Identifying novel transcription factors involved in the inflammatory response by using binding site motif scanning in genomic regions defined by histone acetylation

<div><p>The innate immune response to pathogenic challenge is a complex, multi-staged process involving thousands of genes. While numerous transcription factors that act as master regulators of this response have been identified, the temporal complexity of gene expression changes in response to pathogen-associated molecular pattern receptor stimulation strongly suggest that additional layers of regulation remain to be uncovered. The evolved pathogen response program in mammalian innate immune cells is understood to reflect a compromise between the probability of clearing the infection and the extent of tissue damage and inflammatory sequelae it causes. Because of that, a key challenge to delineating the regulators that control the temporal inflammatory response is that an innate immune regulator that may confer a selective advantage in the wild may be dispensable in the lab setting. In order to better understand the complete transcriptional response of primary macrophages to the bacterial endotoxin lipopolysaccharide (LPS), we designed a method that integrates temporally resolved gene expression and chromatin-accessibility measurements from mouse macrophages. By correlating changes in transcription factor binding site motif enrichment scores, calculated within regions of accessible chromatin, with the average temporal expression profile of a gene cluster, we screened for transcriptional factors that regulate the cluster. We have validated our predictions of LPS-stimulated transcriptional regulators using ChIP-seq data for three transcription factors with experimentally confirmed functions in innate immunity. In addition, we predict a role in the macrophage LPS response for several novel transcription factors that have not previously been implicated in immune responses. This method is applicable to any experimental situation where temporal gene expression and chromatin-accessibility data are available.</p></div

Top ranked motif for each of the eight expression clusters.

<p>Median fold change for each of the eight clusters is represented using blue lines and the values are shown on the left Y axes. Red lines represent Clover raw scores for the top ranked motif and the values are shown on the Y axes on the right. Based on a time-lagged correlation analysis (using optimum lag time for each motif), the correlation between the Clover score and the cluster-median expression levels are: UC1/V$CREBP1_Q2 - <i>R</i> = 0.828 (t = 2.0); UC2/V$IRF_Q6—<i>R</i> = 0.777 (t = 1.0004); UC3/V$IRF_Q6—<i>R</i> = 0.999 (t = 1.343); UC4/V$SP1_Q2_01—<i>R</i> = 0.8965 (t = 2.0); UC5/V$MYF_01—<i>R</i> = -0.900 (t = 1.001) DC1/V$NFY_01—<i>R</i> = 0.992 (t = 0.589); DC2/V$NFY_01—<i>R</i> = 0.916 (t = 2.0); DC3/V$ZFP281_01—<i>R</i> = 0.304 (t = 2.0).</p

Enrichment test results for ChIP-seq tags for specific TFs in HAc-valley regulatory elements within ±5 kbp of TSSs for genes in specific clusters.

<p>Enrichment test results for ChIP-seq tags for specific TFs in HAc-valley regulatory elements within ±5 kbp of TSSs for genes in specific clusters.</p

Clover scores and ChIP-seq counts for TF when motif is not over represented.

<p>An example of observed counts (pane A) and predicted scores (pane B) for TF whose motif was not found to be over represented. Top graph shows normalized counts for IRF1 within the HAc-valley regulatory elements of genes in UC1 (purple line), or within 10kb region centered at TSS for the same genes (green line). Graph below shows predicted binding of those IRF1 as represented by Clover raw scores (red line) superimposed on the UC1 cluster median fold change (blue line).</p

Correlation between Clover scores and observed TF binding.

<p>Plots show relationship between Clover scores (y axes) and ChIP-seq counts (x axes) for motifs for IRF1 (V$IRF1), IRF8 (V$IRF8) and PU.1/SPI1 (V$PU1) for all eight clusters at 0, 2 and 4 h time points. Panes A-C show Clover scores and observed counts for enriched motifs (blue diamonds), correlation for those (black line) and Clover scores and counts for motifs that are not enriched (red squares). Panes D-F show Clover scores and ChIP-seq counts for the SPI1 motif separately for each cluster (three time points for each cluster).</p

AcH4 valleys and active promoter regions.

<p>(A) AcH4 valleys. ChIP-seq signal and smoothed ChIP-seq signal are shown by gray and black lines respectively. Green bars represent the locations of detected AcH4 valleys. (B) Active promoter regions, defined as regions where detected AcH4 valleys (short blue bars shown for different time point) overlap with the ±5,000bp region around TSS (long blue bar).</p

Transcriptional response to LPS.

<p>Heatmap of five upregulated and three downregulated clusters (left) and median cluster fold change (right). Lines show median cluster expression and shaded areas show interquartile range.</p

An example of good correlation between predicted and measured TF binding (for the cluster UC3).

<p>Top 3 graphs show normalized counts for IRF1, IRF8 and SPI1 within the HAc-valley regulatory elements of genes in DC3 (purple line), or within 10kb region centered at TSS for the same genes (green line). Graphs below show predicted binding of those TFs as represented by Clover raw scores (red lines) superimposed on the UC3 cluster median fold change (blue lines).</p

Clover scores and ChIP-seq counts for SMAD1.

<p>Median fold change for clusters is shown by blue lines for UC1 (upper pane) and UC3 (lower pane) and the values are shown on the left Y axes. Red lines represent Clover raw scores for V$SMAD1_01 motif and the values are shown on the Y axes on the right.</p

<i>LYST/Beige</i> overexpression in fibroblasts reduces parasitophorous vacuole size and inhibits parasite growth.

<p>(A) Phase contrast images showing that the PV expansion observed in bg^J/bg^J fibroblasts is inhibited in YAC-bg^J/bg^J fibroblasts overexpressing <i>LYST/Beige</i>. Arrows point to individual PVs. Bar = 10 µm. (B) Analysis of PV size in infected bg^J/bg^J or YAC-bg^J/bg^J fibroblasts, showing the markedly reduced PV expansion in cells overexpressing <i>LYST/Beige</i>. Fibroblasts were infected for 30 min, and PV size was determined microscopically after the indicated time points. The values represent the mean of 50 independent measurements. Asterisks indicate significant differences from the corresponding time points in bg^J/bg^J fibroblasts: * <i>P</i><0.05, ** <i>P</i><0.01 (Student t test). (C) <i>L. amazonensis</i> amastigotes proliferate slowly in wild type and more vigorously in bg^J/bg^J fibroblasts lacking a functional LYST/Beige protein, but not in YAC-bg^J/bg^J fibroblasts that over-express <i>LYST/Beige</i>. Fibroblasts were exposed to <i>L. amazonensis</i> amastigotes for 60 min, washed, and incubated for the indicated periods followed by determination of the number of intracellular parasites. The data corresponds to the mean +/− SD of triplicates. The results shown are representative of several independent experiments. (D) Upregulation of the mutant <i>LYST/Beige</i> transcripts in bg^J/bg^J fibroblasts infected with <i>L. amazonensis</i>. The cells were exposed for 30 min to axenic amastigotes, washed, and incubated for the indicated periods, followed by RNA extraction and analysis by qualitative RT-PCR (left) and quantitative real-time qPCR (right). The data on the right panel represents the mean +/− SD of triplicate determinations. Asterisks indicate significant differences from non-infected samples (NI): * <i>P</i><0.05 (Student t test).</p