Control of Foxp3 induction and maintenance by sequential histone acetylation and DNA demethylation.

Regulatory T (Treg) cells play crucial roles in suppressing deleterious immune response. Here, we investigate how Treg cells are mechanistically induced in vitro (iTreg) and stabilized via transcriptional regulation of Treg lineage-specifying factor Foxp3. We find that acetylation of histone tails at the Foxp3 promoter is required for inducing Foxp3 transcription. Upon induction, histone acetylation signals via bromodomain-containing proteins, particularly targets of inhibitor JQ1, and sustains Foxp3 transcription via a global or trans effect. Subsequently, Tet-mediated DNA demethylation of Foxp3 cis-regulatory elements, mainly enhancer CNS2, increases chromatin accessibility and protein binding, stabilizing Foxp3 transcription and obviating the need for the histone acetylation signal. These processes transform stochastic iTreg induction into a stable cell fate, with the former sensitive and the latter resistant to genetic and environmental perturbations. Thus, sequential histone acetylation and DNA demethylation in Foxp3 induction and maintenance reflect stepwise mechanical switches governing iTreg cell lineage specification.

Combinatorial ETS1-dependent control of oncogenic NOTCH1 enhancers in T-cell leukemia.

Notch activation is highly prevalent among cancers, in particular T-cell acute lymphoblastic leukemia (T-ALL). However, the use of pan-Notch inhibitors to treat cancers has been hampered by adverse effects, particularly intestinal toxicities. To circumvent this barrier in T-ALL, we aimed to inhibit ETS1, a developmentally important T-cell transcription factor previously shown to co-bind Notch response elements. Using complementary genetic approaches in mouse models, we show that ablation of Ets1 leads to strong Notch-mediated suppressive effects on T-cell development and leukemogenesis, but milder intestinal effects than pan-Notch inhibitors. Mechanistically, genome-wide chromatin profiling studies demonstrate that Ets1 inactivation impairs recruitment of multiple Notch-associated factors and Notch-dependent activation of transcriptional elements controlling major Notch-driven oncogenic effector pathways. These results uncover previously unrecognized hierarchical heterogeneity of Notch-controlled genes and points to Ets1-mediated enucleation of Notch-Rbpj transcriptional complexes as a target for developing specific anti-Notch therapies in T-ALL that circumvent the barriers of pan-Notch inhibition.

Stage-specific roles for Zmiz1 in Notch-dependent steps of early T-cell development.

Notch1 signaling must elevate to high levels in order to drive the proliferation of CD4-CD8- double-negative (DN) thymocytes and progression to the CD4+CD8+ double-positive (DP) stage through ß-selection. During this critical phase of pre-T-cell development, which is also known as the DN-DP transition, it is unclear whether the Notch1 transcriptional complex strengthens its signal output as a discrete unit or through cofactors. We previously showed that the protein inhibitor of activated STAT-like coactivator Zmiz1 is a context-dependent cofactor of Notch1 in T-cell leukemia. We also showed that withdrawal of Zmiz1 generated an early T-lineage progenitor (ETP) defect. Here, we show that this early defect seems inconsistent with loss-of-Notch1 function. In contrast, at the later pre-T-cell stage, withdrawal of Zmiz1 impaired the DN-DP transition by inhibiting proliferation, like withdrawal of Notch. In pre-T cells, but not ETPs, Zmiz1 cooperatively regulated Notch1 target genes Hes1, Lef1, and Myc. Enforced expression of either activated Notch1 or Myc partially rescued the Zmiz1-deficient DN-DP defect. We identified residues in the tetratricopeptide repeat (TPR) domain of Zmiz1 that bind Notch1. Mutating only a single residue impaired the Zmiz1-Notch1 interaction, Myc induction, the DN-DP transition, and leukemic proliferation. Similar effects were seen using a dominant-negative TPR protein. Our studies identify stage-specific roles of Zmiz1. Zmiz1 is a context-specific cofactor for Notch1 during Notch/Myc-dependent thymocyte proliferation, whether normal or malignant. Finally, we highlight a vulnerability in leukemic cells that originated from a developmentally important Zmiz1-Notch1 interaction that is hijacked during transformation from normal pre-T cells.