Regulation of early T-lineage gene expression and developmental progression by the progenitor cell transcription factor PU.1

Abstract

The ETS family transcription factor PU.1 is essential for the development of several blood lineages, including T cells, but its function in intrathymic T-cell precursors has been poorly defined. In the thymus, high PU.1 expression persists through multiple cell divisions in early stages but then falls sharply during T-cell lineage commitment. PU.1 silencing is critical for T-cell commitment, but it has remained unknown how PU.1 activities could contribute positively to T-cell development. Here we employed conditional knockout and modified antagonist PU.1 constructs to perturb PU.1 function stage-specifically in early T cells. We show that PU.1 is needed for full proliferation, restricting access to some non-T fates, and controlling the timing of T-cell developmental progression such that removal or antagonism of endogenous PU.1 allows precocious access to T-cell differentiation. Dominant-negative effects reveal that this repression by PU.1 is mediated indirectly. Genome-wide transcriptome analysis identifies novel targets of PU.1 positive and negative regulation affecting progenitor cell signaling and cell biology and indicating distinct regulatory effects on different subsets of progenitor cell transcription factors. Thus, in addition to supporting early T-cell proliferation, PU.1 regulates the timing of activation of the core T-lineage developmental program.

Keywords: Notch; RNA-seq; conditional knockout; hematopoiesis; in vitro development; obligate repressor.

Figure 1.

Deletion of PU.1 in c-Kit⁺ CD27⁺ FLPs results in impaired DN progression and poor survival and recovery of early DN stage T cells. (A) E14.5 B6 and Spi1^fl/fl FLPs were infected with Cre-expressing retroviral supernatant. One day after the infection, Cre⁺ Kit⁺ CD27⁺ cells were sorted and used to start OP9-DL1 cultures. These cultures were harvested on the indicated days and analyzed for DN progression as shown in the figure. FACS plots are representative of two independent experiments. (B) Plot summarizing Cre⁺ Spi1^fl/fl cell counts expressed relative to the number of Cre⁺ B6 cells on day 4 of OP9-DL1 culture, from two independent experiments. Error bars indicate one standard deviation. (C) Failure of Bcl-xL to rescue progression to CD25⁺ (DN2/DN3) stages when PU.1 is deleted. Cells were prepared as in A, except that they were doubly transduced with Cre (NGFR⁺) and Bcl-xL (GFP⁺) retroviruses or controls. Results are from one experiment, representative of two independent experiments.

Figure 2.

PU.1 retards DN progression and enhances proliferation of early T cells. (A) Scheme for cell cycle progression analysis of progeny of PU.1-deleted DN1 cells. B6 and Spi1^fl/fl FLPs were cultured on OP9-DL1 cells for 3 d and infected with Cre-expressing and Bcl-xL-expressing retroviruses. Two days later, Cre⁺ Bcl-xL⁺ DN1 cells were sorted, loaded with CTV dye, and put back in fresh OP9-DL1 cultures. Two days and 3 d later, population phenotypes and CTV fluorescence were determined by FACS analysis. (B) Phenotypes of recultured cells at day 3. (C) Extents of CTV dilution for progeny of labeled DN1 cells, comparing PU.1-deleted DN1 and DN2 progeny with controls. Arrows indicate the day 0 CTV mean fluorescence intensity (MFI) for each population shown in the histogram. (D) All four populations from the histograms in C, superimposed to show the differences in proliferation of various subsets. (E) Relative proliferation of B6 and Spi1^fl/fl DN cell populations measured by the differences in dilution of CTV at day 2 and day 3. CTV MFI for each population was normalized to the initial loading value and then plotted relative to the day 2 control B6 DN1 value. (F) Flowchart showing the design of DN stage progression experiments. E14.5 B6 and Spi1^fl/fl FLPs were cultured on OP9-DL1 for 3 d and infected with a Cre-expressing retrovirus. Cre⁺ DN1, DN2a, and DN2b populations were sorted and used to seed the cultures analyzed 5 d later in G and H. (G) DN stage progression of cells descended from the indicated starting populations, based on CD44 and CD25 expression. The diagram at the right shows populations defined by the four quadrants. (H) Population distributions of progeny subsets descended from the indicated Cre⁺ starting populations, averaged from two independent DN progression experiments as in F and G. Error bars represent one standard deviation.

Figure 3.

Deletion of Spi1 leads to derepression of T-lineage genes. (A) Flowchart for obtaining B6 and Spi1^fl/fl DN1, DN2a, and DN2b cells to determine the effect of loss of PU.1 on the T-lineage developmental program. cDNA was prepared from DN subsets sorted in A, and gene expression changes were measured in the indicated populations using quantitative PCR (qPCR). (B–J) Actin-normalized expression values averaged from two independent experiments are expressed as the fold change relative to the B6 DN1 values (B,D,F,I) or the B6 DN2a values (C,E,G,H,J). The resulting data were plotted for phase 1 and alternate lineage genes (B,C), T-lineage genes (D,E), components of the Notch signaling pathway (F–H), and alternate lineage genes (I,J). Error bars represent one standard deviation. Significant differences at P < 0.05 are indicated with an asterisk (DN1 and DN2) and pound sign (DN2b).

Figure 4.

PU.1 constructs retain target gene specificity and are able to block wild-type (WT) PU.1 function efficiently. (A) Cartoon showing the domain organization of wild-type PU.1 and the various PU.1 constructs used in this study. (B) PU.1-Eng, PU.1-ETS, and PU.1-ΔDEQ can block wild-type PU.1 activity. Adh-2C2 cells were infected with various constructs, as indicated at the top of each panel. After 48 h, diversion to the myeloid lineage was determined by analyzing the surface up-regulation of CD11b. The data in the FACS plots are representative of two independent experiments. (C) Repression of Il7r and Flt3 in progenitors by PU.1-Eng. E14.5 B6 FLPs were transduced with PU.1-Eng and cultured without OP9 stromal cells in medium supplemented with 1 ng/mL SCF and 5 ng/mL each Flt3L and IL-7. Cultures were harvested after 48 h and analyzed by flow cytometry for the indicated markers (representative of two independent experiments). (D) Repression of Itgam and Coro2a depends on the integrity of the PU.1-Eng DNA-binding domain. Plots show gene expression measured by qPCR in sorted FLDN cells 24 h after transduction with empty vector (EV; blue), PU.1 wild type (red), PU.1-Eng (gold), or PU.1-Eng W215G (green). The effects of PU.1-Eng were compared with PU.1 wild type in DN2a and DN2b cells (two experiments), and PU.1-Eng was compared with PU.1-Eng W215G in DN1 and total DN2 cells (two separate experiments). Values (average ± range) are shown relative to Actb on a log₁₀ scale. (E) The lack of effect of PU.1-Eng in Adh.2C2 pro-T cells. Adh.2C2 cells were transduced with the constructs indicated and harvested at 48 h for gene expression analysis by qPCR. Average, Actin-normalized log₁₀ expression values from two independent experiments were used to generate the heat map shown in the figure. All values are row-normalized, and the color scale at the right denotes 30-fold up-regulation (dark red) and 30-fold down-regulation (dark blue) of gene expression.

Figure 5.

PU.1-Eng transduction up-regulates T-lineage-specific gene expression. (A) Cartoon showing alternative mechanisms by which wild-type PU.1 could repress T-cell genes and the use of PU.1-Eng to distinguish between these possibilities. (Left panel) PU.1 could directly repress T-lineage genes by recruiting repressors to silence each gene individually or up-regulate the expression of a repressor “X” that could in turn repress the T-cell program. The effects of obligate repressor PU.1-Eng under the two scenarios are shown in the right panel. (B) Schematic of experiments to generate the data shown in C and D and Supplemental Figure S6, A–F. Bcl2-tg FLDN cells were used to enhance viability under perturbation. They were processed as shown and sorted to purify DN1, DN2a, and DN2b cells for gene expression analysis by qPCR. (C,D) The retroviral supernatants used to express various constructs are indicated above each lane. Sorted DN stage cells from two to three independent experiments were pooled to obtain each set of qPCR data. Average, Actin-normalized log₁₀ expression values from two such pooled data sets were used to generate the heat maps shown in the figure. All values were row-normalized to the control DN2a (C) or control DN1 (D) sample, and the common color scale at the right denotes 30-fold up-regulation (dark red) and 30-fold down-regulation (dark blue) of gene expression. Boldface gene names indicate Notch-activated genes.

Figure 6.

Identification of positive and negative PU.1 target genes by genome-wide analysis. (A) Effects on expression of top-scoring PU.1-Eng-repressed and PU.1-Eng-activated genes in two independent RNA-seq experiments. Data from the two experiments are shown as fold change (log₂ scale) from EV samples in the same experiment. Genes profiled in B–G are identified. (B–G) University of California at Santa Cruz (UCSC) genome browser portraits of three genes down-regulated by PU.1-Eng (B–D) and three genes up-regulated by PU.1-Eng (E–G). Boxed tracks show RNA-seq data from two different experiments (magenta and green) comparing EV, PU.1-Eng, and PU.1-ETS transduced cells with normal reference samples (blue) from DN1, DN2a, DN2b, and DP stages. Red tracks show positions of H3K4me2 histone marks, and brown tracks show binding of PU.1 based on chromatin immunoprecipitation (ChIP) combined with deep sequencing (ChIP-seq) in the same reference cell types. Direction of transcription is right to left in B–D and left to right in E–G. Full scales for vertical axes in B–G are 2.5 reads per million (RPM) for all H3K4me2 ChIP-seq, 5 RPM for all PU.1 ChIP-seq, 1.5 RPM for all RNA-seq tracks in B–D, 5 RPM for all RNA-seq in E and F, and 3 RPM for all RNA-seq in G.

Figure 7.

Characteristics of PU.1 target genes identified by genome-wide analysis. (A) Histogram comparing in vivo PU.1-binding distributions among genes repressed by PU.1-Eng (red), genes up-regulated by PU.1-Eng (light green), and control genes expressed stably and unaffected by PU.1-Eng (dark blue). The X-axis indicates histogram bins and summed ChIP-seq signals for each gene (at top four sites), and the Y-axis shows the fraction of genes in the set in the indicated bin. (B) Developmental indexing of the two independent PU.1-Eng (ENG), PU.1-ETS (ETS), and EV transduced samples as compared with normal reference cells (Zhang et al. 2012). Principal components were based on expression patterns of 173 regulatory genes, as described in the Supplemental Material (Supplemental Table S3). (C,D) Gene set enrichment analyses of the effects of PU.1-Eng on genome-wide transcription relative to normal developmental patterns of expression. The panels shown compare the enrichments of a set of developmentally down-regulated genes (cl. 3, 7, 9, and 23) and a set of developmentally up-regulated genes (cl. 1 and 6) against the full set of expressed loci (Supplemental Table S4J), ranked by fold change caused by PU.1-Eng. (NES) Normalized enrichment score; (positive NES) enriched; (negative NES) depleted. For full results, see Supplemental Table S4. (E–H) Effects of wild-type PU.1 addition or endogenous PU.1 deletion on PU.1-Eng-repressed target genes Gpx1, Lyn, Sh2b2, Neurl3, and Ptpn6 in FLDN1 cells (E,F) and FLDN2a and FLDN2b cells (G,H). Samples were prepared as described for PU.1 wild-type and PU.1-Eng transduced cells in Figure 5 (E,G) and for Cre⁺ control and Spi1^fl/fl cells in Figure 3 (F,H). Gene expression levels determined by qPCR are shown on a log₁₀ scale relative to Actb in E and G and on a linear scale relative to control levels in B6 DN1 or DN2a cells in F and H. Averages and the range of values from two independent experiments are shown.