Transcription Factors Mat2 and Znf2 Operate Cellular Circuits Orchestrating Opposite- and Same-Sex Mating in Cryptococcus neoformans

Abstract

Cryptococcus neoformans is a human fungal pathogen that undergoes a dimorphic transition from a unicellular yeast to multicellular hyphae during opposite sex (mating) and unisexual reproduction (same-sex mating). Opposite- and same-sex mating are induced by similar environmental conditions and involve many shared components, including the conserved pheromone sensing Cpk1 MAPK signal transduction cascade that governs the dimorphic switch in C. neoformans. However, the homeodomain cell identity proteins Sxi1α/Sxi2a encoded by the mating type locus that are essential for completion of sexual reproduction following cell–cell fusion during opposite-sex mating are dispensable for same-sex mating. Therefore, identification of downstream targets of the Cpk1 MAPK pathway holds the key to understanding molecular mechanisms governing the two distinct developmental fates. Thus far, homology-based approaches failed to identify downstream transcription factors which may therefore be species-specific. Here, we applied insertional mutagenesis via Agrobacterium-mediated transformation and transcription analysis using whole genome microarrays to identify factors involved in C. neoformans differentiation. Two transcription factors, Mat2 and Znf2, were identified as key regulators of hyphal growth during same- and opposite-sex mating. Mat2 is an HMG domain factor, and Znf2 is a zinc finger protein; neither is encoded by the mating type locus. Genetic, phenotypic, and transcriptional analyses of Mat2 and Znf2 provide evidence that Mat2 is a downstream transcription factor of the Cpk1 MAPK pathway whereas Znf2 functions as a more terminal hyphal morphogenesis determinant. Although the components of the MAPK pathway including Mat2 are not required for virulence in animal models, Znf2, as a hyphal morphology determinant, is a negative regulator of virulence. Further characterization of these elements and their target circuits will reveal genes controlling biological processes central to fungal development and virulence.

Fungal adhesion protein guides community behaviors and autoinduction in a paracrine manner

“…This paracrine communication regulated by Znf2 is unlikely attributable to sexual pheromones because the deletion of Znf2 does not impair pheromone sensing or production (19). Furthermore, a donor strain overexpressing Mat2 (MAT2 oe ), a transcription factor that induces high levels of pheromone production but not ZNF2 under this condition (19,23), did not elicit the expected responses from adjacent cells ( Fig. 1 C and D).…”

“…This suggests that secreted signal(s) from the nearby α+a coculture elicited the responses. Disruption of Znf2, a master regulator of morphogenesis (19,23), in the α+a donor cells severely reduced the ability of the α+a mixture to elicit the responses from the recipient strain (Fig. 1B and Fig.…”

Analysis ofCryptococcus neoformansSexual Development Reveals Rewiring of the Pheromone-Response Network by a Change in Transcription Factor Identity

“…The target genes of Mat2 contribute to the proper recognition of and subsequent cellular fusion with a mating partner. Among Mat2-PRE targets are genes encoding the previously described, developmentally important transcriptional regulators STE12a, STE12a, SXI1a, and SXI2a (Wickes et al 1997;Yue et al 1999;Chang et al 2001;Lin et al 2010). Via these downstream transcription factors, Mat2 initiates a cascade of regulatory events that are required for subsequent developmental transitions.…”

“…In other systems, studies have shown that regulatory network evolution is directed by changes in (1) transcription-factor identity and/or behavior, (2) transcription-factor binding sites in DNA, and (3) cohorts of regulated genes (Davidson EH et al 2002;Gasch et al 2004;Borneman et al 2007;Sung et al 2009;Booth et al 2010;Wilczynski and Furlong 2010;Baker et al 2011). A recent discovery by Lin et al (2010) implicated the more divergent HMG-domain regulator (Mat2) in pheromone signaling. Although the implication of Mat2 suggests a novel regulatory architecture, divergence of the regulatory network cannot be inferred in the absence of functional homology to the pheromone-response factors of other fungi.…”

Sex-Induced Silencing Operates During Opposite-Sex and Unisexual Reproduction in Cryptococcus neoformans

“…This observation is similar to our finding with respect to SIS in opposite-sex mating (Wang et al 2010). These findings lead us to propose that the potential high mutational burden due to hyperactive transposons during mating necessitates a mechanism like SIS, which is much more robust than silencing occurring during vegetative mitotic growth, to defend against transposition and thereby guard genomic integrity of the progeny.Unisexual and opposite-sex mating involve many shared components, including the Cpk1 MAPK signal transduction cascade that responds to pheromones and governs the dimorphic transition in C. neoformans (Wang and Lin 2011;Hsueh et al 2009;Lin et al 2010). However, the homeodomain cell identity protein Sxi2a encoded by the a mating type locus is present and functions only during a-a mating but not a-a mating.…”