Chromatin triggers tissue-specific immunity in 3D

Abstract

Antigenic and environmental stimuli sensed by innate and adaptive immune cells trigger cognate functions that allow the coordination of the appropriate immune response. In this issue of Immunity, Tzerpos et al. and Jay et al. demonstrate that chromatin architecture dynamics integrate sensory inputs with functional outputs to regulate and direct immune cell responses.

A hallmark of the immune system is to coordinate what it senses with the appropriate effector response. Specialized sensory receptors of immune cells, such as pattern-recognition receptors (PRRs) and cytokine receptors, transduce what is sensed via generic signaling pathways that effect nuclear translocation of signal-responsive transcription factors (SRTFs). The classic paradigm for how non-specific SRTFs impart cell-type-specific gene expression is through selective chromatin accessibility previously established by lineage-determining transcription factors (LDTFs).¹ LDTFs recruit histone acetyltransferases and chromatin remodeling complexes to overcome nucleosomal barriers that determine which genomic targets the SRTFs can bind to.^2,3 Furthermore, LDTF activity is often sufficient to coordinate 3D proximity between distal enhancers and gene promoters prior to pathogen recognition.⁴ However, exceptions to this paradigm exist; for example, SRTFs can promote de novo chromatin accessibility in certain contexts to directly commission latent enhancers for immune function.⁵ These exceptions indicate that immune cells may employ a variety of strategies to coordinate specific effector functions in response to extrinsic stimuli. In this issue of Immunity, complementary studies by Tzerpos et al.⁶ in macrophages and Jay et al.⁷ in T cells uncover chromatin conformations, primed in development and induced by tissue-specific signals, that integrate the sensory inputs with the functional outputs needed for pathogen elimination and tissue repair.

Tzerpos et al.⁶ explored signal coordination strategies used by macrophages from the lens of the transcriptional repressor BACH1, which curiously co-resides with canonical myeloid LDTFs across the genome. While mass spectrometry studies indicated that BACH1 tightly associates with the nucleosome remodeling deacetylase (NuRD) corepressor complex consisting of histone deacetylase (HDAC) activity, genetic deletion of Bach1 counterintuitively resulted in a predominant loss of chromatin accessibility at thousands of BACH1-bound regions, especially at poised enhancers. The authors coined the paradox of chromatin accessibility coinciding with HDAC and repressive remodeling recruitment “pioneering repression”: the conceptual advance being that a repressive transcription factor can prime chromatin accessibility while restraining enhancer or gene activation that might be triggered by the co-residing LDTFs.

To test how BACH1 pioneering repression resolves upon macrophage-activating stimuli, Tzerpos et al. first conducted chromatin immunoprecipitation sequencing (ChIP-seq) for differential BACH1 localization and transcriptional elongation across multiple types of stimuli including heme, lipopolysaccharide (LPS), and IL-4. Strikingly, macrophage activation caused a local redistribution of BACH1, more proximal to gene promoters, at sites enriched for SRTF motifs concordant with the nature of the stimulus. Furthermore, BACH1 redistribution was correlated with heightened transcriptional elongation, suggesting that BACH1’s interaction with specific SRTFs relieved its NuRD-dependent repression at distal poised enhancers, causing expression levels to be increased at cognate genes. To investigate the possibility that BACH1 restricts long-range interactions between poised enhancers and cognate promoters in resting macrophages, the authors profiled higher-order chromatin conformations at BACH1-regulated loci by performing capture Hi-C. Consistent with their enhancer derepression model, stimulus-induced BACH1 redistribution caused new long-distance interactions between poised enhancers formerly bound by BACH1 and nearby promoters of effector genes specific to the nature of the stimulus. Interestingly, spatial proximity between the stimulus-induced BACH1 redistributed sites and its initial binding sites were already established in unstimulated macrophages. This indicates that during macrophage ontogeny, the developmental program commissions these stimulus-responsive immune effector loci to be “spring loaded” for transcriptional bursts, with BACH1-mediated repression being the rate-limiting barrier to gene activation. This architectural mode of poising loci for stimulus responsiveness is reminiscent of Polycomb repressive complexes in embryonic stem cells (ESCs) that spatially constrain Hox genes in 3D hubs for rapid derepression upon developmental cues.⁸ Indeed, Tzerpos et al. found that conditional deletion of Bach1 in macrophages increased long-range chromatin interactions between enhancers and promoters of inflammatory genes, nearly phenocopying the stimulated state in resting macrophages. The loss of BACH1 led to precocious and exaggerated expression of inflammatory genes in vivo, which compromised resolution of systemic inflammation, impaired tissue regenerative capacity, and reduced competitive fitness in specific tissues (Figure 1A).

Figure 1. Alterations in chromatin architecture trigger immune effector functions.

(A) BACH1 restricts enhancer–promoter interactions in resting macrophages, limiting inflammatory gene expression and maintaining a poised state. Upon stimulation, BACH1 occupancy is redistributed, permitting new enhancer–promoter contacts and activation of inflammatory gene programs. During healthy tissue adaptation, inflammatory programs are subsequently attenuated while adaptive programs increase, limiting tissue damage. Loss of BACH1 results in aberrant enhancer–promoter interactions and precocious activation of inflammatory genes in the resting state. Persistence of this inflammatory state impairs macrophage adaptation to tissues, contributing to systemic inflammation.

(B) In DP thymocytes or naive T cells, the Ets1 super-enhancer (Ets1-SE) is poised for contacts with Ets1. Upon stimulation, the Ets1-SE moves into closer proximity with the Ets1 and Fli1 genes, increasing Ets1-Ets1-SE and three-way Ets1-Fli1-SE interactions. Increased Ets1 expression promotes Th1 polarization and contributes to the balance between Th1 and Th2 cell populations. Deletion of the Ets1-SE increases Ets1-Fli1 interactions in DP thymocytes or naive T cells while reducing Ets1-Ets1-SE and three-way Ets1-Fli1-SE interactions. This altered chromatin architecture results in suboptimal Ets1 expression, impairing Th1 differentiation and disrupting the balance between Th1 and Th2 cells, leading to allergy susceptibility.

In an accompanying study, Jay et al. explored the molecular modes by which T cells elicit the appropriate effector function in response to inflammatory signals relayed by innate immune cells.⁷ In contrast to the rapid activation of macrophages which occurs within hours, cytokine-driven differentiation into specialized T helper (Th) subsets takes days, entailing alterations in topologically associating domains (TADs) and enhancer-promoter contacts, with SRTFs having a prominent role in shaping the enhancer repertoire.^9–11 How these alterations in chromatin architecture come about is a major gap in knowledge. The authors aimed to address this gap from the lens of a T cell-specific super-enhancer at the Ets1-Fli1 locus, which is enriched for genetic variants associated with allergy and autoimmune diseases. Deletion of this super-enhancer selectively compromises T cell differentiation into interferon gamma (IFNγ)-producing Th1 cells, which disrupts cross-regulation of type 2 immune responses causing susceptibility to allergic inflammation.¹² However, the mechanistic link between the super-enhancer perturbations, the chromatin architecture of the Ets1-Fli1 locus, and the Th1 effector function remained elusive.

To investigate the role of the super-enhancer on chromatin architecture, Jay et al. turned to optical reconstruction of chromatin architecture (ORCA), a sequential oligo-paint imaging approach that allows the tracing of individual chromatin fiber conformations simultaneously with gene expression profiling. The authors first characterized the ground-state chromatin conformation in developing CD4⁺CD8⁺ (DP) T cells in the thymus. While the most frequent allelic interactions detected were between the Ets1 gene and its super-enhancer, ORCA also revealed frequent three-way interactions between the super-enhancer, Ets1 and Fli1, and five-way interactions with the flanking CCCTC-binding factor (CTCF)-binding sites in the locus that was >5× more prevalent than those previously reported for the Sox2 locus in ESCs. These results indicate that a robust 3D chromatin hub at the Ets1-Fli1 locus is established early in T cell development, potentially poising the locus for signal-responsive effector function in mature T cells.

To determine whether antigen recognition and inflammatory cytokine cues alter this 3D chromatin hub in mature T cells, Jay et al. applied ORCA on in vitro-differentiated Th1 cells. Indeed, the spatial distances within the Ets1-super-enhancer-interacting domain decreased upon Th1 differentiation, leading to substantial increases in frequency of cells exhibiting Ets1-super-enhancer contacts and three-way interactions between the super-enhancer, Ets1, and Fli1, compared to developing DP T cells. Furthermore, the authors found an increased propensity of the super-enhancer to be positioned near the geometric center of each allelic chromatin trace in Th1 cells compared to DP thymocytes. Moreover, the 3D chromatin conformation in Th1 cells was significantly more stable than DP cells as measured by entropy across the traced alleles. These alterations in chromatin architecture in response to cytokine stimulation directly led to an increase in Ets1 expression, which is necessary to promote the Th1 effector state¹² (Figure 1B). The authors next determined the role of the Ets1 super-enhancer on the chromatin architecture of the Ets1-Fli1 locus, established in developing T cells and further augmented in Th1 cells. Deletion of the super-enhancer caused an increase in chromatin conformation entropy with the interaction frequencies shifted from Ets1-super-enhancer contacts toward Ets1-Fli1 contacts with increases in the mean spatial distance between Ets1 and the region where the super-enhancer resides compared to wild-type counterparts for both Th1 and DP cells. The 3D chromatin conformation profiles between super-enhancer-deficient Th1 and DP cells were quantitatively similar, indicating that Th1 cells “look” like thymocytes at this level. While T cells without this super-enhancer can develop, they cannot “mature” due to insufficient Ets1 expression, ultimately resulting in dysregulated type 2 responses and susceptibility to allergic inflammation.^7,12

Taken together, the findings by Tzerpos et al. and Jay et al. illuminate the molecular choreography underlying the design principle of the immune response. In both the innate and the adaptive arms of the immune system, alterations in higher-order chromatin architecture serve as a trigger that integrates what is sensed with the appropriate effector functions to resolve pathogenic invasion and promote tissue repair. An outstanding question in the macrophage story is how NuRD-repressive activity recruited by BACH1 is neutralized at the redistributed site near the gene promoters. It’s possible that the recruited NuRD activity contributes to fine-tuning the expression levels of the respective effector genes. In the Th1 context, it remains unclear whether human genetic variants of the Ets1 super-enhancer associated with allergy and autoimmunity cause defects in cytokine-induced chromatin architecture. If so, investigating the sufficiency of spatial proximity at the locus to rescue the defects in Ets1 expression and Th1 differentiation may avail new therapeutic avenues for allergy and autoimmunity.