Positive feedback between PU.1 and the cell cycle controls myeloid differentiation

Abstract

Regulatory gene circuits with positive-feedback loops control stem cell differentiation, but several mechanisms can contribute to positive feedback. Here, we dissect feedback mechanisms through which the transcription factor PU.1 controls lymphoid and myeloid differentiation. Quantitative live-cell imaging revealed that developing B cells decrease PU.1 levels by reducing PU.1 transcription, whereas developing macrophages increase PU.1 levels by lengthening their cell cycles, which causes stable PU.1 accumulation. Exogenous PU.1 expression in progenitors increases endogenous PU.1 levels by inducing cell cycle lengthening, implying positive feedback between a regulatory factor and the cell cycle. Mathematical modeling showed that this cell cycle-coupled feedback architecture effectively stabilizes a slow-dividing differentiated state. These results show that cell cycle duration functions as an integral part of a positive autoregulatory circuit to control cell fate.

Fig. 1. Cell-cycle lengthening drives PU.1 up-regulation during macrophage development

FLPs (Lin-cKit+CD27+) from E13.5 PU.1-GFP mice were cultured with B- and macrophage-supporting cytokines (SCF, IL-3, IL-7, Flt3L, M-CSF) and analyzed using timelapse imaging or flow cytometry. A) Schematic showing myeloid and lymphoid development from haematopoietic progenitor cells. B) Histograms (left) (left) show PU.1-GFP levels measured after the indicated number of days in culture. Dotted lines give initial PU.1-GFP levels. Flow cytometry plots (right) show CD19, CD11b and Gr-1 levels against PU.1-GFP after six days. C) Merged DIC (gray) and PU.1-GFP fluorescence (green) images of cultured FLPs, taken after the indicated number of hours. Cells with PU.1-GFP time traces shown in F) are marked with correspondingly colored arrowheads. Scale bar = 20 μm. D) Heat map showing PU.1-GFP levels over time for all imaged cells. Rectangles define progenitor (gray), macrophage (blue) and B-cell (red) populations. E) Alternative hypotheses for PU.1-GFP up-regulation in macrophages. The PU.1 synthesis rate for a single cell is given by (Δp/Δt) over the entire observed cell cycle. F) Representative single-cell PU.1-GFP time traces for different cell populations. Data are taken from lineages shown in Fig. S9. Horizontal lines give PU.1-GFP level thresholds for the defined cell populations. G) Histogram (top) showing distribution of PU.1 synthesis rates in progenitors. Scatterplot shows relationship between PU.1 synthesis rates in mother versus daughter cells. Horizontal and vertical lines indicate the threshold for progenitor sub-populations with higher and lower rates of PU.1 synthesis. H) Plots comparing mean PU.1-GFP levels (top), PU.1 synthesis rates (middle) and cell cycle lengths (bottom) in different cell populations. Red crosses indicate boxplot outliers. Bottom error bars represent 95% confidence intervals. Asterisks indicate significantly different means (p<10⁻⁷, one-tailed t-test). Data are representative of three independent experiments.

Figure 2. PU.1 accumulation due to cell-cycle lengthening is important for myeloid differentiation

A) Two hypotheses for the function of high PU.1 levels in differentiating macrophages. B) FLPs were transduced with empty vector (EV), p21, or p27, cultured for four days and analyzed by flow cytometry. Histograms (top) show CellTrace Violet and PU.1-GFP levels for different transduced populations. Gray shaded area indicates slow-dividing cell gate. Flow plots (bottom) show CD11b versus PU.1-GFP levels for different transduced cell populations. C) FLPs transduced with EV or PU.1 antagonist (PU.1-ets) were cultured for 3 days with or without 2.1 μM CDK4/6 inhibitor PD0332991, and analyzed by flow cytometry. Flow plots show CellTrace Violet (top) or CD11b (bottom) versus PU.1-GFP for the different conditions. D) Effects of PD0332991 and PU.1-ets transduction on the percentage of myeloid cells. Bars represent means of two independent experiments, and circles give individual measurements.

Fig. 3. PU.1 up-regulates its own expression during macrophage development by inducing cell-cycle lengthening

FLPs transduced with EV or PU.1 retroviral constructs were sorted and cultured with multi-lineage supporting cytokines (SCF, IL-3, IL-7, Flt3L). A) Histogram showing PU.1-GFP levels (top), and flow plots showing CD11b versus PU.1-GFP levels after four days of culture (bottom). B) Heat maps comparing time evolution of PU.1-GFP levels for imaged EV- or PU.1-transduced cells. C) Representative PU.1-GFP time traces for EV or PU.1-transduced cells, taken from lineage trees shown in Fig. S14. D) Box-plots comparing EV- and PU.1-transduced progenitors, showing percentage of slow dividing cells (top), along with maximal PU.1-GFP levels (middle), and PU.1 synthesis rate (bottom) for both the entire PU.1-transduced progenitor population and slow-dividing progenitors alone. Red crosses indicate outliers. Asterisks indicate significantly different means (% slow dividing, p < 0.05, χ² test, d.f. = 1; maximal PU.1-GFP, one-tailed t-test, p<0.005). Data are representative of two independent experiments. E) EV or PU.1-transduced FcγR2/3^low FLPs were cultured for 2 days, harvested for RNA and analyzed using qRT-PCR. Bar chart shows mRNA level fold change for the indicated genes in PU.1-transduced as compared to EV-transduced cells. Bars represent the means of two independent experiments, and circles represent individual measurements (*-p < 0.1; **-p < 0.01; two-tailed t-test).

Fig. 4. A cell-cycle coupled feedback loop stably maintains a slow-dividing differentiated state

A) Schematic of a cell-cycle coupled positive feedback loop (top), and time traces from stochastic simulations of this circuit architecture (bottom), showing four cells with different initial PU.1 levels but identical rate constants. B) Schematic of a hybrid cell-cycle coupled/transcriptional positive feedback circuit (top), and stochastic simulations of this architecture (bottom), showing maintenance of three stable steady-states. C) Phase diagrams for the two circuit architectures, showing PU.1 synthesis rates (black – hybrid; gray – cell-cycle coupled), as well as dilution rate and cell division rate (same for both models) against PU.1 levels. Red, gray and blue circles denote B, progenitor and macrophage steady-states respectively, and arrows indicate flow of the system. A thorough analysis and discussion of all models is given in the mathematical appendix (22).