Transcription factor regulation and chromosome dynamics during pseudohyphal growth

Abstract

Pseudohyphal growth is a developmental pathway seen in some strains of yeast in which cells form multicellular filaments in response to environmental stresses. We used multiplexed transposon "Calling Cards" to record the genome-wide binding patterns of 28 transcription factors (TFs) in nitrogen-starved yeast. We identified TF targets relevant for pseudohyphal growth, producing a detailed map of its regulatory network. Using tools from graph theory, we identified 14 TFs that lie at the center of this network, including Flo8, Mss11, and Mfg1, which bind as a complex. Surprisingly, the DNA-binding preferences for these key TFs were unknown. Using Calling Card data, we predicted the in vivo DNA-binding motif for the Flo8-Mss11-Mfg1 complex and validated it using a reporter assay. We found that this complex binds several important targets, including FLO11, at both their promoter and termination sequences. We demonstrated that this binding pattern is the result of DNA looping, which regulates the transcription of these targets and is stabilized by an interaction with the nuclear pore complex. This looping provides yeast cells with a transcriptional memory, enabling them more rapidly to execute the filamentous growth program when nitrogen starved if they had been previously exposed to this condition.

FIGURE 1:

Design of multiplexed experiment. Multiplexed Calling Card analysis begins by transforming 28 barcoded Ty5 transposons into 28 Σ1278b strains in which the genomic copy of a TF is tagged with the Ty5-interacting domain of Sir4. Invasive growth and transposon hopping are simultaneously induced by plating on SLAG plates. Ty5 transposition is directed toward TF binding sites in each tagged strain. After 48 h, cells are collected and transposons are mapped on an Illumina HiSeq with the barcode in each transposon identifying the TF that directed it. Locations are mapped to the Σ1278b genome, and significant clusters are calculated.

FIGURE 2:

The transcriptional network of pseudohyphal TFs. (A) The 28 TFs bind a total of 725 targets within the filamentous growth transcriptional network. (B) Percentages of targets bound by the 28 TFs that are required for pseudohyphal growth and whose overexpression can enhance pseudohyphal growth. (C) Genes whose promoters are bound by ≥2 of the 28 tested TFs are more likely to be required for or can enhance pseudohyphal growth than genes that are bound by a single TF or none of the TFs. (D) Fourteen of the 28 TFs showed a nonzero betweenness centrality in the transcriptional network of pseudohyphal growth.

FIGURE 3:

Identification of a novel binding motif for the Flo8-Mfg1-Mss11 complex. (A) Significant DNA-binding motifs for all of the Calling Card tagged TFs in the experiment. (B) The binding distributions of Flo8, Mfg1, and Mss11 are all centered on the same binding motif ±3 base pairs, which is less than the length of the motif. (C) A mutation in the predicted Flo8p-binding site in the CUP9 promoter in a reporter driving DsRed significantly decreases expression in the nitrogen-starved induction condition.

FIGURE 4:

DNA looping occurs at the FLO11 locus. (A) Binding of three activators at FLO11 for three activators (Flo8, Mfg1, and Mss11) shows equal binding in the promoter and terminator. The binding of two repressors (Sok2 and Sfl1) shows higher binding in the promoter. (B) Relative binding of all of the tagged TFs that bind in the promoter of FLO11 correlates with the function of the TF. The y-axis is the phenotypic score for haploid invasive growth for knockouts of each protein, with negative scores meaning that the mutant has a deleterious effect and positive scores meaning that the mutant has an enhanced effect. The x-axis represents the relative binding of the TF in the promoter relative to the terminator. (C) Looping between promoter and terminator will occur when activators are bound and FLO11 is expressed, whereas when repressors are bound, there is no looping and no FLO11 expression. (D) Looping between the FLO11 promoter and terminator depends on nitrogen starvation and is abolished in a Flo8 mutant strain. Error bars are shown as SD among three biological replicates.

FIGURE 5:

DNA looping provides transcriptional memory for activation of pseudohyphal growth. (A) Expression of FLO11 increases more rapidly in cells moved from rich to nitrogen-starved media when they had been pretreated with nitrogen-starved medium 4 h earlier. (B) Looping between the FLO11 promoter and terminator persists in pretreated cells, even after expression of FLO11 has returned to basal expression. (C) Strains with knockouts of nucleoporin protein Nup2 show decreased transcriptional memory for the same pretreatment experiment for FLO11. (D) Looping at FLO11 is inhibited in nitrogen-starved conditions in the mutant Nup2 strain. Error bars are shown as SD among three biological replicates.