Transcriptional establishment of cell-type identity: dynamics and causal mechanisms of T-cell lineage commitment

Abstract

Precursor cell entry into the T-cell developmental pathway can be divided into two phases by the closure of T-lineage commitment. As cells decide against the last alternative options to the T-cell fate, they turn on the transcription factor Bcl11b and silence expression of a group of multipotent progenitor regulatory factors that include hematopoietic transcription factor PU.1. Functional perturbation tests show that Bcl11b is needed for commitment while PU.1 actively participates in keeping open access to alternative fates, until it is silenced; however, PU.1 and Bcl11b both contribute positively to T-cell development. Our recent work reviewed here sheds light on the transcriptional regulatory network that determines the timing and irreversibility of Bcl11b activation, the ways that Notch signaling from the thymic microenvironment restricts the action of PU.1 to prevent it from diverting cells to non-T fates, and the target genes that PU.1 still regulates under the influence of Notch signaling to contribute to T-cell generation. We argue that T-cell development depends on the sequential operation of two interlaced, but mutually antagonistic, gene regulatory networks, one initially supporting expansion before commitment and the other imposing a "terminal" differentiation process on committed cells.

Figure 1

Outline of early T cell development. Top: succession of early intrathymic developmental stages referred to in the text. LMPP: lymphoid-primed multipotent precursor. CLP: common lymphoid precursor. DN: CD4⁻ CD8⁻ surface TCR⁻. ETP (Kit-high DN1): Kit^high CD44^high CD25⁻ DN. DN2a: Kit^high CD44^high CD25⁺ DN. DN2b: Kit^int/+ CD44^high CD25⁺ DN. DN3a: Kit^low CD44^low CD25⁺ DN. DP: CD4⁺ CD8⁺ TCRβ⁺. Blue arrows depict continuous requirement for Notch signaling from ETP through DN3a stage, despite substantial regulatory changes throughout these stages. Curved reflex arrows depict phases of proliferation, either self-renewal or “transit amplifying” type. Lower panel: schematic depicting expression patterns (impressionistic log scale) of three T-lineage transcription factors, GATA-3, TCF-1, and Bcl11b, one pan-hematopoietic transcription factor Runx1/CBFβ, and the multilineage transcription factor PU.1. Other phase 1 transcription factors are regulated similarly to PU.1 (see Fig. 2, Table 1). Figure adapted from (Rothenberg 2012).

Figure 2

Transcription factors that change expression during commitment. Main panel: genes encoding transcriptional regulators that change most in expression between ETP/DN1 stage and newly committed DN2b stage, with fold changes measured by genome-wide RNA-seq analysis (Zhang et al. 2012). Genes annotated as regulatory genes with largest fold decreases in expression or increases in expression (DN2b/DN1 levels <0.5 or >2, FDR<0.05) are arranged on x axis in order of fold change. Values on y axis are log₂-transformed ratios of (expression in DN1)/(expression in DN2b). Axis order is set to show downregulated genes below the x axis and upregulated genes above the x axis. PU.1 (Sfpi1) and Bcl11b are highlighted by red labels. Inset: schematic depiction of expression pattern of Bcl11b as defined at the single-cell level by expression of a Bcl11b-IRES-mCitrine fluorescent protein reporter allele described in text (H.Y.K. and E.V.R., unpublished data).

Figure 3

Feedforward motifs in a gene regulatory network model for T-cell specification. Schematic shows a partial gene regulatory network contributing to the activation and impact of Bcl11b expression. Solid lines: experimental evidence for regulatory impact and direct binding. Dashed lines: evidence for direct binding, possible but not yet demonstrated function at those sites. Dotted lines: evidence for functional effect.

Figure 4

A dominant negative PU.1-engrailed fusion protein construct represses genes that are enriched for natural PU.1 binding targets in early T cells. Top panel: genome-wide analysis of impact of PU.1-engrailed on expression of target genes, as measured by RNA-seq (dark red symbols, A.C., S. D., and E.V.R., unpublished results), plotted against peak levels of endogenous PU.1 binding associated with the same genes in normal DN1 and DN2a cells (blue symbols)(Zhang et al. 2012). The genes shown were selected for >3x upregulation (left side) or >3x downregulation (right side) by PU.1-engrailed relative to empty vector, 24 hr after transduction, and are ordered on the x axis from most upregulated to most downregulated. Note that the genes repressed by PU.1-engrailed expression (toward the right side) tend to include genes linked to stronger PU.1 binding sites in vivo. In contrast, an equal number of genes are upregulated in cells forced to express PU.1-engrailed, but these do not show any enrichment for strong PU.1 binding, consistent with indirect regulation by PU.1 (A.C., S. D., and E.V.R., unpublished results). Bottom panel: Venn diagram outlining the strategy for combining dominant negative perturbation, mapping of natural PU.1 binding site occupancy, and natural developmental expression patterns to identify new PU.1 targets in an unbiased way.

Figure 5

A partial gene regulatory network driven by PU.1 in early T-lineage precursors. The figure summarizes representative linkages from the studies described in the text (Franco et al. 2006; Carotta et al. 2010a; Del Real and Rothenberg 2013)(A.C., S.D., S. L. Nutt, S. Carotta, and E.V.R., unpublished results). Note that combinatorial effects from PU.1 and Notch on certain targets can split the expression patterns seen in vivo (e.g. Lyl1 vs. Flt3). Additional phase 1 genes that can be activated by PU.1 in these cells include Mef2c, Meis1, and many myeloid differentiation genes; however, other genes with “phase 1” expression patterns appear to be direct negative, not positive, regulatory targets of PU.1 (not shown; A.C., S.D., S. L. Nutt, S. Carotta, and E.V.R., unpublished results). The effects depicted are those that survive Notch effects on PU.1 activity itself (Franco et al. 2006; Del Real and Rothenberg 2013). Under these conditions at least, PU.1 appears to damp expression of “T-cell genes” such as Tcf7, Ets1, Zfpm1 through an indirect mechanism, based on results with the obligate repressor dominant negative, suggesting the involvement of a PU.1-activated “Repressor X” function(s) (A.C. & E.V.R., unpublished).

Figure 6

Two sequential gene regulatory networks compete to control the timing of commitment in T-cell precursors. Summary of discussion in text.