Architecture of a lymphomyeloid developmental switch controlled by PU.1, Notch and Gata3

Abstract

Hematopoiesis is a classic system with which to study developmental potentials and to investigate gene regulatory networks that control choices among alternate lineages. T-cell progenitors seeding the thymus retain several lineage potentials. The transcription factor PU.1 is involved in the decision to become a T cell or a myeloid cell, and the developmental outcome of expressing PU.1 is dependent on exposure to Notch signaling. PU.1-expressing T-cell progenitors without Notch signaling often adopt a myeloid program, whereas those exposed to Notch signals remain in a T-lineage pathway. Here, we show that Notch signaling does not alter PU.1 transcriptional activity by degradation/alteration of PU.1 protein. Instead, Notch signaling protects against the downregulation of T-cell factors so that a T-cell transcriptional network is maintained. Using an early T-cell line, we describe two branches of this network. The first involves inhibition of E-proteins by PU.1 and the resulting inhibition of Notch signaling target genes. Effects of E-protein inhibition can be reversed by exposure to Notch signaling. The second network is dependent on the ability of PU.1 to inhibit important T-cell transcription factor genes such as Myb, Tcf7 and Gata3 in the absence of Notch signaling. We show that maintenance of Gata3 protein levels by Myb and Notch signaling is linked to the ability to retain T-cell identity in response to PU.1.

GATA-3 Dose-Dependent Checkpoints in Early T Cell Commitment

“…2E). For more extensive confirmation of the effects of this shRNA on Gata3 RNA and GATA-3 protein in the Adh.2C2 system, also see our recent report (43). Gata3 shRNA thus consistently lowered GATA-3 protein levels, despite its generally mild effect on RNA levels.…”

“…PU.1 and GATA-3 act as antagonists in a network circuit in which GATA-3 restrains the ability of PU.1 to cause myeloid differentiation of DN pro-T cells, even when Spi1 expression itself is not under GATA-3 control (43). PU.1 naturally begins at high levels in ETP and DN2a cells, but falls immediately after GATA-3 reaches its peak (data not shown) (42), and our results suggested that GATA-3 might be required for this decline.…”

“…However, in contrast to cells transduced with Gata3 shRNA, now Zfpm1 (FOG1) was clearly downregulated. This was significant because Zfpm1 exhibits strong binding of GATA-3 in vivo (13) and seems responsive to GATA-3 in a model cell line system (43). If incubation was continued for an additional day before harvesting the cells, detectable reductions in Tcf7, Rag1, Kit , and Zbtb16 (PLZF) were seen, as well as effects on Bcl11b and Ets1 (Fig.…”

Irf4 Regulates the Choice between T Lymphoid-Primed Progenitor and Myeloid Lineage Fates during Embryogenesis

“…These studies indicate that Pu.1 may be repressed gradually following T cell commitment from HSCs. In the thymus, antagonism between Notch and Pu.1 maintains the identity of T cell progenitor and prevents the adoption of myeloid fate (Del Real and Rothenberg, 2013;Franco et al, 2006). However, the underlying molecular mechanism for the downregulation of Pu.1 at the prethymic stage is still unclear.…”

Epigenetic control of hematopoiesis: the PU.1 chromatin connection

“…Importantly and similar to the erythroid lineage, PU.1 expression is required in later T-cell progenitors to become repressed (Anderson et al, 2002), stays repressed in Th2 cells and CD8 T cells, but is reexpressed in Th9 cells (Gerlach et al, 2014). To achieve PU.1 repression in early T cells, the activity of PU.1 is antagonized by Notch, which is essential to push thymocytes into further maturation (Del Real and Rothenberg, 2013). Moreover, T-cell-specific repression of PU.1 expression is thought to be facilitated by binding of Runx1 to a silencer element located between the Sfpi1 promoter and its upstream enhancer elements (Zarnegar et al, 2010).…”