) with a 4 bp flanking sequence on either side (GCGC). The G-G-A loop is followed by the exact complement of the 10-mer and flanking sequence. B. MEME-generated CSI motifs contain a 5′-CAATC-3′ core. Sequences having the highest normalized Z-score values (n = 2585) were input into MEME to generate motifs 1, 2 and 3. The program WEBLOGO was used to generate block logos (http://weblogo.berkeley.edu). The x-axis of each logo represents the position at which a particular base is located. The y-axis represents the information content at a single position. C. In-solution binding to hairpin DNA displays a trend similar to CSI arrays. Individual hairpins representing the Z-scores > 14, 7–8, 4–5, 0 and < 0 (lanes 1–2, 3–4, 5–6, 7–8 and 9–10 respectively) were assayed for binding by Sxi2a-HD by EMSA. Odd-numbered lanes contain only the hairpin DNA, while even-numbered lanes include 50 nM Sxi2a-HD. The corresponding Z-score bins are labelled above each individual set of shifts.

Fig. 4

Sxi2a has similar affinities for the CSI motif and clp19 10-mer. A. CSI data predicted the clp19 10-mer core bound by Sxi2a-HD. The clp19 sequence was parsed into consecutive 10-mer sequences and their corresponding Z-scores were determined based on the CSI data. One of the sequences (ATTGTTTTTC) was omitted due to insufficient statistical depth to generate a Z-score. Representative 10-mer sequences labelled A, B, C and D (lanes 1–2, 3–4, 5–6 and 7–8 respectively) were used as probes in an EMSA to detect their relative binding by Sxi2a-HD. Odd-numbered lanes contain the probe only, while even-numbered lanes include 50 nM purified Sxi2a-HD. B. Sxi2a binds to the clp19 10-mer D (lanes 1 and 2) and the CSI motif 1 (lanes 3 and 4) with similar affinities. To generate the context for this experiment, the clp19 10-mer was replaced with motif 1. Odd-numbered lanes contain the probe only, and even-numbered lanes include 50 nM purified Sxi2a-HD.

Fig. 5

Upstream regions of Sxi-regulated genes that contain the CSI motif are selectively bound by Sxi2a-HD. Even-numbered lanes contain the probe only and odd-numbered lanes contain 50 nM Sxi2a-HD. Probes in lanes 1–8 correspond to the upstream region of Sxi-regulated genes CND05450, CNN02230, CNG03790, CND05810 respectively.

Fig. 6

Sxi1α and Sxi2a contain critical DNA-binding residues that are required for sexual development. A. The homeodomain regions of C. neoformans Sxi1α and Sxi2a are aligned with the homeodomains of proteins from other species and a homeodomain protein consensus sequence (Gehring et al., 1994). A schematic representation above the sequences indicates the locations of three α-helices. Residues shaded in grey are identical or similar to the residue at that position in homeodomain consensus sequence. Sxi1α, α2 (S. cerevisiae), b1-2 (C. cinerea), Meis2 (Mus musculus) and Gsp1 (Chlamydomonas reinhardtii) are members of the HD1 family and contain a 3-amino-acid insertion between helices 1 and 2. Proteins a1 (S. cerevisiae), a2-1 (C. cinerea), Oct1 (Homo sapiens), en (engrailed, D. melanogaster), bW (U. maydis) and Sxi2a fall into the HD2 family. Some key residues known to be critical for DNA binding are in bold (N51, R53 and R55). B. Mutant alleles of SXI1α do not induce sexual development. In panels 1–5, wild-type Sxi1α, empty vector, Sxi1α N51A, Sxi1α R53A and Sxi1α R55A were each transformed into an α sxi1αΔ strain and were crossed by an a strain. C. Mutant alleles of SXI2a do not induce sexual development. In panels 1–5, wild-type Sxi2a, empty vector, Sxi2a N51A, Sxi2a R53A and Sxi2a R55A were transformed into an a sxi2aΔ strain and were crossed by an α strain. Panels show the periphery of a spot on V8 medium photographed at 100× magnification.

Fig. 7

Sxi2a-HD mutants exhibit little or no DNA binding but are generally folded in solution. A. Lanes 1–13 contain 75 nM clp19 probe. Lanes 2–4, 5–7, 8–10 and 11–13 contain 5, 10 or 20 nM purified Sxi2a-HD wild type, N51A, R53A and R55A respectively. B. The CD spectra of purified Sxi2a-HD wild type (black continuous line), N51A (dotted line), R53A (long dashed line) and R55A (short dashed line) are plotted. The x-axis refers to the wavelength at which data were collected (in nm), and the y-axis refers to the molar ellipticity (in degree cm² decimol⁻¹).