Scientists at the Salk Institute say they have discovered that the Foxp3 protein is essential for creating the unique chromatin architecture of regulatory T cells and, in turn, promoting their immune suppressive function. Their study, “Foxp3 orchestrates reorganization of chromatin architecture to establish regulatory T cell identity,” appears in Nature Communications.

“Chromatin conformation reorganization is emerging as an important layer of regulation for gene expression and lineage specification. Yet, how lineage-specific transcription factors contribute to the establishment of cell type-specific 3D chromatin architecture in the immune cells remains unclear, especially for the late stages of T cell subset differentiation and maturation. Regulatory T cells (Treg) are mainly generated in the thymus as a subpopulation of T cells specializing in suppressing excessive immune responses,” the investigators wrote.

“Here, by comprehensively mapping 3D chromatin organization during Treg cell differentiation, we show that Treg-specific chromatin structures were progressively established during its lineage specification, and highly associated with Treg signature gene expression. Additionally, the binding sites of Foxp3, a Treg lineage specifying transcription factor, were highly enriched at Treg-specific chromatin loop anchors. Further comparison of the chromatin interactions between wide-type Tregs versus Treg cells from Foxp3 knock-in/knockout or newly-generated Foxp3 domain-swap mutant mouse revealed that Foxp3 was essential for the establishment of Treg-specific 3D chromatin architecture, although it was not dependent on the formation of the Foxp3 domain-swapped dimer.

“These results highlighted an underappreciated role of Foxp3 in modulating Treg-specific 3D chromatin structure formation.”

Body’s peacekeepers

“Regulatory T cells are the peacekeepers in our body,” said Ye Zheng, PhD, NOMIS Center for Immunobiology and Microbial Pathogenesis, and co-senior author of the study. “Having regulatory T cells telling other cells to calm down is crucial in maintaining a healthy body. Fully understanding the influence of Foxp3 on how these peacekeepers develop teaches us about how our immune system functions—and dysfunctions in disease.”

Scientists have long known that Foxp3 is key to regulatory T cell development, but only as an on-off switch for regulatory T cell genes. Zheng believed this view of Foxp3 as a simple genetic switch did not capture the full picture. The complexity and influence of chromatin architecture on cellular identity prompted Zheng to turn to Jesse Dixon, MD, PhD, a chromatin architecture expert, assistant professor at Salk, and co-senior author of the study, to explore the relationship between Foxp3 and regulatory T cells at this higher, structural level.

The researchers mapped the 3D chromatin architecture of regulatory T cells to see whether Foxp3 was changing the chromatin architecture in regulatory T cells to expose genes necessary for the cells’ function. To capture the unique relationship between Foxp3 and regulatory T cells, they compared regulatory T cell chromatin architecture to another T cell subtype, the effector T cell. According to Zheng, effector T cells are like regulatory T cells’ opposites—they instigate attacks and instruct other immune cells to fight.

A metaphase spread of chromosomes (blue). [Salk Institute]
As the researchers compared the architecture of regulatory and effector T cells, they noticed there were many unique Foxp3 binding regions only present in regulatory T cells—affirming the special relationship between Foxp3 and the peacekeeping immune cells.

“Regulatory and effector T cells follow an almost identical route of differentiation until Foxp3 gets involved,” said Dongsung Lee, PhD, co-first author and former postdoctoral researcher in Dixon’s lab. “Comparing regulatory and effector T cells gave us a clear picture of Foxp3’s impact on regulatory T cell identity, since Foxp3 is only seen in regulatory T cells.”

DNA loops

They also found that regulatory T cells had distinct chromatin architecture features called DNA loops. They saw genes that bind to Foxp3 were pulled physically closer to genes that control regulatory T cell identity, so that Foxp3 could easily promote the expression of identity-forming genes.

Ye Zheng, PhD, (left), and Zhi Liu, PhD [Salk Institute]
Ye Zheng, PhD, (left), and Zhi Liu, PhD [Salk Institute]
“We wanted to see whether Foxp3 was benefiting from DNA loops that the regulatory T cell chromatin structure was already making, or if Foxp3 was in some way creating those characteristic loops,” said Zhi Liu, PhD, co-first author and former postdoctoral researcher in Zheng’s lab. “We found that Foxp3 was necessary in creating the loops, and therefore necessary in creating the chromatin architecture unique to regulatory T cells.”

Foxp3 was playing a far more fundamental and extensive role in regulatory T cell development than expected. Previous research pointed to two Foxp3 proteins pairing up in a special way to create these DNA loops. The team found that these pairs were not necessary to create the characteristic loops, suggesting other Foxp3 protein-containing complexes could be involved.

The findings demonstrate that beyond serving as a genetic on-off switch, Foxp3 oversees greater genetic structural change within regulatory T cells. The presence of Foxp3 orchestrates chromatin architecture changes that, in turn, guide the functional success of the peacekeeping immune cells.

“Now that we know Foxp3 plays a greater role in regulatory T cell function, we may be able to find ways to turn up and down Foxp3 to regulate immunosuppression,” pointed out Dixon. “If we turn up Foxp3, we could see more immunosuppression, which could treat autoimmunity. If we turn down Foxp3, we could see less immunosuppression, which could be helpful in fighting cancerous tumors, since normally regulatory T cells infiltrate tumors and suppress the action of other immune cells.”

More research is needed to understand how Foxp3 works with other proteins to create DNA loops in regulatory T cells. As the researchers uncover more details of the relationship between Foxp3 and regulatory T cells, they hope Foxp3 becomes a possible target for therapies that modulate immunosuppression.

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