Dynamic chromatin architecture identifies new autoimmune-associated enhancers for IL2 and novel genes regulating CD4+ T cell activation
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eLife assessment
This study presents a useful characterization of 3D chromosome conformation changes in activated T lymphocytes, linking risk variants for autoimmune disease to putative target genes. The study employs solid methods and approaches and demonstrates the utility of using chromatin conformation to understand gene regulatory processes. However, the same data modality (chromatin conformation) was previously generated by another group in the same model system, and a more in-depth comparison of results would have improved the utility of this study.
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Abstract
Genome-wide association studies (GWAS) have identified hundreds of genetic signals associated with autoimmune disease. The majority of these signals are located in non-coding regions and likely impact cis -regulatory elements (cRE). Because cRE function is dynamic across cell types and states, profiling the epigenetic status of cRE across physiological processes is necessary to characterize the molecular mechanisms by which autoimmune variants contribute to disease risk. We localized risk variants from 15 autoimmune GWAS to cRE active during TCR-CD28 costimulation of naïve human CD4+ T cells. To characterize how dynamic changes in gene expression correlate with cRE activity, we measured transcript levels, chromatin accessibility, and promoter-cRE contacts across three phases of naive CD4+ T cell activation using RNA-seq, ATAC-seq, and HiC. We identified ∼1,200 protein-coding genes physically connected to accessible disease-associated variants at 423 GWAS signals, at least one-third of which are dynamically regulated by activation. From these maps, we functionally validated a novel stretch of evolutionarily conserved intergenic enhancers whose activity is required for activation-induced IL2 gene expression in human and mouse, and is influenced by autoimmune-associated genetic variation. The set of genes implicated by this approach are enriched for genes shown by high-throughput CRISPR screens to control CD4+ T cell proliferation and function, and we pharmacologically validated 8 implicated genes as novel regulators of T cell activation. These studies directly show how autoimmune variants and the genes they regulate influence processes involved in CD4+ T cell proliferation and activation.
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eLife assessment
This study presents a useful characterization of 3D chromosome conformation changes in activated T lymphocytes, linking risk variants for autoimmune disease to putative target genes. The study employs solid methods and approaches and demonstrates the utility of using chromatin conformation to understand gene regulatory processes. However, the same data modality (chromatin conformation) was previously generated by another group in the same model system, and a more in-depth comparison of results would have improved the utility of this study.
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Reviewer #1 (Public Review):
Summary:
The authors profile gene expression, chromatin accessibility, and chromosomal architecture (by Hi-C) in activated CD4 T cells and use this information to link non-coding variants associated with autoimmune diseases with putative target genes. They find over 1000 genes physically linked with autoimmune disease loci in these cells, many of which are upregulated upon T cell activation. Focusing on IL2, they dissect the regulatory architecture of this locus, including the allelic effects of GWAS variants. They also intersect their variant-to-gene lists with data from CRISPR screens for genes involved in CD4 T cell activation and expression of inflammatory genes, finding enrichments for regulators. Finally, they showed that pharmacological inhibition of some of these genes impacts T-cell activation.
This …
Reviewer #1 (Public Review):
Summary:
The authors profile gene expression, chromatin accessibility, and chromosomal architecture (by Hi-C) in activated CD4 T cells and use this information to link non-coding variants associated with autoimmune diseases with putative target genes. They find over 1000 genes physically linked with autoimmune disease loci in these cells, many of which are upregulated upon T cell activation. Focusing on IL2, they dissect the regulatory architecture of this locus, including the allelic effects of GWAS variants. They also intersect their variant-to-gene lists with data from CRISPR screens for genes involved in CD4 T cell activation and expression of inflammatory genes, finding enrichments for regulators. Finally, they showed that pharmacological inhibition of some of these genes impacts T-cell activation.
This is a solid study that follows a well-established canvas for variant-to-gene prioritisation using 3D genomics, applying it to activated T cells. The authors go some way in validating the lists of candidate genes, as well as exploring the regulatory architecture of a candidate GWAS locus. Jointly with data from previous studies performing variant-to-gene assignment in activated CD4 T cells (and other immune cells), this work provides a useful additional resource for interpreting autoimmune disease-associated genetic variation.
Suggestions for improvement:
Autoimmune disease variants were already linked with genes in CD28-stimulated CD4 T cells using chromosome conformation capture, specifically Promoter CHi-C and the COGS pipeline (Javierre et al., Cell 2016; Burren et al., Genome Biol 2017; Yang et al., Nat Comms 2020). The authors cite these papers and present a comparative analysis of their variant-to-gene assignments (in addition to scRNA-seq eQTL-based assignments). Furthermore, they find that the Burren analysis yields a higher enrichment for gold standard genes.
The obvious question that the authors don't venture into is why the results are quite different. In principle, this could be due to the differences between:
(a) the cell stimulation procedure
(b) the GWAS datasets used
(c) the types of assay (Hi-C vs Capture Hi-C)
(d) approaches for defining gene-linked regions (loops vs neighbourhoods)
(e) how the GWAS signals at gene-linked regions are aggregated (e.g., the flavours of COGS in Javierre and Burren vs the authors' approach).Re (a), I'm not sure the authors make it explicitly clear in the main text that the Capture Hi-C-based studies also use *stimulated* CD4 T cells, particularly in the section "Comparative predictive power...". So the cells used are pretty much the same, and the differences likely arise from points (b) to (e).
It would be useful for the community to understand more clearly what is driving these differences, ideally with some added data. Could the authors, for example, take the PCHi-C data from Javierre/Burren and use their GWAS data and variant-to-gene assignment algorithms?
In addition, given that the authors use Hi-C, a popular method for V2G prioritisation for this type of data is currently ABC (Nasser et al, Nature 2021). Could the authors provide a comparative analysis with respect to the V2G assignments in the paper and, if they see it appropriate, also run ABC-based GWAS integration on their own Hi-C data?
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Reviewer #2 (Public Review):
Summary:
There is significant interest in characterizing the mechanisms by which genetic mutations linked to autoimmunity perturb immune processes. Pahl et al. collect information on dynamic accessible regions, genes, and 3D contacts in primary CD4+ T cell samples that have been stimulated ex vivo. The study includes a variety of analyses characterizing these dynamic changes. With TF footprinting they propose factors linked to active regulatory elements. They compare the performance of their variant mapping pipeline that uses their data versus existing datasets. Most compelling there was a deep dive into additional study of regulatory elements nearby the IL2 gene. Finally, they perform a pharmacological screen targeting several genes they suggest are involved in T cell proliferation.
Strengths:
The work done …
Reviewer #2 (Public Review):
Summary:
There is significant interest in characterizing the mechanisms by which genetic mutations linked to autoimmunity perturb immune processes. Pahl et al. collect information on dynamic accessible regions, genes, and 3D contacts in primary CD4+ T cell samples that have been stimulated ex vivo. The study includes a variety of analyses characterizing these dynamic changes. With TF footprinting they propose factors linked to active regulatory elements. They compare the performance of their variant mapping pipeline that uses their data versus existing datasets. Most compelling there was a deep dive into additional study of regulatory elements nearby the IL2 gene. Finally, they perform a pharmacological screen targeting several genes they suggest are involved in T cell proliferation.
Strengths:
The work done characterizing elements at the IL2 locus is impressive.
Weaknesses:
- Missing critical context to evaluate claims. There are extensive studies performed on resting and activated immune cell states (CD4+ T cells and other cell types) and some at multiple time points or concentrations of stimuli that collect ATAC-seq and/or RNA-seq that have been ignored by this study. How do conclusions from previous studies compare to what the authors conclude here? It is impossible to evaluate the claims without this additional context. These are a few studies I am familiar with (the authors should perform a more comprehensive search to be sure they're not ignoring existing observations) that would be important to compare/contrast conclusions:
o Alasoo, K. et al. Shared genetic effects on chromatin and gene expression indicate a role for enhancer priming in immune response. Nat. Genet. 50, 424-431 (2018).
o Calderon, D., Nguyen, M.L.T., Mezger, A. et al. Landscape of stimulation-responsive chromatin across diverse human immune cells. Nat Genet 51, 1494-1505 (2019).
o Gate, R.E., Cheng, C.S., Aiden, A.P. et al. Genetic determinants of co-accessible chromatin regions in activated T cells across humans. Nat Genet 50, 1140-1150 (2018).
o Glinos, D.A., Soskic, B., Williams, C. et al. Genomic profiling of T-cell activation suggests increased sensitivity of memory T cells to CD28 costimulation. Genes Immun 21, 390-408 (2020).
o Gutierrez-Arcelus, M., Baglaenko, Y., Arora, J. et al. Allele-specific expression changes dynamically during T cell activation in HLA and other autoimmune loci. Nat Genet 52, 247-253 (2020).
o Kim-Hellmuth, S. et al. Genetic regulatory effects modified by immune activation contribute to autoimmune disease associations. Nat. Commun. 8, 266 (2017).
o Ye, C. J. et al. Intersection of population variation and autoimmunity genetics in human T cell activation. Science 345, 1254665 (2014).- As a general point, I appreciate it when each claim includes a corresponding effect size and p-value, which helps me evaluate the strength of significance of supporting evidence.
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Reviewer #3 (Public Review):
Summary:
This paper used RNAseq, ATACseq, and Hi-C to assess gene expression, chromatin accessibility, and chromatin physical associations for native CD4+ T cells as they respond to stimulation through TCR and CD28. With these data in hand, the author identified 423 GWAS signals to their respective target genes, where most of these were not in the proximal promoter, but rather distal enhancers. The IL-2 gene was used as an example to identify new distal cis-regulatory regions required for optimal IL-2 gene transcription. These distal elements interact with the proximal IL2 promoter region. When the distal enhancer contained an autoimmune SNP, it affected IL-2 gene transcription. The authors also identified genetic risk variants that were associated with genes upon activation. Some of these regulate …
Reviewer #3 (Public Review):
Summary:
This paper used RNAseq, ATACseq, and Hi-C to assess gene expression, chromatin accessibility, and chromatin physical associations for native CD4+ T cells as they respond to stimulation through TCR and CD28. With these data in hand, the author identified 423 GWAS signals to their respective target genes, where most of these were not in the proximal promoter, but rather distal enhancers. The IL-2 gene was used as an example to identify new distal cis-regulatory regions required for optimal IL-2 gene transcription. These distal elements interact with the proximal IL2 promoter region. When the distal enhancer contained an autoimmune SNP, it affected IL-2 gene transcription. The authors also identified genetic risk variants that were associated with genes upon activation. Some of these regulate proliferation and cytokine production, but others are novel.
Strengths:
This paper provides a wealth of data related to gene expression after CD4 T cells are activated through the TCR and CD28. An important strength of this paper is that these data were intensively analyzed to uncover autoimmune disease SNPs in cis-acting regions. Many of these could be assigned to likely target genes even though they often are in distal enhancers. These findings help to provide a better understanding concerning the mechanism by which GWAS risk elements impact gene expression.
Another strength of this study was the proof-of-principle studies examining the IL-2 gene. Not only were new cis-acting enhancers discovered, but they were functionally shown to be important in regulating IL-2 expression, including susceptibility to colitis. Their importance was also established with respect to such distal enhancers harboring disease-relevant SNPs, which were shown to affect IL-2 transcription.
The data from this study were also mined against past CRISPR screens that identified genes that control aspects of CD4 T cell activation. From these comparisons, novel genes were identified that function during T cell activation.
Weaknesses:
A weakness of this study is that few individuals were analyzed, i.e., RNAseq and ATACseq (n=3) and HiC (n=2). Thus, the authors may have underestimated potentially relevant risk associations by their chromatin capture-based methodology. This might account for the low overlap of their data with the eQTL-based approach or the HIEI truth set.
Impact:
This study indicates that defining distal chromatin interacting regions helps to identify distal genetic elements, including relevant variants, that contribute to gene activation.
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