Understanding how regulatory T cells (Tregs) develop and work is key to determining how they might be manipulated to encourage the destruction of cancer cells or prevent autoimmunity. Cell behavior is influenced by chromatin architecture—the 3D shape of chromosomes—and which genes are accessible to proteins that promote regulatory T cell development. Studies involving engineered mice, led by a team led by Salk professor Ye Zheng, PhD, and assistant professor Jesse Dixon, MD, PhD, have now shown that a protein called Foxp3 is essential for creating the unique chromatin architecture of regulatory T cells during their development and, in turn, promoting their immune suppressive function.

“Regulatory T cells are the peacekeepers in our body,” said Zheng. “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.” Zheng is co-senior author of the team’s published report in Nature Communications, which is titled, “Foxp3 orchestrates reorganization of chromatin architecture to establish regulatory T cell identity.” In their paper, the scientists wrote, “We compared the 3D chromatin structures of Treg cells and their precursors and revealed that the 3D chromatin architecture of Treg cells was gradually established during Treg lineage specification, and that changes in chromatin interactions align with the trajectory of Treg development.”

The instructions to make a cell are encoded in DNA strands that are wrapped in proteins and RNA, and wound together into a 3D structure called chromatin. Changes to this 3D architecture have a critical influence on the identity of a cell. Altering chromatin architecture can expose or conceal stretches of genetic code that are responsible for the behavior of the entire cell. “Chromatin conformation reorganization is emerging as an important layer of regulation for gene expression and lineage specification,” the authors wrote. “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 are specialized immune cells that suppress the immune response and prevent the body from attacking its own cells. “Tregs are mainly generated in the thymus as a subpopulation of T cells specializing in suppressing excessive immune responses,” the authors explained. This process of T cell development in the thymus occurs through different stages, and 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, an expert on regulatory T cells, thought this view of Foxp3 as a simple genetic switch did not capture the full picture. And as the authors noted, the role of chromatic architecture hasn’t been well understood “… it remains elusive how the Treg 3D chromatin architecture is established during their lineage specification, how it influences gene expression in Tregs, and whether and how Foxp3 contributes to Treg-specific chromatin interactions.”

The complexity and influence of chromatin architecture on cellular identity prompted Zheng to turn to Dixon, a chromatin architecture expert, to explore the relationship between Foxp3 and regulatory T cells at this higher, structural level.

From left: Dongsung Lee and Jesse Dixon. [Salk Institute]
From left: Dongsung Lee and Jesse Dixon. [Salk Institute]
For their reported study, 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. The authors further explained, “To examine whether Treg cells have a unique 3D chromatin structure to support their distinct gene expression profile and function, we performed a pairwise comparison of the 3D chromatin structure of splenic Treg and Tcon [conventional T] cells.”

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. “By comparing Treg cells and their closely related conventional T cells, we identified chromatin structures unique to Treg cells,” they noted.

“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.”

The investigators 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.

“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.” The team stated,“ … the binding sites of Foxp3, a Treg lineage specifying transcription factor, were highly enriched at Treg-specific chromatin loop anchors …”

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’s study, which included experiments in cells from engineered GFP knock-in Foxp3 knockout (Foxp3-KIKO) mice, and in wild-type (WT) cells, found that these pairs were not necessary to create the characteristic loops, suggesting other Foxp3 protein-containing complexes could be involved. “Since Foxp3 by itself does not function as a chromatin loop anchor to stabilize chromatin interactions, it likely cooperates with other proteins,” they further noted.

The findings demonstrated 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. “This study presented a model of how lineage-specific TFs function during the late or terminal stage of cell differentiation,” the authors stated. “Once the 3D genome structure is formed in mature Tregs, the contribution of Foxp3 in the maintenance of Treg 3D chromatin structure seems to be relatively minor. Instead, Foxp3 acts as a TF to activate or repress gene expression by leveraging the promoter-enhancer proximity facilitated by the Treg-specific chromatin looping structure.

“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,” commented Dixon, co-senior author of the study. “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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