Pbx1 Function Research Articles

Loss of PBX1 function in Leydig cells causes testicular dysgenesis and male sterility

Leydig cells are essential components of testicular interstitial tissue and serve as a primary source of androgen in males. A functional deficiency in Leydig cells often causes severe reproductive disorders; however, the transcriptional programs underlying the fate decisions and steroidogenesis of these cells have not been fully defined. In this study, we report that the homeodomain transcription factor PBX1 is a master regulator of Leydig cell differentiation and testosterone production in mice. PBX1 was highly expressed in Leydig cells and peritubular myoid cells in the adult testis. Conditional deletion of Pbx1 in Leydig cells caused spermatogenic defects and complete sterility. Histological examinations revealed that Pbx1 deletion impaired testicular structure and led to disorganization of the seminiferous tubules. Single-cell RNA-seq analysis revealed that loss of Pbx1 function affected the fate decisions of progenitor Leydig cells and altered the transcription of genes associated with testosterone synthesis in the adult testis. Pbx1 directly regulates the transcription of genes that play important roles in steroidogenesis (Prlr, Nr2f2 and Nedd4). Further analysis demonstrated that deletion of Pbx1 leads to a significant decrease in testosterone levels, accompanied by increases in pregnenolone, androstenedione and luteinizing hormone. Collectively, our data revealed that PBX1 is indispensable for maintaining Leydig cell function. These findings provide insights into testicular dysgenesis and the regulation of hormone secretion in Leydig cells.Graphical abstract

Spindle function and Wnt pathway inhibition by PBX1 to suppress tumor progression via downregulating DCDC2 in colorectal cancer

PBX1 is a transcription factor that regulates a variety of genes, involved in intracellular lipid metabolism, cell proliferation, and other pathways. The promoting and inhibiting function of PBX1 in various cancer types was extensively discussed, however, there have been no studies on PBX1 proteins in colorectal cancer (CRC). This study aimed to reveal the anti-tumor function of PBX1 in CRC and the underlying molecular mechanism. Bioinformatics analysis revealed that PBX1 is downregulated in CRC, indicating that is a potential antioncogene in CRC. Overexpression of PBX1 suppresses tumor growth and metastasis in vitro and in vivo. Mechanistically, we found that PBX1 acted as a transcription factor that suppressed DCDC2 expression and inhibited spindle function. Moreover, the PBX1-DCDC2 axis controlled the Wnt pathway in CRC cells. Overexpression of DCDC2 restored CRC proliferation, metastasis abilities and Wnt pathway. In conclusion, this study suggests that PBX1 acts as a transcription factor to suppress DCDC2 expression and inhibit cell proliferation and metastasis by disrupting spindle function and the Wnt pathway in CRC.

Meis1, Hi1α, and GATA1 are integrated into a hierarchical regulatory network to mediate primitive erythropoiesis.

During development, erythroid cells are generated by two waves of hematopoiesis. In zebrafish, primitive erythropoiesis takes place in the intermediate cell mass region, and definitive erythropoiesis arises from the aorta-gonad mesonephros. TALE-homeoproteins Meis1 and Pbx1 function upstream of GATA1 to specify the erythroid lineage. Embryos lacking Meis1 or Pbx1 have weak gata1 expression and fail to produce primitive erythrocytes. Nevertheless, the underlying mechanism of how Meis1 and Pbx1 mediate gata1 transcription in erythrocytes remains unclear. Here we show that Hif1α acts downstream of Meis1 to mediate gata1 expression in zebrafish embryos. Inhibition of Meis1 expression resulted in suppression of hif1a expression and abrogated primitive erythropoiesis, while injection with in vitro-synthesized hif1α mRNA rescued gata1 transcription in Meis1 morphants and recovered their erythropoiesis. Ablation of Hif1α expression either by morpholino knockdown or Crispr-Cas9 knockout suppressed gata1 transcription and abrogated primitive erythropoiesis. Results of chromatin immunoprecipitation assays showed that Hif1α associates with hypoxia-response elements located in the 3'-flanking region of gata1 during development, suggesting that Hif1α regulates gata1 expression in vivo. Together, our results indicate that Meis1, Hif1α, and GATA1 indeed comprise a hierarchical regulatory network in which Hif1α acts downstream of Meis1 to activate gata1 transcription through direct interactions with its cis-acting elements in primitive erythrocytes.

PBX1 promotes the cell proliferation via JAK2/STAT3 signaling in clear cell renal carcinoma

PBX1 was abnormally overexpressed and its intracellular localization was found to be frequently amplified in many types of cancer, including renal clear cell carcinoma.PBX1 displays oncogenic activity in several different types of cells, but little is known about how signaling pathways are altered, and the function of PBX1 in renal clear cell carcinoma has not been well investigated. In this study, we demonstrate that the expression of PBX1 was significantly upregulated in 30 pairs of human tissues compared to adjacent normal tissues and the overall survival rate of PBX1-high group was significantly worse than that of PBX1-low group. Furthermore, JAK2 expression is significantly correlated to PBX1 expression in human clinical specimen and PBX1 knockdown inhibits STAT3 phosphorylation and reduced transcription of STAT3 target genes Cyclin D1. More interestingly, PBX1 knockdown inhibits ccRCC cell viability, proliferation and cell cycle progression in vivo and in vitro. Thus, our results demonstrate that PBX1 plays an oncogenic role in ccRCC via JAK2/STAT3 pathway and indicate its potential application for the treatment of human ccRCC in future.

Transcriptional Regulatory Landscape of TCF3-PBX1-Positive Leukemia and Novel Targeted Treatments

IntroductionThe oncogenic TCF3-PBX1 (TP; also known as E2A-PBX1) fusion gene results from a translocation between chromosomes 1 and 19 in pre-B-cell acute leukemia (pre-B-ALL). Both TCF3 and PBX1 function as transcription factors (TF), and their fusion generates a unique transcriptional landscape in t(1;19) leukemias which differs from other pre-B-ALL subtypes. Here we explored the transcriptional regulatory landscape of t(1;19)-leukemia genome-wide and sought novel targeted therapy options.Materials & MethodsWe modeled the oncogenicity of the fusion protein in leukemic cells by either expressing (Nalm6-TP, induced for 16 hours) or silencing (697-shTP, down to appr. 40 %) the fusion. Proliferation, apoptosis and cell cycle were studied using fluorometric reactions and flow cytometry. Patient samples (TP, n=4; other subtypes, n=18) and various cell line models were subjected to global nuclear run-on sequencing (GRO-seq), which provides a genome-wide map of nascent (primary) transcription. All transcripts that were altered after overexpression or silencing of the fusion-TF in cell models were inspected from the patient GRO-seq samples. TCF3-PBX1-regulated genomic regions were studied for enrichment of TF binding motifs and altered signaling pathways. Mature RNA levels and potential novel long non-coding RNAs were further validated by qPCR. Gene expression differences between t(1;19) and other subtypes were compared using a curated microarray data set containing 1304 pre-B-ALL samples retrieved from 15 different data sets from the Gene Expression Omnibus. ARACNE, a network inference algorithm, and GRO-seq were used to identify TFs correlating strongly with the t(1;19) subtype and to find novel drug targets. Drugs were tested in cell culture using t(1;19)-positive cell line models and patient samples either alone or in combination with known leukemia therapies.ResultsGRO-seq analysis allowed elucidation of the regulatory landscape downstream of the TCF3-PBX1 fusion protein. Directly regulated enhancer RNAs were matched to the cis regulated genes, and vice versa, to clarify enhancer-gene relations. As an example, correspondingly regulated enhancer regions were located for WNT16 and ANKS1B, two genes that are known targets of TCF3-PBX1, and that were found consistently upregulated in the studied sample sets. EBF3, a tumor suppressor gene, was one of the top hits in the network inference analysis and was also found consistently regulated by TCF3-PBX1. One of the identified druggable target was RORB which was directly upregulated by TCF3-PBX1. An inhibitor targeting RORB decreased viability of TCF3-PBX1-positive cell lines and cells from a t(1;19) patient. The effect was especially prominent when the inhibitor was combined with a low dose (1 nM) of vincristine, yielding a marked synergistic effect.ConclusionsOur results provide the first genome-wide transcriptional regulatory landscape of TCF3-PBX1 leukemia. We also identified novel putative druggable targets and a potential inhibitor for this leukemia subtype. DisclosuresHeckman:Celgene: Research Funding; Pfizer: Research Funding.

Inhibition of non-small-cell lung cancer cell proliferation by Pbx1.

Lung cancer is one of the most deadly human cancers and continues to be a major unsolved health problem worldwide. Here, we evaluate the function of Pbx1 in the proliferation of non-small-cell lung cancer (NSCLC). In contrast with its known proliferative function, we found that Pbx1 inhibits the proliferation of lung cancer cells. In particular, Pbx1-specific RNA interference resulted in increased proliferation in lung cancer cells. In addition, histone H3 phosphorylation was also increased following inhibition of Pbx1 expression. In contrast, Pbx1 overexpression repressed the proliferation of lung cancer cells and inhibited DNA synthesis. Collectively, our data indicate that Pbx1 inhibits proliferation in lung cancer cells, suggesting a complex role for Pbx1 in modulating the proliferation of cancer cells and making this protein a potential new target for lung cancer therapy.

The Internal-Tandem-Duplication of Flt3 Mutation Generates Growth Inhibitory Signals in Mouse Hematopoietic Progenitor Cells by Down-Regulating Pbx1 Expression Via P21cdkn1a.

Abstract 2340Internal-Tandem-Duplication (ITD) Flt3 mutations are found in 30% of patients with AML; however, Flt3-inhibitors are not significantly efficacious in the therapy. We have reported that ITD-Flt3 down-regulates Pbx1 expression, a transcriptional factor that regulates the self-renewal of hematopoietic stem cells, in mouse bone marrow c-kit+-Sca-1+-Lin− (KSL) cells, and that Pbx1 expression levels are reduced in patients with ITD-Flt3+AML compared with those lacking ITD-Flt3 (ASH 2011). However, the function of Pbx1 in ITD-Flt3 signaling remains unknown. In this report, we investigate the function of Pbx1 in hematopoietic progenitor cells transformed by ITD-Flt3.ITD-Flt3 transduction in mouse marrow cells led to a decrease in Pbx1 mRNA expression levels in KSL cells (80±8% reduction compared with normal KSL cells, P<0.01, N=3), which coincided with the growth-factor-independent expansion of KSL cells. However, the transduction of shRNAs specific for Pbx1 (Pbx1-shRNA) in ITD-Flt3+ bone marrow cells led to a reduction in the number of CFU-GM colonies proliferating in the absence of growth factors compared with control shRNA-transduced ITD-Flt3+ cells (25 ± 9% reduction, P<0.05, N=3). Similarly, Pbx1-shRNA induced a decrease in the proliferation of ITD-Flt3+Ba/F3 cells upon cytokine deprivation (42±1% reduction, N=4, P<0.001). These results indicate that although Pbx1 is down-regulated by ITD-Flt3, the factor-independent proliferation of ITD-Flt3+cells is functionally supported by Pbx1. In contrast, p21cdkn1a (p21)expression was up-regulated by ITD-Flt3 in both KSL cells (5.0±1.5-fold, P<0.05, N=3) and Ba/F3 cells (8.7±1.8-fold, P<0.05, N=3). However, the gene deletion of p21 resulted in an enhancement of ITD-Flt3+CFU-GM colony proliferation (2.0±0.3-fold increase, P<0.01, N=8), thereby indicating that an ITD-Flt3-mediated increase in p21 expression negatively modulates the aberrant proliferation of ITD-Flt3+ cells. Moreover, the loss of p21 upregulated Pbx1 mRNA expression in ITD-Flt3+KSL cells (2.1 ± 0.5-fold in p21−/−compared with p21+/+, P<0.05, N=3). However, the cell cycle fractionation of ITD-Flt3+KSL cells using Hoechst33314 and Pyronin-Y staining indicated that Pbx1 expression was reduced in G1 phase cells compared with cells in the G0 phase (0.2±0.1-fold, P<0.01, N=3), thereby suggesting that p21 negatively regulates Pbx1 in ITD-Flt3+cells, while the up-regulation of Pbx1 was not due to the cell cycle progression induced by p21 deletion. The transduction of Pbx1-shRNA in ITD-Flt3+p21−/−bone marrow cells led to a decrease in the cytokine-independent hyperproliferation of CFU-GM colonies induced by p21 deletion compared with ITD-Flt3+p21−/− cells containing the control shRNA (shRNA-1: 43±4% reduction; shRNA-2: 41±7% reduction, P<0.05, N=3). Similarly, anti-p21-shRNA treatment increased the growth-factor-independent proliferation of ITD-Flt3+Ba/F3 cells compared with the control-shRNA cells (5.3±0.3-fold increase, P<0.001, N=4), whereas the transduction of shRNA specific for Pbx1 in the ITD-Flt3+Ba/F3 cells harboring anti-p21-shRNA partially abrogated the enhanced proliferation that was induced by p21 knockdown (29±1% reduction compared with the control, P<0.001, N=4).Our data indicates that expression of Pbx1, which supports the growth-factor-independent proliferation of ITD-Flt3+ cells, was down-regulated by p21, which was elevated in the presence of ITD-Flt3. The enhanced proliferation of ITD-Flt3+ cells induced by p21 deletion was partially abrogated by knocking down Pbx1, thereby indicating that the down-regulation of Pbx1 is secondary to the up-regulation of p21 that is induced by ITD-Flt3, which resulted in the growth inhibition of ITD-Flt3+cells. The data demonstrates that ITD-Flt3 can generate growth inhibitory signals by reducing the Pbx1 expression levels via p21.This implies that Flt3-inhibitors may destroy the Pbx1/p21 growth inhibitory signaling that is generated by ITD-Flt3, thereby contributing to subsequent recurrence of ITD-Flt3+AML cells. Disclosures:No relevant conflicts of interest to declare.

The SLE-associated Pbx1-d isoform acts as a dominant-negative transcriptional regulator

Pbx1 is a transcription factor involved in multiple cellular processes, including the maintenance of self-renewal of hematopoietic progenitors. We have shown that the CD4(+) T-cell expression of a novel splice isoform of Pbx1, Pbx1-d, is associated with lupus susceptibility in the NZM2410 mouse and in lupus patients. The function of Pbx1 in T cells is unknown, but the splicing out of the DNA-binding domain in Pbx1-d predicts a dominant-negative function. In support of this hypothesis, we have shown that Pbx1-d transduction accelerates differentiation of MC3T3-E1 osteoblast pregenitors and mimics the effect of short hairpin RNA silencing of Pbx1. Conversely, Pbx1-d transduction reduced the expression of Sox3, a gene strongly transactivated by Pbx1, and Pbx1-d did not bind the Sox3 promoter. These results constitute a first step towards the understanding on how Pbx1-d contributes to systemic autoimmunity in the NZM2410 mouse model as well as in lupus patients.

PBX1: A Novel Stage-Specific Regulator of Adipocyte Development

Although adipocyte terminal differentiation has been extensively studied, the early steps of adipocyte development and the embryonic origin of this lineage remain largely unknown. Here we describe a novel role for the pre-B-cell leukemia transcription factor one (PBX1) in adipocyte development using both mouse embryonic stem cells (mESCs) and human multipotent adipose-derived stem (hMADS) cells. We show that Pbx1(-/-) mESCs are unable to generate adipocytes, despite normal expression of neuroectoderm and neural crest (NC) markers. Early adipocyte lineage markers are not induced in Pbx1(-/-) mESCs, suggesting that Pbx1 controls the generation and/or the maintenance of adipocyte progenitors (APs) from the NC. We further characterize the function of PBX1 in postnatal adipogenesis and show that silencing of PBX1 expression in hMADS cells reduces their proliferation by preventing their entry in the S phase of the cell cycle. Furthermore, it promotes differentiation of hMADS cells into adipocytes and partially substitutes for glucocorticoids and rosiglitazone, two key proadipogenic agents. These effects involve direct modulation of PPARγ activity, most likely through regulation of the biosynthesis of PPARγ natural endogenous ligand(s). Together, our data suggest that PBX1 regulates adipocyte development at multiple levels, promoting the generation of NC-derived APs during embryogenesis, while favoring APs proliferation and preventing their commitment to the adipocyte lineage in postnatal life.

PBX1 is dispensable for neural commitment of RA-treated murine ES cells

Experimentation with PBX1 knockout mice has shown that PBX1 is necessary for early embryogenesis. Despite broad insight into PBX1 function, little is known about the underlying target gene regulation. Utilizing the Cre–loxP system, we targeted a functionally important part of the homeodomain of PBX1 through homozygous deletion of exon-6 and flanking intronic regions leading to exon 7 skipping in embryonic stem (ES) cells. We induced in vitro differentiation of wild-type and PBX1 mutant ES cells by aggregation and retinoic acid (RA) treatment and compared their profiles of gene expression at the ninth day post-reattachment to adhesive media. Our results indicate that PBX1 interactions with HOX proteins and DNA are dispensable for RA-induced ability of ES to express neural genes and point to a possible involvement of PBX1 in the regulation of imprinted genes.Electronic supplementary materialThe online version of this article (doi:10.1007/s11626-008-9162-5) contains supplementary material, which is available to authorized users.

Identification of a new type of PBX1 partner that contains zinc finger motifs and inhibits the binding of HOXA9-PBX1 to DNA

PBX1 belongs to the TALE-class of homeodomain protein and has a wide functional diversity during development. Indeed, PBX1 is required for haematopoiesis as well as for multiple developmental processes such as skeletal patterning and organogenesis. It has furthermore been shown that PBX1 functions as a HOX cofactor during development. More recent data suggest that PBX1 may act even more broadly by modulating the activity of non-homeodomain transcription factors. To better understand molecular mechanisms triggered by PBX1 during female genital tract development, we searched for additional PBX1 partners that might be involved in this process. Using a two hybrid screen, we identified a new PBX1 interacting protein containing several zinc finger motifs that we called ZFPIP for Zinc Finger PBX1 Interacting Protein. We demonstrated that ZFPIP is expressed in embryonic female genital tract but also in other PBX1 expression domains such as the developing head and the limb buds. We further showed that ZFPIP is able to bind physically and in vivo to PBX1 and moreover, that it prevents the binding of HOXA9/PBX complexes to their consensus DNA site. We suggest that ZFPIP is a new type of PBX1 partner that could participate in PBX1 function during several developmental pathways.