The interplay between the master transcription factor PU.1 and miR-424 regulates human monocyte/macrophage differentiation

Abstract

We describe a pathway by which the master transcription factor PU.1 regulates human monocyte/macrophage differentiation. This includes miR-424 and the transcriptional factor NFI-A. We show that PU.1 and these two components are interlinked in a finely tuned temporal and regulatory circuitry: PU.1 activates the transcription of miR-424, and this up-regulation is involved in stimulating monocyte differentiation through miR-424-dependent translational repression of NFI-A. In turn, the decrease in NFI-A levels is important for the activation of differentiation-specific genes such as M-CSFr. In line with these data, both RNAi against NFI-A and ectopic expression of miR-424 in precursor cells enhance monocytic differentiation, whereas the ectopic expression of NFI-A has an opposite effect. The interplay among these three components was demonstrated in myeloid cell lines as well as in human CD34+ differentiation. These data point to the important role of miR-424 and NFI-A in controlling the monocyte/macrophage differentiation program.

Fig. 1.

Role of miR-424 in monocyte/macrophage differentiation. miRNA TaqMan microRNA assays (Applied Biosystem) on total RNA from: (A) CD34+ cells induced to differentiate to the monocytic lineage (samples taken at the indicated days) and (B) cells from an APL patient, before (−TPA) and 48 h after TPA treatment (+TPA). miR-25, whose levels do not change in differentiation, is used as control. (C) Ten micrograms of RNA, from untreated NB4 cells (lanes 0) or from the same cells treated with TPA for the indicated times, was analyzed by Northern blot with the probes indicated on the side of each image. Endogenous spliceosomal U2 snRNA and miR-25 were used as loading controls. (D Upper) Schematic representation of the lentiviral construct for miR-424 expression (Lenti-424); (Lower) Northern blot analysis of 10 μg of total RNA extracted from NB4 cells infected with the empty vector (lane Vector) or with Lenti-424 (lane Lenti-424) and incubated for 48 h. miR-424 signals were normalized for U2 snRNA hybridization and the values, expressed as fractions with respect to mock-treated cells (Vector), are indicated below each lane. (E) Percentage of CD11b- or CD14-positive cells in NB4 cells ectopically expressing either the empty vector or Lenti-424. (F) Morphological analysis of NB4 cells 7 days after infection with the empty vector or with Lenti-424. In all histograms, the values represent the means ± SEM from triplicates.

Fig. 2.

NFI-A is a miR-424 target. (A) Total proteins, from NB4 cells grown for the indicated hours in the absence (lanes C) or presence of TPA (lanes TPA), were analyzed by Western blot with anti-NFI-A antibodies. Signals were normalized for GAPDH and the values, expressed as fractions with respect to time 0, are indicated below. (B) qRT-PCR analysis of NFI-A mRNA levels in NB4 cells treated with TPA for the indicated times. The histograms represent the means ± SEM from triplicates. (C) Western blot analysis of proteins extracted from cells infected with the empty vector of with the Lenti-424. Signals were normalized for GAPDH, and the values, expressed as fractions with respect to mock-treated cells (Vector), are indicated below. (D) Schematic representation of the constructs used in the luciferase assay. The sequences shown below indicate: the putative miR-424 target site on the wild-type 3′UTR (construct 3′NFI-Awt), its mutated derivative (construct 3′NFI-Amut), and the pairing regions of miR-424. (E) Northern blot analysis of HeLa cells infected with the empty vector (lane C) or with Lenti-424 (lane 424). (F) Cells were infected with Lenti-424 (HeLa-424) or with the control vector (HeLa-C) and then transfected with either 3′NFI-Awt (black boxes) or 3′NFI-Amut (white boxes). Renilla luciferase activity was normalized to Firefly luciferase activity, and then the forward luciferase-containing constructs were normalized to the mutated controls. Error bars represent the SEM from triplicates.

Fig. 3.

NFI-A knockdown and overexpression. NB4 cells were infected with the empty lentivector (Vector) or with a lentiviral construct expressing siRNAs against NFI-A (siNFI-A). (A) Proteins were extracted 48 h after infection and 50 μg analyzed by Western blot with anti-NFI-A and control anti-GAPDH antibodies. Signals were normalized for GAPDH and the values, expressed as fractions with respect to mock-treated cells (Vector), are indicated below each lane. (B) CD11b- or CD14-positive cells were analyzed by FACS; the values indicate the fold induction of TPA-induced positive vs. untreated cells. (C) Expression levels of M-CSFr mRNA measured by qRT-PCR in NB4 cells infected with the siNFI-A lentiviral construct or with an empty vector. The values indicate the fold induction of TPA-induced cells vs. untreated ones. (D) Schematic representation of Lenti-HA-NFIA and Western analysis with an anti-HA antibody of its ectopic expression in NB4 cells. (E–H) NB4 cells were infected with the empty lentiviral vector (Vector) or with the Lenti-HA-NFIA. After the infection, half of the culture was treated with (+TPA) and half without TPA (−TPA). Cells were analyzed by FACS for CD11b (F) and CD14 (G) expression, by qRT-PCR for M-CSFr expression (H), and by Wright–Giemsa staining for morphology (E). For each image, the histograms represent the means ± SEM from triplicates.

Fig. 4.

PU.1 binds the miR-424 promoter. (A) Schematic representation of the miR-424 genomic region and of the constructs containing the wild-type (WT) or mutant (ΔPU.1) miR-424 promoter fused to the premiR-126 coding region. The transcriptional start sites (TSS) are indicated by open arrows. The sequence and location of the PU.1-binding site are indicated by the dashed box, whereas the prom/1 arrows point to the regions amplified by qPCR in the ChIP experiments. (B) NB4 cells were treated with TPA for the indicated times. Fifty micrograms of total protein was analyzed by Western blot with anti-PU.1 antibody. Signals were normalized for GAPDH, and the values, expressed as fractions with respect to time 0, are indicated below. (C) Chromatin from cells at different times of induction was immunoprecipitated with anti-PU.1 antibodies, and the recovered DNA was submitted to qPCR with prom/1 oligos (miR-424). Oligonucleotides corresponding to the −14-kb URE3′ region of the PU.1 promoter were used as positive control (URE3′). An unrelated genomic region (UR) was amplified as a negative control (UR). The histograms represent the means ± SEM from three independent experiments. (D) NB4 cells were infected with a lentiviral construct expressing siRNAs against PU.1 (siPU.1) or with an empty vector. Fifty micrograms of proteins was analyzed by Western blot analysis with anti-PU.1. Signals were normalized for GAPDH, and the values, expressed as fractions with respect to mock-treated cells (Vector), are indicated below. (E) Northern blot analysis of RNA from control cells (lanes −) or cells treated with TPA for 48 h (lanes +) and either infected with an empty vector (lanes Vector) or with Lenti-siPU.1 (lanes siPU.1). miR-424 signals were normalized for U2 snRNA hybridization, and the values, expressed as fractions with respect to TPA-minus samples, are indicated below. (F) NB4 cells were electroporated with WT or ΔPU.1 constructs (A), and the amount of miR-126 was measured with the Applied Biosystems TaqMan MicroRNA Assay. The values were normalized against the U6 snRNA. The histograms represent the means ± SEM from triplicates.

Fig. 5.

Role of PU.1, miR-424, and NFI-A in normal hematopoiesis. (A) Monocyte/macrophage differentiation of human CD34+ cells. Samples were collected at the indicated days after induction. (Top) FACS analysis of CD14⁺ cells; (Middle) TaqMan microRNA assays of miR-424 (Applied Biosystems). The values are normalized with the U6 snRNA, and error bars represent standard errors from triplicates. (Bottom) Western blot analysis on total proteins (15 μg) with antibodies against the indicated proteins. (B) CD34+ cells, induced to differentiate toward the monocyte/macrophage lineage, were infected with Lenti-424 and sorted for the GFP marker carried by the vector. Growth in hematopoietic progenitor cell–monocyte (HPC M) medium. (Left), CD14 expression (Center), and morphological analysis at day 7 (Right) are shown. (C) CD34+ cells, induced to differentiate toward the monocyte/macrophage lineage, were infected with Lenti-HA-NFIA (Upper) or Lenti-siPU.1 (Lower) and sorted for the GFP marker carried by the vector. (Left) Growth in HPC M culture; (Center) CD14+ expression at day 10 (Right) and day 8 (Lower). (Right) Morphological analyses at day 10; representative fields are shown together with the percentage of mature cells. In all histograms, the values represent the means ± SEM from three separate experiments. *, P < 0.01 compared with control.