Coordination of Myeloid Differentiation with Reduced Cell Cycle Progression by PU.1 Induction of MicroRNAs Targeting Cell Cycle Regulators and Lipid Anabolism

Abstract

During macrophage development, myeloid progenitor cells undergo terminal differentiation coordinated with reduced cell cycle progression. Differentiation of macrophages from myeloid progenitors is accompanied by increased expression of the E26 transformation-specific transcription factor PU.1. Reduced PU.1 expression leads to increased proliferation and impaired differentiation of myeloid progenitor cells. It is not understood how PU.1 coordinates macrophage differentiation with reduced cell cycle progression. In this study, we utilized cultured PU.1-inducible myeloid cells to perform genome-wide chromatin immunoprecipitation sequencing (ChIP-seq) analysis coupled with gene expression analysis to determine targets of PU.1 that may be involved in regulating cell cycle progression. We found that genes encoding cell cycle regulators and enzymes involved in lipid anabolism were directly and inducibly bound by PU.1 although their steady-state mRNA transcript levels were reduced. Inhibition of lipid anabolism was sufficient to reduce cell cycle progression in these cells. Induction of PU.1 reduced expression of E2f1, an important activator of genes involved in cell cycle and lipid anabolism, indirectly through microRNA 223. Next-generation sequencing identified microRNAs validated as targeting cell cycle and lipid anabolism for downregulation. These results suggest that PU.1 coordinates cell cycle progression with differentiation through induction of microRNAs targeting cell cycle regulators and lipid anabolism. KEYWORDS E2F1, microRNA, PU.1, cell cycle, differentiation, lipid synthesis, myeloid cells A central problem in biology is understanding how cell division is regulated in response to developmental and environmental cues. During macrophage development, proliferating myeloid progenitor cells undergo terminal differentiation that is coordinated with reduced cell cycle progression (1). However, terminally differentiated macrophages are not necessarily permanently cell cycle arrested, as shown by evidence that epidermal Langerhans cells and brain microglia can reenter the cell cycle to self-renew (2, 3). Thus, cell cycle arrest and terminal differentiation, while normally coincident, can be independently and actively regulated. Much remains to be learned about the mechanisms by which cell-type-specific transcription factors coordinate cell cycle arrest with differentiation.

A role for ATP Citrate Lyase in cell cycle regulation during myeloid differentiation

“…BN cells are myeloid precursor cells that are impaired for differentiation as a consequence of low PU.1 expression, and proliferate continuously in culture in response to granulocyte-macrophage colony-stimulating factor (GM-CSF) (26, 32). We previously showed that induction of PU.1 in inducible BN (iBN) cells resulted in downregulation of Acly encoding ATP Citrate Lyase (ACL), and that this regulation was likely indirectly mediated through induction of microRNAs (27). BMS303141 (BMS) is an effective inhibitor of ACL activity (33) and was sufficient to inhibit cell cycle progression in cultured BN cells (27).…”

“…We previously showed that induction of PU.1 in inducible BN (iBN) cells resulted in downregulation of Acly encoding ATP Citrate Lyase (ACL), and that this regulation was likely indirectly mediated through induction of microRNAs (27). BMS303141 (BMS) is an effective inhibitor of ACL activity (33) and was sufficient to inhibit cell cycle progression in cultured BN cells (27). We set out to determine if supplementation of BN cells with acetyl-CoA could rescue BMS inhibition of cell cycle.…”

“…The BN and iBN inducible cell lines were previously described (25, 27). BN cells were cultured in Iscove’s modified Dulbecco’s medium (IMDM) (Wisent, St-Bruno, QC), with 1 ng/ml GM-CSF (Peprotech, QC), and additionally supplemented with 10% fetal bovine serum (FBS) (Wisent, St-Bruno, QC), penicillin (100 U/ml)/streptomycin (100 μg/ml) (Mediatech, Manassas, VA), L-glutamine (2 mmol/L) (Mediatech), 2-mercaptoethanol (5 x 10 -5 M) (Sigma-Aldrich, St. Louis, MO).…”

“…Our laboratory showed that cell cycle progression is inhibited by induction of PU.1 expression in a cultured myeloid cell line expressing low PU.1 concentration (Spi1 BN/BN ) (25, 26). Induction of PU.1 in inducible cells (iBN cells) resulted in macrophage differentiation and induction of microRNAs that targeted acetyl-CoA metabolism including Acly encoding ACL (27). We found that chemical inhibition of ACL activity was sufficient to block cell cycle progression in cultured BN cells (27).…”

“…Induction of PU.1 in inducible cells (iBN cells) resulted in macrophage differentiation and induction of microRNAs that targeted acetyl-CoA metabolism including Acly encoding ACL (27). We found that chemical inhibition of ACL activity was sufficient to block cell cycle progression in cultured BN cells (27).…”