TET proteins regulate T cell and iNKT cell lineage specification in a TET2 catalytic dependent manner

Abstract

TET proteins mediate DNA demethylation by oxidizing 5-methylcytosine to 5-hydroxymethylcytosine (5hmC) and other oxidative derivatives. We have previously demonstrated a dynamic enrichment of 5hmC during T and invariant natural killer T cell lineage specification. Here, we investigate shared signatures in gene expression of Tet2/3 DKO CD4 single positive (SP) and iNKT cells in the thymus. We discover that TET proteins exert a fundamental role in regulating the expression of the lineage specifying factor Th-POK, which is encoded by Zbtb7b. We demonstrate that TET proteins mediate DNA demethylation - surrounding a proximal enhancer, critical for the intensity of Th-POK expression. In addition, TET proteins drive the DNA demethylation of site A at the Zbtb7b locus to facilitate GATA3 binding. GATA3 induces Th-POK expression in CD4 SP cells. Finally, by introducing a novel mouse model that lacks TET3 and expresses full length, catalytically inactive TET2, we establish a causal link between TET2 catalytic activity and lineage specification of both conventional and unconventional T cells.

MiR-10b-5p Impairs TET2-Mediated Inhibition of PD-L1 Transcription Thus Promoting Immune Evasion and Tumor Progression in Glioblastoma

“…TET2 mutations is also found to be associated with lineage specification of conventional and unconventional T cells, such as invariant natural killer T cells, myeloid cell, and B-cell, indicating previse biological role of TET2 in immune response (Aijo et al 2022). TET2 deficiency in regulatory T cells resulted in a lethal hyperproliferation, promoted Foxp3 destabilization, and caused conversion into IL-17-producing cells in the spleen and mesenteric lymph nodes (Nakatsukasa et al 2019).…”

Protocol to isolate mature thymic T cell subsets using fluorescence-activated cell sorting for assessing gene expression by RNA-seq and transcription factor binding across the genome by CUT&RUN

“…Then, details are provided to prepare libraries for RNA-seq in order to assess gene expression and CUT&RUN, in order to assess transcription factor binding, from 500.000 FACS sorted CD4 SP cells. 1 However, we have also used this protocol to isolate CD8 SP cells and iNKT cells. 2 , 3 We have isolated total RNA and performed RNA-seq.…”

“…We have not generated any new genome-wide datasets or code for this study. We have used datasets from our published article 1 ( Äijö et al., 2022 ) that are available at GEO: GSE206450 .…”

Deciphering the TET3 interactome in primary thymic developing T cells

“… 34 , 35 Moreover, TET2 and TET3 fine-tune the expression of the transcription factor ThPOK both in CD4 SP and in iNKT cells, by regulating DNA demethylation of the locus and binding of the transcription factor GATA3. 36 While in the aforementioned T cell subsets at least 2 TET proteins must be deleted to observe impactful phenotypes, we have previously reported that deficiency of TET3 alone is sufficient to impact iNKT cell lineage specification in the thymus and promote skewing toward iNKT cells that express RORγt and secrete IL-17, known as NKT17 cells. 15 , 37 , 38 …”

“…In the present study, we focus on TET3, the most highly expressed of the TET proteins in thymic murine T cell subsets 11 , 36 and yet the least studied among the TET proteins. We seek to discover the interacting partners that mediate recruitment of TET3 across the genome in developing thymic T cells and to decipher the biological implications of these interactions.…”