1. During the homeostatic proliferation of CD8+ T cells, how the downstream JAK/STAT signaling pathway alters chromatin state to regulate gene expression and promote cell proliferation has not been well answered.  We have discovered that two key factors in immune CD8+ T cells—TCF1, a cell fate determinant, and CTCF, a chromatin structure regulatory protein—collaborate to regulate the homeostatic proliferation of naïve CD8+ T cells by altering chromatin accessibility and 3D structure. CTCF is an important chromatin structural protein, generally considered an insulator located at the boundaries of chromatin topological domains. TCF1, the fate determinant for CD8+ T cells, is a crucial transcription factor involved in T cell development, differentiation, and cancer immunotherapy efficacy. The study found that CTCF not only acts as an insulator but also interacts with TCF1 to regulate key gene expression by increasing chromatin accessibility and enhancer-promoter interactions, thereby maintaining the homeostatic proliferation of CD8+ T cells. This study was published in Nature Immunology in 2022 (PMID: 35882936).
2. Upon antigen stimulation, naïve CD8+ T cells differentiate into effector CD8+ T cells. However, few studies have explored whether changes in the 3D chromatin structure influence this differentiation process. We found that CTCF plays a critical role in shaping these chromatin structure changes and regulating gene expression. Using multi-omics approaches, including RNA-seq, ATAC-seq, CUT&RUN, and HiC, we discovered that changes in CTCF during CD8+ T cell differentiation are significantly correlated with the expression of genes involved in immune activation and the cell cycle. Using the hichub algorithm, we systematically compared regions of 3D chromatin interaction between naïve and effector CD8+ T cells. Many regions near genes related to immune function and stemness exhibited significant structural changes, driven by CTCF binding alterations. Further studies involving CTCF knockout revealed that the differentiation of effector CD8+ T cells was inhibited, with the cells remaining in a naïve state and showing transcriptional features of memory T cells. Analysis of chromatin insulation boundaries associated with CTCF revealed two functions of CTCF during immune cell activation: (1) within topologically associated domains (TADs), CTCF interacts with enhancers and related transcription factors (such as AP-1 and Tbx21) to promote the expression of immune-related genes; (2) at TAD boundaries, CTCF ensures the structural stability within TADs through its insulating role, facilitating the expression of stemness genes (such as Tcf1 and Id3) in naïve CD8+ T cells, thereby regulating their transcriptional state. This study was published in Journal of Experimental Medicine in 2023 (PMID: 36752796).

3, Naïve CD8+ T cells are a class of immune cells that have not been activated, while memory CD8+ T cells are formed after responding to antigen invasion, preparing for future antigen challenges. How the 3D chromatin structure changes during this transition affects the secondary antigen response. This issue involves 3D structural changes across multiple cell states, requiring more complex algorithms to identify patterns. The applicant developed a new algorithm called multi-HiCHub to systematically and comprehensively compare 3D chromatin structural changes between different cell types. This algorithm can accurately capture dynamic patterns in chromatin structure. When comparing hichub 3D structural changes in naïve, effector, and memory CD8+ T cells, hichub and differential gene expression (DEG) were classified into distinct change pattern categories and sorted in similar order. The results showed that the hichub change patterns were highly consistent with the differential gene expression changes within them, demonstrating the superior precision of the hichub method over other 3D chromatin structure analysis methods. Further analysis of the dynamic patterns in multi-HiCHub revealed that memory T cells not only inherit the stemness gene structures of naïve T cells (e.g., the structure near Il7r) but also inherit the effector gene structures of effector T cells (e.g., the structure near the chemokine Ccl gene family). This provides a chromatin structural basis for memory T cells to respond rapidly to secondary antigen invasion. Moreover, CTCF knockout experiments showed that memory T cells failed to undergo cell division upon antigen stimulation, further demonstrating the importance of chromatin structure for memory T cells. This study was published in PNAS in 2023 (PMID: 38085779).

CTCF,3D genome,transcriptional regulation,CD8+ T cells,epigenetics