Diversification of DNA binding specificities enabled SREBP transcription regulators to expand the repertoire of cellular functions that they govern in fungi

Abstract

The Sterol Regulatory Element Binding Proteins (SREBPs) are basic-helix-loop-helix transcription regulators that control the expression of sterol biosynthesis genes in higher eukaryotes and some fungi. Surprisingly, SREBPs do not regulate sterol biosynthesis in the ascomycete yeasts (Saccharomycotina) as this role was handed off to an unrelated transcription regulator in this clade. The SREBPs, nonetheless, expanded in fungi such as the ascomycete yeasts Candida spp., raising questions about their role and evolution in these organisms. Here we report that the fungal SREBPs diversified their DNA binding preferences concomitantly with an expansion in function. We establish that several branches of fungal SREBPs preferentially bind non-palindromic DNA sequences, in contrast to the palindromic DNA motifs recognized by most basic-helix-loop-helix proteins (including SREBPs) in higher eukaryotes. Reconstruction and biochemical characterization of the likely ancestor protein suggest that an intrinsic DNA binding promiscuity in the family was resolved by alternative mechanisms in different branches of fungal SREBPs. Furthermore, we show that two SREBPs in the human commensal yeast Candida albicans drive a transcriptional cascade that inhibits a morphological switch under anaerobic conditions. Preventing this morphological transition enhances C. albicans colonization of the mammalian intestine, the fungus’ natural niche. Thus, our results illustrate how diversification in DNA binding preferences enabled the functional expansion of a family of eukaryotic transcription regulators.

High-Throughput Affinity Measurements of Transcription Factor and DNA Mutations Reveal Affinity and Specificity Determinants

“…In many other cases, double mutant cycle analysis reveals functional consequences that could not be predicted purely from static structures. Here, R262Y, located at a known DNA backbone contact, was the only mutant at either position 262 or 263 still capable of binding DNA, consistent with phylogenetic data showing that the human bHLH family member SREBP1 contains a tyrosine at the orthologous position and that this mutation enhances conformational flexibility to allow binding to varied E-box sequences (del Olmo Toledo, et al, 2018;Parraga, et al, 1998;Fig. S25).…”

“…4B,S15). Several mutations to critical DNA-contacting residues, R262Y and H255N, both of which are observed in the phylogenetic record (Kim, et al, 1995;Shimizu, et al, 1997;del Olmo Toledo, et al, 2018;Figure 2A) appeared to increase the variance in measured affinities, suggesting that these mutations may primarily alter specificity (Fig. 4B).…”

The Regulatory Proteins Rtg1/3 Govern Sphingolipid Homeostasis in the Human-Associated Yeast Candida albicans

“…The C. albicans wild-type reference strain and the rtg3 deletion mutant were grown in liquid YPD medium at 37 C for 2 hours in the presence or absence of either caffeine or myriocin. Cell collection, RNA purification, library preparation, sequencing, read mapping and differential gene expression analysis were carried out as we recently described (Bö hm et al, 2017;Del Olmo Toledo et al, 2018). We obtained between 22 and 58 million reads per sample which were aligned to the C. albicans genome using START (v2.5.2b) with default parameters.…”

A Fungal Transcription Regulator of Vacuolar Function Modulates Candida albicans Interactions with Host Epithelial Cells

“…The chromatin immunoprecipitation procedure was carried out as described previously ( 6 , 57 ). Processing of raw data was conducted following the procedures outlined in other report ( 59 ).…”

“…We obtained 41 to 48 million reads per sample (paired-end, 150-nt reads). Read processing, quality control, mapping, and differential gene expression analyses followed standard computational procedures described by our laboratory ( 59 , 67 ). Briefly, Illumina adaptor sequences were removed using the Trimmomatic tool (v.0.39) ( 68 ).…”