Genome-wide nucleosome and transcription factor responses to genetic perturbations reveal chromatin-mediated mechanisms of transcriptional regulation

Abstract

Epigenetic mechanisms contribute to gene regulation by altering chromatin accessibility through changes in transcription factor (TF) and nucleosome occupancy throughout the genome. Despite numerous studies focusing on changes in gene expression, the intricate chromatin-mediated regulatory code remains largely unexplored on a comprehensive scale. We address this by employing a factor-agnostic, reverse-genetics approach that uses MNase-seq to capture genome-wide TF and nucleosome occupancies in response to the individual deletion of 201 transcriptional regulators in Saccharomyces cerevisiae, thereby assaying nearly one million mutant-gene interactions. We develop a principled approach to identify and quantify chromatin changes genome-wide, observing differences in TF and nucleosome occupancy that recapitulate well-established pathways identified by gene expression data. We also discover distinct chromatin signatures associated with the up- and downregulation of genes, and use these signatures to reveal regulatory mechanisms previously unexplored in expression-based studies. Finally, we demonstrate that chromatin features are predictive of transcriptional activity and leverage these features to reconstruct chromatin-based transcriptional regulatory networks. Overall, these results illustrate the power of an approach combining genetic perturbation with high-resolution epigenomic profiling; the latter enables a close examination of the interplay between TFs and nucleosomes genome-wide, providing a deeper, more mechanistic understanding of the complex relationship between chromatin organization and transcription.

Figure 1:. Genome-wide landscape of chromatin occupancy for 201 transcriptional regulator mutants

(A) Functional classification of the 201 mutants in our MNase-seq dataset. Labels are derived from SGD. (B) Schematic of chromatin occupancy profiling of 201 yeast mutants via MNase-seq. MNase digests unprotected DNA, leaving DNA fragments bound by nucleosomes or DNA-binding proteins such as TFs. The resulting fragments are purified, sequenced, and plotted as a function of length, allowing nucleosome and TF occupancy to be visualized. (C) Genome-wide map of chromatin changes in response to genetic perturbations. Loci with significant chromatin changes in response to a deletion are highlighted in purple. Each row represents a mutant, while the x-axis represents genomic locations (bp) grouped by chromosome. (D) Deletion of CBF1 results in a loss of TF occupancy and inward shifting of nucleosomes at the MET10 locus (in contrast, this effect is not seen when BAS1 or MBP1, for example, are deleted). (E) Deletion of ROX1 results in a disruption and loss of nucleosome occupancy at the ANB1 locus. (F) Deletion of ARG80 and ARG81 both result in a loss of TF occupancy at the promoter in the ARG8 locus.

Figure 2:. Chromatin occupancy profiling simultaneously reveals nucleosome and TF changes.

(A) Example of changing nucleosome occupancy at the PHO89 locus when PHO85 is deleted. (B) Example of changing TF occupancy in the promoter region of MET10 when CBF1 is deleted. (C) Chromatin changes at the GAL1-10 locus as a result of deleting the repressor GAL80. For both GAL10 and GAL1, nucleosomes exhibit decreased occupancy in the gene body while TFs exhibit increased occupancy in the promoter. (D) Barplots displaying the log2 fold change (log2(FC)) in nucleosome and TF occupancy at both the GAL10 and GAL1 genes as a result of deleting GAL80.

Figure 3:. Chromatin changes are associated with gene expression changes in the context of genetic perturbation.

(A) Scatter plot of all mutants plotted by their gene expression change (x-axis) and chromatin change (y-axis). Colored points represent genes whose chromatin changes as a result of a single-gene perturbation were above the Laplacian significance threshold (see Methods): blue points are genes that are downregulated in terms of gene expression (log2(FC) ≤ −0.5), red points are genes that are upregulated (log2(FC) ≥ 0.5), and green points are genes without a significant change in gene expression (−0.5 < log2(FC) < 0.5). Triangles represent direct interactions between the deleted TF and its target gene (binding site). (B) The number of upregulated, downregulated, and low differential expression interactions with significant chromatin changes. (C–H) Scatter plots of individual mutants and their target genes plotted by their gene expression change and chromatin change. Significant interactions are labeled as described in (A). (I–K) Heatmaps of TF and nucleosome occupancy changes and their associated gene expression changes for known biological pathways.

Figure 4:. Distinct chromatin signatures between upregulated and downregulated genes.

(A) Violin plots comparing the log2(FC) of TF occupancy at promoters, nucleosome occupancy within gene bodies, and gene expression between upregulated and downregulated genes. Upregulated genes are characterized by a gain in TF occupancy at promoters and a loss in nucleosome occupancy within gene bodies, whereas downregulated genes are characterized by a loss in TF occupancy at promoters. Significance of comparisons was computed using a standard t-test. (B) Upregulated and downregulated genes linked by their TF occupancy, nucleosome occupancy, and gene expression changes. (C) Violin plots comparing the log2(FC) of TF occupancy at promoters, nucleosome occupancy within gene bodies, and gene expression between established activators (n = 82) and repressors (n = 25). Plots are split between direct (top) and indirect (bottom) interactions, where direct genes indicate changes involving a direct interaction annotated by motif (FIMO), or binding site (MacIsaac et al. and/or Rossi et al.) whereas indirect genes indicate an indirect interaction. (D) Interactions split by activator vs. repressor mutants linked by their TF occupancy, nucleosome occupancy, and gene expression. (E) Top 20 upregulated mutant-gene interactions based on gene expression. Interactions are sorted by TF occupancy change. (F) Top 20 downregulated mutant-gene interactions based on gene expression. Interactions are again sorted by TF occupancy change.

Figure 5:. Relationship between gene expression changes and chromatin features.

Each point represents a significant mutant-gene interaction. Change in gene expression (y-axis) as a function of change in (A) TF occupancy in the promoter, (B) nucleosome occupancy in the gene body, (C) nucleosome disorganization in the promoter and gene body, (D) nucleosome occupancy in the promoter, and (E) nucleosome occupancy at the polyadenylation site (PAS). (F) Change in gene expression as a function of the NET-seq transcription rate (Churchman and Weissman 2012). (G) Multiple linear regression model incorporating all chromatin features and transcription rate to predict change in gene expression. Points are colored based on change in gene expression (red upregulated, blue downregulated). All R values are Pearson correlations.

Figure 6:. Analyzing changes in TF occupancy elucidates the mechanisms of pioneer TFs.

(A) MNase fragment plot at the QCR10 locus. Top panel shows the control, when Cbf1, Hap3, and Hap5 are all available to bind in the promoter. Subsequent panels show what happens when one of those TFs is deleted. The HAP3 mutant retains much TF binding, the HAP5 mutant has less TF binding, and the CBF1 mutant has no TF binding (and an upstream nucleosome has shifted into the promoter), suggesting that Cbf1 exhibits pioneering activity at this locus. (B) Barplot of TFs and the number of promoters at which they exhibit pioneering activity. (C) Barplots representing the change in TF occupancy at various gene promoters. Individual bars represent separate TFs, and are colored blue to indicate the TF labeled as pioneering. (D) Comparison of expression change of genes between deleted pioneer TFs and non-pioneer TFs.

Figure 7:. Chromatin dynamics from genetic perturbations recapitulate transcriptional regulatory networks (TRNs).

(A) TRN based on gene expression as a result of deleting BAS1 (∣ log2(FC) ∣ > 0.85). Colored edges represent upregulation (red) or downregulation (blue). Edges representing direct or indirect effects are denoted solid or dashed, respectively. The central node representing the mutant is colored purple; target genes are orange if they are significantly changed in both their chromatin and expression, or gray if they are only significantly different in one of the two. (B) TRN reconstructed based on chromatin information alone. Colored edges are based on predicted up/downregulation from chromatin features. (C) Chromatin TRN of bas1Δ with a spatial layout based on the mechanistic context of TF and nucleosome changes. (D) Chromatin TRN of cse2Δ. (E) Chromatin TRN of gal80Δ.