Differential Regulation of White-Opaque Switching by Individual Subunits of Candida albicans Mediator

Abstract

The multisubunit eukaryotic Mediator complex integrates diverse positive and negative gene regulatory signals and transmits them to the core transcription machinery. Mutations in individual subunits within the complex can lead to decreased or increased transcription of certain subsets of genes, which are highly specific to the mutated subunit. Recent studies suggest a role for Mediator in epigenetic silencing. Using white-opaque morphological switching in Candida albicans as a model, we have shown that Mediator is required for the stability of both the epigenetic silenced (white) and active (opaque) states of the bistable transcription circuit driven by the master regulator Wor1. Individual deletions of eight C. albicans Mediator subunits have shown that different Mediator subunits have dramatically diverse effects on the directionality, frequency, and environmental induction of epigenetic switching. Among the Mediator deletion mutants analyzed, only Med12 has a steady-state transcriptional effect on the components of the Wor1 circuit that clearly corresponds to its effect on switching. The MED16 and MED9 genes have been found to be among a small subset of genes that are required for the stability of both the white and opaque states. Deletion of the Med3 subunit completely destabilizes the opaque state, even though the Wor1 transcription circuit is intact and can be driven by ectopic expression of Wor1. The highly impaired ability of the med3 deletion mutant to mate, even when Wor1 expression is ectopically induced, reveals that the activation of the Wor1 circuit can be decoupled from the opaque state and one of its primary biological consequences.

Analysis of the Candida albicans Phosphoproteome

“…Cdk8/Ssn3 is a kinase that is part of the CDK8 module of Mediator, and it has been shown to modulate Mediator and transcription factor activity in ways that impact hyphal growth, switching, metabolism, and stress resistance (42,43,47,82). To directly determine if Cdk8 catalyzes the phosphorylation of Mediator subunits, we performed in vitro kinase assays using purified Mediator or mixtures of Mediator subcomplexes.…”

“…Wild-type C. albicans Mediator complex was purified from cTTR03 through 6ϫHis-3ϫFlag-tagged Med8 (42). To allow for the affinity purification of Mediator complex from mutant strains, one or both copies of MED8 were fused to a sequence encoding a C-terminal 6ϫHis-3ϫFlag tag.…”

“…To allow for the affinity purification of Mediator complex from mutant strains, one or both copies of MED8 were fused to a sequence encoding a C-terminal 6ϫHis-3ϫFlag tag. In addition, Med8 was tagged in backgrounds lacking different Mediator components, including AZC52 (med12⌬/⌬ [42]), AZC63(med9⌬/⌬ [42]), cTTR01 (med15⌬/⌬ [42]), and SN913 (ssn3⌬/⌬ [43]) using PCR products amplified by primer pair LM025/LM014 (44) from the plasmids pFA6a-6ϫHis-3ϫFlag-HIS1 and pFA6a-6ϫHis-3ϫFlag-ARG4. These two template plasmids were generated by replacing the SAT1 marker on pFA6a-6ϫHis-3ϫFlag-SAT1 (44) between the AscI and PmeI sites with HIS1 and ARG4 markers subcloned from pFA6a-TAP-HIS1 and pFA6a-TAP-ARG4 (45), respectively.…”

Fungal Mediator Tail Subunits Contain Classical Transcriptional Activation Domains

“…The regulatable CaMAL2 promoter was used to mitigate the toxicity of some constructs in the C. albicans reporter strain cRC106 (19). Our earlier work has shown that the CaMAL2 promoter functions independently of deletions in the Mediator tail module in C. albicans (20). Specifically, the CaACT1 promoter in CIp-LexA (19) was replaced by the CaMAL2 promoter by amplifying the CaMAL2 promoter from pSFS2 (21) using ZL179/ZL180.…”

Pivotal Role for a Tail Subunit of the RNA Polymerase II Mediator Complex CgMed2 in Azole Tolerance and Adherence in Candida glabrata

“…Furthermore, while the Mediator complex has conventionally been considered a transcriptional coactivator complex, acting as a dynamic interface between eukaryotic transcription factors and RNA polymerase II, recent evidence suggests a direct interaction between the Med5 and Med17 constituents of the Mediator complex with the histone H4 tail (41). Consistently, Mediator is required for the stability of the epigenetically repressed (white) and epigenetically active (opaque) morphological states of C. albicans (42). Hence, it is plausible that desilencing of several genes coding for members of the 23 EPA gene family, including EPA1 and EPA7, may account for the hyperadherence to the Lec2 cells observed upon deletion of the CgMED2 gene in C. glabrata ( Fig.…”