Human cytomegalovirus infection coopts chromatin organization to diminish TEAD1 transcription factor activity
- Center for Autoimmune Genomics and Etiology, Division of Human Genetics, Cincinnati Children's Hospital Medical Center, United States
- Department of Molecular Genetics, Biochemistry and Microbiology, University of Cincinnati, United States
- Immunology Graduate Program, University of Cincinnati College of Medicine, United States
- Department of Medicine, Division of Infectious Diseases, Brigham and Women's Hospital, Harvard Medical School, United States
- Department of Microbiology, Harvard Program in Virology, Harvard Medical School, United States
- Center for Integrated Solutions to Infectious Diseases, Broad Institute of Harvard and MIT, United States
- Department of Medicine, University of Pittsburgh School of Medicine, United States
- Department of Pediatrics, University of Cincinnati College of Medicine, United States
- Research Service, Cincinnati VA Medical Center, United States
- Division of Allergy and Immunology, Cincinnati Children's Hospital Medical Center, United States
- Division of Biomedical Informatics, Cincinnati Children's Hospital Medical Center, United States
- Division of Developmental Biology, Cincinnati Children’s Hospital Medical Center, United States
Figures
Figure 1 with 1 supplement
Experimental design.
Schematic overview of the experimental design. (A) Human foreskin fibroblasts or human retinal epithelial cells were infected with the TB40/E strain of human cytomegalovirus (HCMV) at a multiplicity of infection of 5 and 10, respectively. Uninfected and HCMV-infected cells were harvested 48 hr post-infection. (B) Gene expression, chromatin accessibility, histone marks of active regulatory elements (H3K27ac), transcription factor occupancy (TEAD1 and CTCF), and chromatin looping were measured genome-wide using RNA-seq, ATAC-seq, ChIP-seq, and HiChIP, respectively. Differential analyses were employed to identify HCMV-dependent functional events on a genome-wide scale.
Figure 1—figure supplement 1
Principal component analysis of ATAC-seq replicates of uninfected and human cytomegalovirus (HCMV)-infected fibroblasts and retinal epithelial cells.
Figure 2 with 2 supplements
Extensive human cytomegalovirus (HCMV)-mediated alterations to human chromatin accessibility.
Venn diagram comparing ATAC-seq peaks in uninfected vs. HCMV-infected fibroblasts (A) and retinal epithelial cells (B). ATAC-seq signal comparison in uninfected and HCMV-infected fibroblasts (C) and retinal epithelial cells (D).
Figure 2—figure supplement 1
Comparison of differentially accessible chromatin across cell types.
Human cytomegalovirus (HCMV)-induced alterations to chromatin were compared between ARPE and HFF cells. An upset plot indicates the number of altered regions that were shared or distinct between the two cell types.
Figure 2—figure supplement 2
Comparison of HiChIP signal between replicates of uninfected and human cytomegalovirus (HCMV)-infected cells.
Axes represent genomic bin location.
Figure 3
Human cytomegalovirus (HCMV) infection alters the accessibility of chromatin containing TEAD DNA-binding motifs and avoids altering CTCF-containing sites.
Systematic prediction of human transcription factors (TFs) with HCMV-altered binding. (A) Left panel: TF-binding site motif enrichment comparison within ATAC-seq peaks that are unchanged with infection (X-axis) vs. peaks that are closed with infection (Y-axis). Right panel: same analysis comparing peaks that are unchanged with infection (X-axis) and peaks that are opened with infection (Y-axis). Each dot represents a human TF-binding site motif. Motifs are color-coded by TF family (see key). (B) Same analysis in retinal epithelial cells. (C) Percent of predicted binding sites for TEAD and CTCF in ATAC-seq peak regions unchanged with infection and regions closed by HCMV infection. (D) Same analysis in retinal epithelial cells.
Figure 4
Human cytomegalovirus (HCMV) infection leads to widespread coincident loss of chromatin accessibility, TEAD1 binding, H3K27ac marks, and chromatin looping.
(A) HiChIP and ChIP-seq signal in the context of differentially accessible chromatin regions (ATAC-seq). Regions are split into those containing ATAC-seq signal that is unchanged (top), closed upon infection (middle), or opened upon infection (bottom). The corresponding normalized reads counts are depicted for (left to right): ATAC-seq, HiChIP, and ChIP-seq for TEAD1, CTCF, and H3K27ac. Each row in the heatmaps represents a single genomic locus. (B) Genome browser images showing depletion of TEAD1, H3K27ac marks, and chromatin looping interactions proximal to Hippo pathway genes FRMD6 and RASSF2. Solid boxes highlight differential TEAD1-binding sites. The FRMD6 dashed box highlights a promoter, and the RASSF2 dashed box highlights an enhancer. Chromatin looping interactions lost upon infection are highlighted in blue.
Figure 5 with 2 supplements
Human cytomegalovirus (HCMV) infection alters Hippo signaling gene and protein expression levels.
(A) Differentially expressed genes in fibroblasts with HCMV infection. (B) KEGG pathway enrichment analysis of differentially expressed genes. Pathways relevant to infection are highlighted with blue boxes. Key developmental pathways are highlighted with red boxes. (C) Differentially expressed genes within the Hippo pathway. (D) Gene expression profiles (transcripts per million [TPM] values) of all four TEAD family transcription factors with and without HCMV infection. (E) Western blots of established TEAD1 targets THBS1 and CCN1 using whole-cell lysates of uninfected and HCMV-infected cells. GAPDH is used as a control.
-
Figure 5—source data 1
Raw image data without labels.
- https://cdn.elifesciences.org/articles/101578/elife-101578-fig5-data1-v1.zip
-
Figure 5—source data 2
Raw image data with labels.
- https://cdn.elifesciences.org/articles/101578/elife-101578-fig5-data2-v1.zip
Figure 5—figure supplement 1
Heatmap of ChIP-seq peaks for TEAD1, CTCF, and H3K27ac between replicates in uninfected and human cytomegalovirus (HCMV)-infected fibroblasts.
Figure 5—figure supplement 2
Heatmap of gene expression profiles between replicates of uninfected and human cytomegalovirus (HCMV)-infected fibroblasts.
Figure 6 with 2 supplements
Human cytomegalovirus (HCMV) impairs TEAD1 activity through multiple distinct mechanisms.
(A) Representative Western blots for HCMV proteins IE1/2, YAP1, pYAP1, TEAD1, and the H3K27ac mark from whole-cell lysates of uninfected and HCMV-infected cells. GAPDH was used as a loading control. Additional Western blots (biological triplicates) are provided in Figure 6—figure supplement 1, along with quantifications and p-values. (B) Western blots using cytosolic and nuclear fractions obtained from uninfected and HCMV-infected cells indicating the localization of YAP1 and pYAP1. GAPDH and Histone H3 were used as controls for cytoplasmic and nuclear fractions, respectively. (C) Agarose gel image of RT-PCR products of TEAD1 exon 6 splicing events. The full-length TEAD1 targeted region is 91 bp long; it is 79 bp without exon 6. Jurkat cells, which have approximately equal expression of TEAD1 with and without exon 6 (Choi et al., 2022), were used as a control. (D) Model depicting four distinct mechanisms by which HCMV reduces the activity of the TEAD1 transcription factor. (E) Enrichment of phenotype-associated genetic variants at HCMV-altered TEAD1-binding events. Enrichment values calculated by the RELI algorithm are presented for TEAD1-binding events for the lobe attachment (A) and ocular cup (B) phenotypes. Enrichment for TEAD1-binding loss with HCMV infection is statistically significant for each assessment except for the group for lobe attachment (shown in purple). For each bar, dots represent RELI enrichment results from different ancestral groups as defined in the original GWAS studies.
-
Figure 6—source data 1
Raw image files without labels.
- https://cdn.elifesciences.org/articles/101578/elife-101578-fig6-data1-v1.zip
-
Figure 6—source data 2
Raw image files without labels.
- https://cdn.elifesciences.org/articles/101578/elife-101578-fig6-data2-v1.zip
Figure 6—figure supplement 1
Western blots of uninfected and human cytomegalovirus (HCMV)-infected fibroblasts in biological triplicates for TEAD1, H3K27ac, YAP1, pYAP1, and HCMV Immediate Early proteins IE1/2.
For the HCMV IE1/2 Western blot, β-Actin (red color) was used as a loading control. For TEAD1, H3K27ac, YAP1, and pYAP1, GAPDH was used as a control (red color). Quantification of Western blot signal intensities for each protein is shown next to the blot images. Signal quantification was performed using Emperia Studio software and p-values were calculated using two-tailed t-test on GraphPad Prism software. NS, not significant.
-
Figure 6—figure supplement 1—source data 1
Raw image files without labels.
- https://cdn.elifesciences.org/articles/101578/elife-101578-fig6-figsupp1-data1-v1.zip
-
Figure 6—figure supplement 1—source data 2
Raw image files with labels.
- https://cdn.elifesciences.org/articles/101578/elife-101578-fig6-figsupp1-data2-v1.zip
Figure 6—figure supplement 2
TEAD1 alternative splicing event summary.
(A) Visualization of the two significant TEAD1 splicing changes in human cytomegalovirus (HCMV)-infected and uninfected cells. The splicing model of these events shows the percent spliced in (PSI) values, reported as percentages, for uninfected and HMCV-infected cells. The red splice junction indicates the splicing outcome that is upregulated in HCMV infection. (B) Sashimi plots depicting TEAD1 splice junctions and read coverage from exons 4 to 7, with the significant splicing changes of exon 6 (E6) skipping and partial inclusion of intron 5 (I5) highlighted in gray (C). DNA sequence flanking TEAD1 exon 6 (green). RT-PCR primers are depicted in red.
Author response image 1
Constitutive TEAD1 expression reduces expression of two HCMV late genes at 72 and 96 hours after infection.
A-C. Primary human foreskin fibroblasts with and without constitutive TEAD1 expression were infected with pp150-GFP HCMV at a multiplicity of infection (MOI) of 0.3 or 1 and assessed 48 hours post infection. A. HCMV positive cells were quantified by measuring the percent of cells that were GFP positive. B. The percentages of immediate early (IE1/IE2) positive cells were quantified by flow cytometry. C. The mean florescence intensity of immediate early positive cells was quantified by flow cytometry. D. Primary human foreskin fibroblasts with and without constitutive TEAD1 expression were infected with pp150-GFP HCMV at an MOI of 1 and assessed by Western blot at various time point post infection. UL44 and pp65 are expressed late in the cascade of HCMV gene expression. TEAD1 expression levels and uncropped Westerns are provided in Supplemental Figure S8
Tables
Table 1
Genomic regions with extensive loss of TEAD1-binding events upon human cytomegalovirus (HCMV) infection.
In an unbiased analysis, a 300-kb window was drawn around the transcription start site of each gene with differential expression upon HCMV infection. The number of TEAD1-binding loss events within this window was then counted. All results with five or more TEAD1 loss events are provided in this table. Many of these genes encode members of the Hippo, TGF-beta, and WNT signaling pathways (see text).
| Chr | Start | End | Number of TEAD1 loss events | Gene |
|---|---|---|---|---|
| chr15 | 67,200,000 | 67,500,000 | 19 | SMAD3 |
| chr14 | 51,800,000 | 52,100,000 | 17 | FRMD6 |
| chr3 | 30,500,000 | 30,800,000 | 14 | TGFBR2 |
| chr11 | 12,500,000 | 12,800,000 | 11 | TEAD1 |
| chr8 | 128,600,000 | 128,900,000 | 11 | MYC |
| chr1 | 218,400,000 | 218,700,000 | 10 | TGFB2 |
| chr12 | 1,600,000 | 1,900,000 | 10 | WNT5B |
| chr19 | 47,600,000 | 47,900,000 | 10 | BBC3 |
| chr5 | 141,900,000 | 142,200,000 | 10 | FGF1 |
| chr11 | 111,500,000 | 111,800,000 | 9 | PPP2R1B |
| chr2 | 202,700,000 | 203,000,000 | 9 | FZD7 |
| chr6 | 7,600,000 | 7,900,000 | 9 | BMP6 |
| chr4 | 126,100,000 | 126,400,000 | 8 | FAT4 |
| chr7 | 100,600,000 | 100,900,000 | 8 | SERPINE1 |
| chr7 | 116,800,000 | 117,100,000 | 8 | WNT2 |
| chr8 | 28,200,000 | 28,500,000 | 8 | FZD3 |
| chr11 | 69,300,000 | 69,600,000 | 7 | CCND1 |
| chr17 | 42,500,000 | 42,800,000 | 7 | FZD2 |
| chr20 | 33,900,000 | 34,200,000 | 7 | GDF5 |
| chr3 | 55,400,000 | 55,700,000 | 7 | WNT5A |
| chr6 | 132,100,000 | 132,400,000 | 7 | CTGF |
| chr17 | 73,400,000 | 73,700,000 | 6 | LLGL2 |
| chr2 | 208,500,000 | 208,800,000 | 6 | FZD5 |
| chr20 | 49,200,000 | 49,500,000 | 6 | PARD6B |
| chr7 | 5,400,000 | 5,700,000 | 6 | ACTB |
| chr1 | 39,800,000 | 40,100,000 | 5 | BMP8A |
| chr17 | 79,300,000 | 79,600,000 | 5 | ACTG1 |
| chr22 | 46,200,000 | 46,500,000 | 5 | WNT7B |
| chr4 | 95,500,000 | 95,800,000 | 5 | BMPR1B |
| chr5 | 167,600,000 | 167,900,000 | 5 | WWC1 |
Additional files
-
Supplementary file 1
ATAC-seq data QC results.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp1-v1.xlsx
-
Supplementary file 2
ATAC-seq differential peak analysis results.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp2-v1.xlsx
-
Supplementary file 3
Chromatin looping interaction statistics.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp3-v1.xlsx
-
Supplementary file 4
HiChIP data QC results.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp4-v1.xlsx
-
Supplementary file 5
Chromatin looping event coordinates.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp5-v1.xlsx
-
Supplementary file 6
Pairwise statistical comparison of datasets generated in this study using RELI.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp6-v1.xlsx
-
Supplementary file 7
ChIP-seq data QC results.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp7-v1.xlsx
-
Supplementary file 8
ChIP-seq differential peak analysis results.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp8-v1.xlsx
-
Supplementary file 9
RNA-seq data QC results.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp9-v1.xlsx
-
Supplementary file 10
HCMV gene expression levels (TPMs).
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp10-v1.xlsx
-
Supplementary file 11
RNA-seq DEG analysis results.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp11-v1.xlsx
-
Supplementary file 12
Regions of intense TEAD1-binding activity loss near differentially expressed genes.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp12-v1.xlsx
-
Supplementary file 13
Hippo pathway genes from KEGG.
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp13-v1.xlsx
-
Supplementary file 14
Phenotype-associated genetic variant overlap analysis results (RELI algorithm applied to GWAS).
- https://cdn.elifesciences.org/articles/101578/elife-101578-supp14-v1.xlsx
-
MDAR checklist
- https://cdn.elifesciences.org/articles/101578/elife-101578-mdarchecklist1-v1.docx
Download links
A two-part list of links to download the article, or parts of the article, in various formats.
Downloads (link to download the article as PDF)
Open citations (links to open the citations from this article in various online reference manager services)
Cite this article (links to download the citations from this article in formats compatible with various reference manager tools)
Human cytomegalovirus infection coopts chromatin organization to diminish TEAD1 transcription factor activity
eLife 13:RP101578.
https://doi.org/10.7554/eLife.101578.3
