Master Transcription Factor PU Research Articles

DNA selection by the master transcription factor PU.1

The master transcriptional regulator PU.1/Spi-1 engages DNA sites with affinities spanning multiple orders of magnitude. To elucidate this remarkable plasticity, we have characterized 22 high-resolution co-crystallographic PU.1/DNA complexes across the addressable affinity range in myeloid gene transactivation. Over a purine-rich core (such as 5'-GGAA-3') flanked by variable sequences, affinity is negotiated by direct readout on the 5' flank via a critical glutamine (Q226) sidechain and by indirect readout on the 3' flank by sequence-dependent helical flexibility. Direct readout by Q226 dynamically specifies PU.1's characteristic preference for purines and explains the pathogenic mutation Q226E in Waldenström macroglobulinemia. The structures also reveal how disruption of Q226 mediates strand-specific inhibition by DNA methylation and the recognition of non-canonical sites, including the authentic binding sequence at the CD11b promoter. A re-synthesis of phylogenetic and structural data on the ETS family, considering the centrality of Q226 in PU.1, unifies the model of DNA selection by ETS proteins.

FBW7 Inhibits Myeloid Differentiation in Acute Myeloid Leukemia via GSK3-Dependent Ubiquitination of PU.1.

Glycogen synthase kinase 3β (GSK3β), an ubiquitously expressed serine/threonine kinase is reported to be overexpressed and hyperactivated in cancers including acute myeloid leukemia (AML) where it promotes self-renewal, growth, and survival of AML cells. Therefore, GSK3β inhibition results in AML cell growth inhibition and myeloid differentiation. Here we identified master transcription factor PU.1 of monocyte-macrophage differentiation pathway as potential GSK3β target. We demonstrate that GSK3β phosphorylates PU.1 at Ser41 and Ser140 leading to its recognition and subsequent ubiquitin-mediated degradation by E3 ubiquitin ligase FBW7. This GSK3-dependent degradation of PU.1 by FBW7 inhibited monocyte-macrophage differentiation. We further showed that a phospho-deficient PU.1 mutant (PU.1-S41, S140A) neither bound to FBW7 nor was degraded by it. Consequently, PU.1-S41, S140A retained its transactivation, DNA-binding ability and promoted monocyte-macrophage differentiation of U937 cells even without phorbol 12-myristate 13-acetate (PMA) treatment. We further showed that FBW7 overexpression inhibited both PMA as well as M-CSF-induced macrophage differentiation of myeloid cell lines and peripheral blood mononuclear cells (PBMC) from healthy volunteers, respectively. Contrarily, FBW7 depletion promoted differentiation of these cells even without any inducer suggesting for a robust role of GSK3β-FBW7 axis in negatively regulating myeloid differentiation. Furthermore, we also recapitulated these findings in PBMCs isolated from patients with leukemia where FBW7 overexpression markedly inhibited endogenous PU.1 protein levels. In addition, PBMCs also showed enhanced differentiation when treated with M-CSF and GSK3 inhibitor (SB216763) together compared with M-CSF treatment alone. IMPLICATIONS: Our data demonstrate a plausible mechanism behind PU.1 restoration and induction of myeloid differentiation upon GSK3β inhibition and further substantiates potential of GSK3β as a therapeutic target in AML.

Intrinsic disorder controls two functionally distinct dimers of the master transcription factor PU.1.

Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The hematopoietic master regulator PU.1 presents a well-defined model of the most common configuration of intrinsically disordered regions (IDRs) in transcription factors. We report that the structured DNA binding domain (DBD) of PU.1 regulates gene expression via antagonistic dimeric states that are reciprocally controlled by cognate DNA on the one hand and by its proximal anionic IDR on the other. The two conformers are mediated by distinct regions of the DBD without structured contributions from the tethered IDRs. Unlike DNA-bound complexes, the unbound dimer is markedly destabilized. Dimerization without DNA is promoted by progressive phosphomimetic substitutions of IDR residues that are phosphorylated in immune activation and stimulated by anionic crowding agents. These results suggest a previously unidentified, nonstructural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly cationic DBDs.

Intrinsic Disorder Directs Two Distinct Dimers of the Master Transcription Factor PU.1

Transcription factors comprise a major reservoir of conformational disorder in the eukaryotic proteome. The transcription factor PU.1 presents a well-defined model of Type I intrinsic disorder, the most common configuration of intrinsically disordered regions (IDRs) in transcription factors in which the DNA-binding domain (DBD) is the only structured domain. PU.1 is also a master regulator of hematopoiesis, the physiologic process by which all blood cell lineages develop ultimately from a single population of stem cells in the bone marrow. Dose-response experiments using synthetic reporters and native PU.1 target genes show that the structured DBD of PU.1 regulates gene expression in live cells via a negatively cooperative DNA-bound dimer. Formation of the bound dimer is antagonized, in the absence of DNA, by a conformationally distinct dimeric state under the control of its proximal acidic IDR. The two conformers involve distinct surfaces on the DBD without disorder-to-order contributions from the tethered IDRs. Spectroscopic and calorimetric characterizations show that, unlike DNA-bound complexes, charge-charge contributions from the flanking IDRs make critical contributions in realizing a conformationally destabilized dimer. Dimerization without DNA is further promoted by phosphomimetic (Ser → Asp) substitutions of IDR residues that are physiologically phosphorylated in B-cell activation, and stimulated by acidic crowding agents. The data suggests a novel non-structural role for charged IDRs in conformational control by mitigating electrostatic penalties that would mask the interactions of highly basic DBDs without DNA. As PU.1 demonstrates, this mechanism renders thermodynamically insulated dimeric states that mutually antagonize to regulate the concentration of functionally active monomer at the protein/DNA level.

Molecular Self-Titration as a Mechanism of Gene Regulation

In systems biology, molecular titration is a mechanism for introducing sensitivity of genetic networks to their regulatory proteins. Since most eukaryotic transcription factors lack chemical control by low-molecular-weight hormones or metabolites, transcription factors have evolved various strategies, such as cooperative DNA binding and sequestering partner proteins, to generate sensitivity in the expression of the genes that they regulate. The ETS superfamily of transcription factors, which are broadly distributed in the animal kingdom, regulate target genes in an active monomeric state. On the one hand, many ETS members, such as the master transcription factor PU.1, are involved in critical cell-fate decisions and signaling pathways that require highly sensitive output responses, yet no coherent mechanism exists for generating the required sensitivity. On the other hand, dimerization of ETS proteins in both DNA-bound and free states via their DNA-binding domain has been reported, but its purpose and biophysical nature remain obscure. We propose that dimerization serves as a sensitivity-generating mechanism by molecular self-titration: self-association sequesters free ETS proteins in inactive dimers, while dimerization in the DNA-bound state inhibits transactivation by the active protein/DNA complex. With the positive auto-regulation of the PU.1 (Spi1) gene generating self-amplification of PU.1, homodimerization would provide the minimal negative feedback needed to prevent explosion and maintain stable dynamics of PU.1 target genes in the absence of additional interactions. We are defining the thermodynamic and structural parameters of the PU.1 homodimer in the free and DNA-bound states. The data show that dimerization involves previously unexplored molecular surfaces that are distinct from the “orthosteric” protein/DNA contact interface and offer insight into how molecular self-titration would participate in the dynamics of PU.1-dependent gene expression.

Mechanisms of in vivo binding site selection of the hematopoietic master transcription factor PU.1.

The transcription factor PU.1 is crucial for the development of many hematopoietic lineages and its binding patterns significantly change during differentiation processes. However, the ‘rules’ for binding or not-binding of potential binding sites are only partially understood. To unveil basic characteristics of PU.1 binding site selection in different cell types, we studied the binding properties of PU.1 during human macrophage differentiation. Using in vivo and in vitro binding assays, as well as computational prediction, we show that PU.1 selects its binding sites primarily based on sequence affinity, which results in the frequent autonomous binding of high affinity sites in DNase I inaccessible regions (25–45% of all occupied sites). Increasing PU.1 concentrations and the availability of cooperative transcription factor interactions during lineage differentiation both decrease affinity thresholds for in vivo binding and fine-tune cell type-specific PU.1 binding, which seems to be largely independent of DNA methylation. Occupied sites were predominantly detected in active chromatin domains, which are characterized by higher densities of PU.1 recognition sites and neighboring motifs for cooperative transcription factors. Our study supports a model of PU.1 binding control that involves motif-binding affinity, PU.1 concentration, cooperativeness with neighboring transcription factor sites and chromatin domain accessibility, which likely applies to all PU.1 expressing cells.

T helper 9 cell development is dependent on the T cell maturation stage (152.13)

Abstract T helper 9 (Th9) cells, a novel CD4 T cell subset, seem to play a complex role in the outcome of specific immune responses. Whilst they may enhance a pro-inflammatory Th17 cell-driven immune response they are also able to prevent allograft rejection by intermediating T cell tolerance. Here, we have analyzed the Th9 cell differentiation requirements from human naive and memory CD4 T cells. Neonatal CD4 T cells required more stringent Th9 differentiation conditions when compared to adult CD4 T cells reflecting a different cytokine requirement for the expression of the Th9 cell master transcription factor PU.1. The requirements for Th9 cell differentiation weakened from neonatal through adult naive to memory T cells. Moreover, the phenotype of developing cells became gradually more heterogeneous even under stringent Th9-inducing conditions from neonatal through adult naive to memory CD4 T cells. Whereas neonatal CD4 T cells differentiated into “pure” Th9 cells producing exclusively IL-9, adult naive or memory CD4 T cells developed into effectors consisting of IL-9-producing cells as well as of cells capable of IL-4, IFN-γ, IL-5, or IL-17 co-production together with IL-9. The data suggest that the conditions for Th9 cell development and therefore for IL-9 production are continuously changing during life which may indicate a growing contributive role of IL-9 in specific immune responses with advancing age.

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

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.