I: TCF-1 and chromatin accessibility: When our lab started in 2015, it was known that the transcription factor TCF-1 is required for T cell development. However, the precise mechanisms driving this process remained unknown. In our lab’s first publication which is cited ~ 200 times, we discovered that TCF-1 controls T cell fate through reprogramming silent chromatin in progenitors (1). TCF-1 has different domains including the highly structured DNA-binding domain (termed HMG) in addition to a poorly structured intrinsically disordered region (IDR). Although the DNA binding domain of TCF-1 has been widely studied, our knowledge of this protein’s IDR and whether it plays any role in T cell biology remains limited. IDRs inherent structural flexibility allows them to adopt multiple conformations, facilitating interactions with various molecular partners. Therapeutically, targeting IDRs offers the potential to modulate protein interactions and functions in an adaptable way. In a follow-up study (2), we discovered that a 43 amino-acid region within TCF-1’s IDR (termed L1) is required for T cell development and endows TCF-1 the ability to bind silent chromatin. Remarkably, the L1 domain could be functionally replaced with a heterologous disordered domain of another transcription factor. Given the critical role of TCF-1 in tumor immunology, our identification of the functional region within TCF-1’s IDR not only redefines how we perceive the structural and functional dynamics of TCF-1 but also opens new avenues to modulate this protein for effective immunotherapeutic strategies.

1. Johnson JL, Georgakilas G, Petrovic J, Kurachi M, Cai S, Harly C, Pear WS, Bhandoola A, Wherry EJ, Vahedi G. Lineage-Determining Transcription Factor TCF-1 Initiates the Epigenetic Identity of T Cells. Immunity. 2018;48(2):243-57. Cited > 200 times.
2. Goldman N, Chandra A, Johnson I, Sullivan MA, Patil AR, Vanderbeck A, Jay A, Zhou Y, Ferrari EK, Mayne L, Aguilan J, Xue HH, Faryabi RB, John Wherry E, Sidoli S, Maillard I, Vahedi G. Intrinsically disordered domain of transcription factor TCF-1 is required for T cell developmental fidelity. Nature Immunology. 2023;24(10):1698-710.

II: TCF-1 and chromatin intermingling: Although it is known that three-dimensional (3D) chromatin organization plays a pivotal role in cell fate determination, the contribution of TCF-1 on the global control of 3D genome remained unclear. Utilizing advanced genomics and imaging technologies, we discovered that TCF-1 dismantles boundaries of insulated neighborhoods in T cell progenitors, increasing long-range interactions among enhancers and promoters of critical T cell genes (3). Mechanistically, the TCF-1 dependent gain in long-range interactions is linked to the recruitment of the cohesin-loading factor NIPBL to TCF-1 dependent active enhancers. This work is the first to show TCF-1 is a global genome organizer in T cells, which opens a new direction to investigate other T cell transcription factors contributing to chromosome structure.

3. Wang W, Chandra, A., Goldman, N., Yoon, S., Ferrari, E., Nguyen, S.C., Joyce, E.F., Vahedi, G. TCF-1 promotes chromatin interactions across topologically associating domains in T cell progenitors. Nature Immunology. 2022.

III: Multi-enhancer hubs: Recent advances in genomics and imaging technologies attest to the formation of spatial clusters of enhancers, called multi-enhancer hubs. Despite numerous efforts profiling multi-enhancer hubs across diverse developmental programs, the functional relevance of these structures is poorly understood. We applied a mathematical analysis on multi-enhancer interactions which directed us towards a highly interacting multi-enhancer hub at a locus harboring Ets1 and Fli1 transcription factors. Strikingly, the ~1 Mbp DNA sequence in humans encompasses ~100 independent noncoding SNPs associated with a broad spectrum of immune-mediated diseases. Yet, it remains a mystery why this locus has been associated with many complex immune diseases (3). We began to address this question using the CRISPR/Cas9 technology to delete a ~25 kbp noncoding region at the Ets1-Fli1 locus in mice which allowed us to investigate the functional and mechanistic relevance of multi-enhancer formation for immune responses (4). Our work revealed a paradigm that the multi-enhancer connectivity at the Ets1 locus can act as a dimmer, adjusting the expression level of this gene with substantial biological and clinical consequences such as allergic inflammation. This study made a giant leap forward for our understanding of the functional relevance of multi-way enhancer interactions in immune responses, which may lead to novel therapeutic interventions in allergic diseases. 

3. Fasolino M, Goldman N, Wang W, Cattau B, Zhou Y, Petrovic J, Link VM, Cote A, Chandra A, Silverman M, Joyce EF, Little SC, Consortium H, Kaestner KH, Naji A, Raj A, Henao-Mejia J, Faryabi RB, Vahedi G. Genetic Variation in Type 1 Diabetes Reconfigures the 3D Chromatin Organization of T Cells and Alters Gene Expression. Immunity. 2020;52(2):257-74 e11.
4. Chandra A, Yoon S, Michieletto MF, Goldman N, Ferrari EK, Abedi M, Johnson I, Fasolino M, Pham K, Joannas L, Kee BL, Henao-Mejia J, Vahedi G. Quantitative control of Ets1 dosage by a multi-enhancer hub promotes Th1 cell differentiation and protects from allergic inflammation. Immunity. 2023;56(7):1451-67 e12.

IV: Modeling Type 1 Diabetes: The inhibition of proinflammatory cytokines holds the promise for favorable clinical outcomes in many autoimmune diseases. However, there remains a profound lack of understanding concerning the precise timing, specific cellular contexts, and identity of aberrant cytokines contributing to the progression of autoimmunity. A major goal of our lab is to test the hypothesis that the identity of aberrant cytokines is encoded in the epigenome of immune cells residing in relevant lymph nodes. Leveraging the strengths of our environment at Penn, we focused on testing this hypothesis in the context of Type 1 Diabetes (T1D) which is an autoimmune disease where T cells destroy insulin producing beta cells. Since 2016, we have been a member of the Human Pancreas Analysis Program (HPAP) at Penn. Our initial contribution to the program related the computational analysis of the first major single-cell RNA-seq study in islets of individuals with T1D (5) and a follow-up work (6). We also demonstrated the proof-of-concept for using machine learning and single-cell expression analysis in human islets for early detection of T1D (7).

To reconstruct the cytokine environment in T1D, our lab simultaneously measured gene expression and chromatin accessibility in 1 million+ single immune cells from pancreatic lymph nodes of individuals with T1D collected by HPAP. These data represent the largest existing multiome profiling of the human immune system in health and disease. This study led us to discover heightened gene activity of the tumor necrosis factor (TNF) pathway and subsequent chromatin remodeling in central memory CD4+ T cells in the pancreatic lymph nodes of T1D and non-diabetic islet-autoantibody positive donors. The manuscript reporting these data is currently in revision in Nature Immunology and is also available on bioRxiv (8). Our ambitious ongoing work aims to model T1D progression using machine learning and the single-cell immune profiling data across tissues. If successful, our study can pinpoint pre-symptomatic individuals who could benefit from immunomodulation techniques, transforming the lives of millions at risk.

5. Fasolino M, Schwartz GW, Patil AR, Mongia A, Golson ML, Wang YJ, Morgan A, Liu C, Schug J, Liu J, Wu M, Traum D, Kondo A, May CL, Goldman N, Wang W, Feldman M, Moore JH, Japp AS, Betts MR, Consortium H, Faryabi RB, Naji A, Kaestner KH, Vahedi G. Single-cell multi-omics analysis of human pancreatic islets reveals novel cellular states in type 1 diabetes. Nature Metabolism. 2022;4(2):284-99. Cited > 80 times.

6. Patil AR, Schug J, Naji A, Kaestner KH, Faryabi RB, Vahedi G. Single-cell expression profiling of islets generated by the Human Pancreas Analysis Program. Nature Metabolism. 2023;5(5):713-5.

7. Patil AR, Schug J, Liu C, Lahori D, Descamps HC, Human Pancreas Analysis C, Naji A, Kaestner KH, Faryabi RB, Vahedi G. Modeling type 1 diabetes progression using machine learning and single-cell transcriptomic measurements in human islets. Cell Reports Medicine. 2024;5(5):101535.

8. Abedi M, Rai, P., Zhou, Y., Liu, C., Johnson, I., Chandra, A., Fasolino, M., Rostami, S., Kaestner, K., Naji, A., Faryabi, R. #, Vahedi, G. # Aberrant TNF signaling in pancreatic lymph nodes of patients with Type 1 Diabetes. biorXiv. 2024.

V: Functional assessment and optical reconstruction of multi-way enhancer hubs. Since we published the functional assessment of a 25kbp noncoding deletion in the Ets1-Fli1 locus and its link to allergic inflammation (4), we have engineered this locus using four additional mouse strains. Moreover, we have implemented the optical reconstruction of chromatin architecture (ORCA) to visualize multi-way interactions at the single-allele level. We will dedicate major efforts to better understand the dynamics of multi-way enhancer interactions by combining CRISPR screening, microscopy, mouse engineering, and cellular immunology at the Ets1-Fli1 and additional multi-enhancer hub loci.