pre-B-cell Leukemia Homeobox 1 Research Articles

Regulation of the STAT3 pathway by lupus susceptibility gene Pbx1 in T cells

Systemic lupus erythematosus (SLE) is a chronic autoimmune disease in which poorly characterized genetic factors lead to the production of proinflammatory or autoreactive T cells. Pre-B cell leukemia homeobox 1 (PBX1) is a transcription factor whose dominant negative isoform (PBX1-D) is overexpressed in the CD4+ T cells of SLE patients and lupus-prone mice. Pbx1-D overexpression favors the expansion of proinflammatory T cells and impairs regulatory T (Treg) cell development. Here we show that Pbx1 deficiency and Pbx1-D overexpression decreased STAT3 expression and activation in T cells. Accordingly, Pbx1 deficiency in T cells and Pbx1-D overexpression reduced STAT3-dependent TH17 cell polarization in vitro, but it had no effect in vivo at steady state. STAT3-dependent follicular helper T (TFH) cell polarization in vitro and splenic TFH cell frequency were not affected by either Pbx1 deficiency or Pbx1-D overexpression. Pbx1 deficiency also increased the expression of cell cycle arrest and pro-apoptotic genes, with an increased apoptosis in T cells. Our results suggest a complex interplay between PBX1 and STAT3, which may contribute to lupus pathogenesis through dysregulation of the cell cycle and apoptosis.

Molecular Mechanisms of Lupus Susceptibility Allele PBX1D.

Pre-B cell leukemia homeobox 1 (PBX1) controls chromatin accessibility to a large number of genes in various cell types. Its dominant negative splice isoform, PBX1D, which lacks the DNA and Hox-binding domains, is expressed more frequently in the CD4+ T cells from lupus-prone mice and patients with systemic lupus erythematosus than healthy control subjects. PBX1D overexpression in CD4+ T cells impaired regulatory T cell homeostasis and expanded inflammatory CD4+ T cells. In this study, we showed that PBX1 message expression is downregulated by activation in CD4+ T cells as well as in B cells. PBX1D protein was less stable than the normal isoform, PBX1B, and it is degraded through the ubiquitin-proteasome-dependent pathway. The DNA binding domain lacking in PBX1D has two putative ubiquitin binding sites, K292 and K293, that are predicted to be in direct contact with DNA. Mutation of K292-293 reduced PBX1B stability to a level similar to PBX1D and abrogated DNA binding. In addition, contrary to PBX1B, PBX1D is retained in the cytoplasm without the help of the cofactors MEIS or PREP1, indicating a different requirement for nuclear translocation. Overall, these findings suggest that multiple post-transcriptional mechanisms are responsible for PBX1D loss of function and induction of CD4+ T cell inflammatory phenotypes in systemic lupus erythematosus.

Exosomes from LSD1 knockdown breast cancer cells activate osteoclastogenesis and inhibit osteoblastogenesis

Bone metastasis is a common and incurable complication of breast cancer. Lysine-specific demethylase 1 (LSD1), a histone demethylase, plays an important role in the metastasis of breast cancer. However, the role of LSD1 in bone metastasis of breast cancer is unclear. We hypothesized that exosomes from LSD1 knockdown breast cancer cells promote bone metastasis by remodeling bone microenvironment. To verify this hypothesis, exosomes from LSD1 knockdown Estrogen receptor-positive cancer cell lines, MCF7 and T47D, were isolated, and the effects of these exosomes on osteoblast and osteoclast differentiation were investigated. Interestingly, exosomes from LSD1 knockdown breast cancer cells inhibited osteoblast differentiation and promoted osteoclast differentiation. Mechanistically, miR-6881-3p was decreased in the exosomes from LSD1 knockdown cells, and miR-6881-3p suppressed the expression of pre-B-cell leukemia homeobox 1 (PBX1) and additional sex combs like-2 (ASXL2), two genes with essential functions in osteoblast and osteoclast differentiations respectively. Transfection of miR-6881-3p into LSD1 knockdown cells reversed the effects of the exosomes on osteoblast and osteoclast differentiations. Our study reveals important roles of LSD1 on the regulation of exosomal miRNAs and the formation of favorable bone microenvironment for metastasis.

Targeting SPHK1/PBX1 Axis Induced Cell Cycle Arrest in Non-Small Cell Lung Cancer.

Non-small cell lung cancer (NSCLC) accounts for 85~90% of lung cancer cases, with a poor prognosis and a low 5-year survival rate. Sphingosine kinase-1 (SPHK1), a key enzyme in regulating sphingolipid metabolism, has been reported to be involved in the development of NSCLC, although the underlying mechanism remains unclear. In the present study, we demonstrated the abnormal signature of SPHK1 in NSCLC lesions and cell lines of lung cancers with a potential tumorigenic role in cell cycle regulation. Functionally, ectopic Pre-B cell leukemia homeobox-1 (PBX1) was capable of restoring the arrested G1 phase induced by SPHK1 knockdown. However, exogenous sphingosine-1-phosphate (S1P) supply had little impact on the cell cycle arrest by PBX1 silence. Furthermore, S1P receptor S1PR3 was revealed as a specific switch to transport the extracellular S1P signal into cells, and subsequently activated PBX1 to regulate cell cycle progression. In addition, Akt signaling partially participated in the SPHK1/S1PR3/PBX1 axis to regulate the cell cycle, and the Akt inhibitor significantly decreased PBX1 expression and induced G1 arrest. Targeting SPHK1 with PF-543 significantly inhibited the cell cycle and tumor growth in preclinical xenograft tumor models of NSCLC. Taken together, our findings exhibit the vital role of the SPHK1/S1PR3/PBX1 axis in regulating the cell cycle of NSCLC, and targeting SPHK1 may develop a therapeutic effect in tumor treatment.

M6 A-mediated regulation of PBX1-GCH1 axis promotes gastric cancer proliferation and metastasis by elevating tetrahydrobiopterin levels.

BackgroundMethyltransferase 3 (METTL3)‐mediated N6‐methyladenosine (m6A) RNA modification has been demonstrated to be a potential factor in promoting gastric cancer (GC). METTL3 regulates a series of signaling pathways by modifying various mRNAs. This study aimed to identify novel METTL3‐mediated signaling pathways and explored possible targets for use in the clinical setting of gastric cancer.MethodsTo investigate the proliferation and metastatic capacity of GC cell lines with METTL3 knockdown, a xenograft, lung metastasis, and popliteal lymph node metastasis model was used. The m6A‐modified RNA immunoprecipitation (Me‐RIP) sequence was utilized to explore the target mRNAs of METTL3. Cell counting kit 8 and transwell assays were performed to investigate the promoting function of pre‐B cell leukemia homeobox 1 (PBX1) and GTP cyclohydrolase 1 (GCH1). Western blotting and chromatin immunoprecipitation were employed to confirm the involvement of the METTL3‐PBX1‐GCH1 axis. ELISA and liquid chromatography‐mass spectrometry were used to explore the biological function of tetrahydrobiopterin (BH4).ResultsKnockdown of METTL3 suppressed xenograft tumor growth and lung/lymph node metastasis in vivo. Mechanistically, we found that METTL3 combined with and stabilized PBX1 mRNAs. Chromatin immunoprecipitation (ChIP) and further experiments suggested that PBX1 acted as a transcription factor inducing GCH1 expression. Moreover, the METTL3‐PBX1‐GCH1 axis increased BH4 levels in GC cells, thereby promoting tumor progression.ConclusionsThis study suggested that METTL3 enzymes promote tumor growth and lung/lymph node metastasis via METTL3‐PBX1‐GCH1 axis increasing BH4 levels in GC.

Oncogenic Heterogeneous Nuclear Ribonucleoprotein D-like Promotes the Growth of Chronic Myeloid Leukemia Cells through Post-Transcriptional Regulation of Pre-B-Cell Leukemia Homeobox 1

Heterogeneous nuclear ribonucleoproteins (hnRNPs) represent a large family of RNA-binding proteins. Heterogeneous nuclear ribonucleoprotein D-like (HNRPDL) is a member of this family, which is able to bind RNA thus regulating multiple steps of mRNA metabolism. Chronic myeloid leukemia (CML) originates from hematopoietic stem cells acquiring BCR-ABL fusion gene. The specific inhibitor against BCR-ABL (e.g. imatinib mesylate, IM) has improved CML management though single agent is not a cure yet. Thus the delineation of growth/survival mechanisms of CML stem/progenitor cells is still needed. We have reported the aberrant expression of HNRPDL in CML CD34+ cells; however, the function of HNRPD and its underlying mechanism remain unclear yet.In the present study, we showed that HNRPDL silencing significantly inhibited the growth and the colony-forming cell (CFC) production of K562, MEG-01 and BV173 cells. Moreover, the CFC production of CML CD34+ cells was suppressed by HNRPDL silencing (ranging from 36% to 73%, P=0.014, n=6). Then BaF3/BCR-ABL and HNRPDL silenced BaF3/BCR-ABL cells were injected into lethally irradiated mice; though most mice developed leukemia (8/8 vs. 7/8, 1×105 cells/mouse), HNRPDL silencing attenuated BCR-ABL induced leukemia analyzed with Kaplan-Meier method (P<0.0001). In addition, less leukemic cells were detected in both bone marrow (24±2% vs. 6±2%, P=0.002) and spleen (12±1% vs. 3±2%, P=0.012) of the leukemic mice in HNRPDL silenced group than those in control group. Conversely, HNRPDL overexpression conferred BaF3 cells hyper-responsiveness upon murine IL-3 treatment and induced lethal leukemia in mice (8/8, 1×106cells/mouse). HNRPDL and BCR-ABL cooperatively promoted the growth of BaF3 cells as well. Lastly, we found that HNRPDL silencing sensitized K562 cells upon IM treatment as more apoptotic cells were induced; in contrast, HNRPDL overexpression protected K562 cells from IM treatment.To investigate how HNRPDL acts, microarray data comparing HNRPDL silenced K562 cells with their control have been generated. Q-RT-PCR was performed to validate that the expression of pre-B-cell leukemia homeobox 1 (PBX1) was significantly decreased in HNRPDL silenced K562 and CML CD34+ cells (n=3) than their control cells. We found that the PBX1 expression decreased more rapidly in HNRPDL silenced cell than control cells upon Actinomycin D treatment. Sequence analysis showed that PBX1 mRNA contained a HNRPDL consensus binding motif (5'- ACUAGC-3') in its 3'-untranslated region (3'-UTR). A piece of 3'-UTR only containing this consensus motif was sub-cloned in the 3'-end of a luciferase reporter vector, which conferred elevated reporter activity than control vector (3-fold, P=0.001, n=4). The elevated reported activity was entirely abolished when a mutant motif (5'-UAACAA-3') introduced in the reporter vector. In addition, the enhanced reporter activity was decreased in HNRPDL silenced cells than their control. Importantly, the PBX1 transcript expression was higher in CML CD34+ cells (n=10) compared with that in normal control cells (n=7) significantly (4-fold, P=0.019). PBX1 silencing inhibited the growth of K562 cells and sensitized them to IM treatment; while PBX1 overexpression “rescued” the suppressed growth and sensitized IM response caused by HNRPDL silencing. Taken together, our data has shown that HNRPDL plays a pivotal role in the growth and IM response of CML cells partially through its post-transcriptional regulation of PBX1. DisclosuresNo relevant conflicts of interest to declare.

Oncogenic heterogeneous nuclear ribonucleoprotein D-like modulates the growth and imatinib response of human chronic myeloid leukemia CD34+ cells via pre-B-cell leukemia homeobox 1.

Chronic myeloid leukemia (CML) originates from normal hematopoietic stem cells acquiring BCR-ABL fusion gene, specific BCR-ABL inhibitors (e.g., imatinib mesylate, IM) have greatly improved patient management. However, some patients are still suffering from relapse and drug resistance, which urges better understanding of the growth/survival mechanisms of CML stem/progenitor cells. In the present study, the role and its underlying mechanism of heterogeneous nuclear ribonucleoprotein D-like (HNRPDL) in CML cells were investigated. Firstly, overexpression of HNRPDL promoted the growth of murine BaF3 cells in vitro and induced leukemia in vivo, which was enhanced by co-expression of BCR-ABL. Conversely, HNRPDL silencing inhibited colony-forming cell (CFC) production of CML CD34+ cells and attenuated BCR-ABL induced leukemia. In addition, HNRPDL modulated imatinib response of K562 cells and HNRPDL silencing sensitized CML CD34+ cells to imatinib treatment. Mechanistically, we found the stability of pre-B-cell leukemia homeobox 1 (PBX1) mRNA was sustained by HNRPDL through its binding to a specific motif (ACUAGC) in 3'-untranslated region (3'-UTR) of PBX1. The expression of PBX1 was significantly higher in CML CD34+ cells than that in control cells and PBX silencing inhibited the growth of CML cells and sensitized them to imatinib treatment. In contrast, overexpression of PBX1 elevated the CFC production of normal hematopoietic CD34+ cells and "rescued" HNRPDL silencing induced growth inhibition and imatinib sensitization. Taken together, our data have demonstrated that HNRPDL transforms hematopoietic cells and a novel HNRPDL/PBX1 axis plays an important role in human CML CD34+ cells.

A hydrophobic residue in the TALE homeodomain of PBX1 promotes epithelial-to-mesenchymal transition of gastric carcinoma.

Pre-B-cell leukemia homeobox 1 (PBX1) was originally identified as a proto-oncogene in human leukemia. Although this protein has been shown to contribute to cellular development and tumorigenesis, the role of PBX1 in gastric carcinoma (GC) remains unclear. In this study, we observed increased expression of PBX1 in GC tissues compared with adjacent normal tissues. This increase in PBX1 expression levels negatively correlated with HOXB9 mRNA expression and was also associated with malignancy and metastasis. PBX1 promoted proliferation and metastasis of GC cells both in vitro and in vivo. These phenomena were also accompanied by epithelial-to-mesenchymal transition (EMT). Additionally, we observed that PBX1 promotes the expression of tumor growth and angiogenic factors. A structural model of the PBX1-HOX complex revealed that hydrophobic binding between PBX1 and the hexapeptide motif might be required for EMT induction. This analysis also demonstrated that the Phe252 residue in the first helix of the TALE homeodomain is involved in the latter hydrophobic binding reaction. In vitro data from PBX1 mutants suggest that PBX1 cannot promote tumorigenesis of GC cells via EMT induction when Phe252 residues lose hydrophobicity. It is likely that the presence of this residue is essential in facilitating hydrophobic binding with the hexapeptide motif. These findings suggest that PBX1 may be a potential target for GC treatment and this study provides a platform to elucidate the molecular mechanisms that underpin the role of PBX1 in GC tumorigenesis.

Overexpression of lipid metabolism genes and PBX1 in the contralateral breasts of women with estrogen receptor-negative breast cancer

Risk biomarkers for estrogen receptor (ER)-negative breast cancer have clear value for breast cancer prevention. We previously reported a set of lipid metabolism (LiMe) genes with high expression in the contralateral unaffected breasts (CUBs) of ER-negative cancer cases. We now further examine LiMe gene expression in both tumor and CUB, and investigate the role of Pre-B-cell leukemia homeobox-1 (PBX1) as a candidate common transcription factor for LiMe gene expression. mRNA was extracted from laser-capture microdissected epithelium from tumor and CUB of 84 subjects (28 ER-positive cases, 28 ER-negative cases, 28 healthy controls). Gene expression was quantitated by qRT-PCR. Logistic regression models were generated to predict ER status of the contralateral cancer. Protein expression of HMGCS2 and PBX1 was measured using immunohistochemistry. The effect of PBX1 on LiMe gene expression was examined by overexpressing PBX1 in MCF10A cells with or without ER, and by suppressing PBX1 in MDA-MB-453 cells. The expression of DHRS2, HMGCS2, UGT2B7, UGT2B11, ALOX15B, HPGD, UGT2B28 and GLYATL1 was significantly higher in ER-negative versus ER-positive CUBs, and predicted ER status of the tumor in test and validation sets. In contrast, LiMe gene expression was significantly lower in ER-negative than ER-positive tumors. PBX1 overexpression in MCF10A cells up-regulated most LiMe genes, but not in MCF10A cells overexpressing ER. Suppressing PBX1 in MDA-MB-453 cells resulted in decrease of LiMe gene expression. Four binding sites of PBX1 and cofactor were identified in three lipid metabolism genes using ChIP-qPCR. These data suggest a novel role for PBX1 in the regulation of lipid metabolism genes in benign breast, which may contribute to ER-negative tumorigenesis.

The PBX1 lupus susceptibility gene regulates CD44 expression.

PBX1-d is novel splice isoform of pre-B-cell leukemia homeobox 1 (PBX1) that lacks its DNA-binding and Hox-binding domains, and functions as a dominant negative. We have shown that PBX1-d expression in CD4+ T cells is associated with systemic lupus erythematosus (SLE) in a mouse model as well as in human subjects. More specifically, PBX1-d expression leads to the production of autoreactive activated CD4+ T cells, a reduced frequency and function of Foxp3+ regulatory T (Treg) cells and an expansion of follicular helper T (Tfh) cells. Very little is known about the function of PBX1 in T cells, except that it directly regulates the expression of miRNAs associated with Treg and Tfh homeostasis. In the present study, we show that PBX1 directly regulated the expression of CD44, a marker of T cell activation. Two PBX1 binding sites in the promoter directly regulated CD44 expression, with PBX1-d driving a higher expression than the normal isoform PBX1-b. In addition, mutations in each of the two binding sites had different effects of PBX1-b and PBX1-d. Finally, we showed that an enhanced recruitment of co-factor MEIS by PBX1-d over PBX1-b, while there was no difference for co-factor PREP1 recruitment. Therefore, this study demonstrates that the lupus-associated PBX1-d isoform directly transactivates CD44, a marker of CD44 activation and memory, and that it has different DNA binding and co-factor recruitment relative to the normal isoform. Taken together, these results confirm that PBX1 directly regulates genes related to T cell activation and shows that the lupus-associated isoform PBX1-d has unique molecular functions.

Abstract 1985: PBX1 regulated lipid metabolism gene expression and epithelial-mesenchymal transition independent of estrogen receptor

Abstract Introduction: Pre-B-cell leukemia homeobox-1 (PBX1) is a member of the three amino acid loop extension (TALE) family of homeodomain proteins that bind to DNA and regulate gene transcription by forming heterodimeric transcription complexes with Meis and Prep1. PBX1 is involved in cell fate determination during organogenesis and contributes to oncogenic activity in breast cancer. As a pioneer factor, PBX1 was found to drive ER signaling in ER+ breast cancer by remodeling the chromatin and increasing DNA accessibility. But the role of PBX1 in benign breast and ER- cancer cells is not clear. In our previous studies, we identified and validated that the expression of a set of lipid metabolism genes was higher in the contralateral breast of ER- tumor. Bioinformatic analysis on lipid metabolism gene promoter regions and revealed that PBX1 may act as a potential transcription factor to co-regulate those genes. In this study, we further investigate the function of PBX1 in ER- cells. Methods: Among the ER- cell lines, we infected cell lines expressing low endogenous PBX1 (MCF10A and MDA-MB-231) with PBX1 gene in lentiviral vector. We also infected cell lines expressing high endogenous PBX1 (MDA-MB-453 and SK-BR-3) with PBX1-shRNA to knockdown PBX1. The expression of lipid metabolism genes was detected by qRT-PCR. Markers for epithelial-to-mesenchymal transition (EMT) including E-cadherin, vimentin, β-catenin and α-SMA were detected using Western blot. The effects of overexpression or knock-down of PBX1 on proliferation, migration, and invasion were measured using IncuCyte live cell imaging system. The expression of PBX1 protein was measured in benign contralateral breast and in the matching tumor using mmnunohistochemistry. Results: Over-expression of PBX1 in ER- cell lines (MCF10A and MDA-MB-231) up-regulated lipid metabolism genes and promoted cell migration and invasion by inducing EMT (increased vimentin and decreased E-cadherin and β-catenin). In contrast, knocking-down PBX1 using shRNA in ER- cell lines (MDA-MB-453 and SK-BR-3) suppressed lipid metabolism gene expression. PBX1 was more highly expressed in benign tissues associated with ER- tumors compared to ER+ tumors. In tumor tissue, on the contrary, ER+ tumors shower higher PBX1 expression levels than ER- tumors. Conclusion: PBX1 is a master regulator of lipid metabolism genes. PBX1 promoted cell migration and invasion by inducing EMT. PBX1 may play different roles and interact with different co-factors in ER+ tumors and in benign tissues associated with ER- tumors. Citation Format: Ali Shidfar, Liannian Liu, Vamsi Parini, MiRan Choi, David Ivancic, Megan E. Sullivan, Demirkan B. Gursel, Seema A. Khan, Jun Wang. PBX1 regulated lipid metabolism gene expression and epithelial-mesenchymal transition independent of estrogen receptor. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 1985. doi:10.1158/1538-7445.AM2015-1985

Prep1 and Meis1 competition for Pbx1 binding regulates protein stability and tumorigenesis

Pbx-regulating protein-1 (Prep1) is a tumor suppressor, whereas myeloid ecotropic viral integration site-1 (Meis1) is an oncogene. We show that, to perform these activities in mouse embryonic fibroblasts, both proteins competitively heterodimerize with pre-B-cell leukemia homeobox-1 (Pbx1). Meis1 alone transforms Prep1-deficient fibroblasts, whereas Prep1 overexpression inhibits Meis1 tumorigenicity. Pbx1 can, therefore, alternatively act as an oncogene or tumor suppressor. Prep1 posttranslationally controls the level of Meis1, decreasing its stability by sequestering Pbx1. The different levels of Meis1 and the presence of Prep1 are followed at the transcriptional level by the induction of specific transcriptional signatures. The decrease of Meis1 prevents Meis1 interaction with Ddx3x and Ddx5, which are essential for Meis1 tumorigenesis, and modifies the growth-promoting DNA binding landscape of Meis1 to the growth-controlling landscape of Prep1. Hence, the key feature of Prep1 tumor-inhibiting activity is the control of Meis1 stability.

Functional Regulation of Pre-B-cell Leukemia Homeobox Interacting Protein 1 (PBXIP1/HPIP) in Erythroid Differentiation

Pre-B-cell leukemia homeobox interacting protein 1 or human PBX1 interacting protein (PBXIP1/HPIP) is a co-repressor of pre-B-cell leukemia homeobox 1 (PBX1) and is also known to regulate estrogen receptor functions by associating with the microtubule network. Despite its initial discovery in the context of hematopoietic cells, little is yet known about the role of HPIP in hematopoiesis. Here, we show that lentivirus-mediated overexpression of HPIP in human CD34(+) cells enhances hematopoietic colony formation in vitro, whereas HPIP knockdown leads to a reduction in the number of such colonies. Interestingly, erythroid colony number was significantly higher in HPIP-overexpressing cells. In addition, forced expression of HPIP in K562 cells, a multipotent erythro-megakaryoblastic leukemia cell line, led to an induction of erythroid differentiation. HPIP overexpression in both CD34(+) and K562 cells was associated with increased activation of the PI3K/AKT pathway, and corresponding treatment with a PI3K-specific inhibitor, LY-294002, caused a reduction in clonogenic progenitor number in HPIP-expressing CD34(+) cells and decreased K562 cell differentiation. Combined, these findings point to an important role of the PI3K/AKT pathway in mediating HPIP-induced effects on the growth and differentiation of hematopoietic cells. Interestingly, HPIP gene expression was found to be induced in K562 cells in response to erythroid differentiation signals such as DMSO and erythropoietin. The erythroid lineage-specific transcription factor GATA1 binds to the HPIP promoter and activates HPIP gene transcription in a CCCTC-binding factor (CTCF)-dependent manner. Co-immunoprecipitation and co-localization experiments revealed the association of CTCF with GATA1 indicating the recruitment of CTCF/GATA1 transcription factor complex onto the HPIP promoter. Together, this study provides evidence that HPIP is a target of GATA1 and CTCF in erythroid cells and plays an important role in erythroid differentiation by modulating the PI3K/AKT pathway.

RARγ is Essential for Retinoic Acid Induced Chromatin Remodeling and Transcriptional Activation in Embryonic Stem Cells

We have utilized retinoic acid receptor γ (gamma) knockout (RARγ(-/-)) embryonic stem (ES) cells as a model system to analyze RARγ mediated transcriptional regulation of stem cell differentiation. Most of the transcripts regulated by all-trans retinoic acid (RA) in ES cells are dependent upon functional RARγ signaling. Notably, many of these RA-RARγ target genes are implicated in retinoid uptake and metabolism. For instance, Lrat (lecithin:retinol acyltransferase), Stra6 (stimulated by retinoic acid 6), Crabp2 (cellular retinoic acid binding protein 2), and Cyp26a1 (cytochrome p450 26a1) transcripts are induced in wild type (WT), but not in RARγ(-/-) cells. Transcripts for the transcription factors Pbx1 (pre-B cell leukemia homeobox-1), Wt1 (Wilm's tumor gene-1), and Meis1 (myeloid ecotropic viral integration site-1) increase upon RA treatment of WT, but not RARγ(-/-) cells. In contrast, Stra8, Dleu7, Leftb, Pitx2, and Cdx1 mRNAs are induced by RA even in the absence of RARγ. Mapping of the epigenetic signature of Meis1 revealed that RA induces a rapid increase in the H3K9/K14ac epigenetic mark at the proximal promoter and at two sites downstream of the transcription start site in WT, but not in RARγ(-/-) cells. Thus, RA-associated increases in H3K9/K14ac epigenetic marks require RARγ and are associated with increased Meis1 transcript levels, whereas H3K4me3 is present at the Meis1 proximal promoter even in the absence of RARγ. In contrast, at the Lrat proximal promoter primarily the H3K4me3 mark, and not the H3K9/K14ac mark, increases in response to RA, independently of the presence of RARγ. Our data show major epigenetic changes associated with addition of the RARγ agonist RA in ES cells.

TG-interacting Factor (TGIF) Downregulates SOX3 Gene Expression in the NT2/D1 Cell Line

SOX3 is a member of the Sox gene family implicated in brain formation and cognitive function. It is considered to be one of the earliest neural markers in vertebrates, playing a role in specifying neuronal fate. Recently, we have established the first link between TALE (three-amino-acid loop extension) proteins, PBX1 (pre-B-cell leukemia homeobox 1) and MEIS1 (myeloid ecotropic viral integration site 1 homologue), and the expression of the human SOX3 gene. Here we present the evidence that TGIF (TG-interacting factor) is an additional TALE superfamily member involved in the regulation of human SOX3 gene expression in NT2/D1 cells by direct interaction with the consensus binding site that is conserved in primate orthologue promoters. Functional analysis demonstrated that mutation of the TGIF binding site resulted in the activation of SOX3 promoter. TGIF overexpression downregulates SOX3 promoter activity and decreases endogenous SOX3 protein expression in both uninduced and retinoic acid (RA)-induced NT2/D1 cells. Up to now, this is the first transcription factor identified as a negative regulator of SOX3 gene expression. The obtained results further underscore the significance of TALE proteins as important transcriptional regulators of SOX3 gene expression.

PBX1 (pre-B-cell leukemia homeobox 1)

Review on PBX1 (pre-B-cell leukemia homeobox 1), with data on DNA, on the protein encoded, and where the gene is implicated.

Pre-B-cell leukemia homeobox 1 (PBX1) shows functional and possible genetic association with bone mineral density variation

Bone mineral density (BMD) is one of the major determinants of risk for osteoporotic fracture. Multiple studies reveal that peak bone mass is under strong genetic influence. One of the major susceptibility loci for peak spine BMD has been mapped to chromosome 1q21-q23 in the Caucasian population. We have previously replicated this finding in Southern Chinese pedigrees and detected a maximum multipoint log of odds (LOD) score of 2.36 in this region. To further fine-map this region, 380 single-nucleotide polymorphic (SNP) markers were genotyped in 610 sibpairs from 231 families. Several markers were identified in the association analysis as important candidates underlying BMD variation. Among them, successful replication was demonstrated for SNPs in pre-B-cell leukemia homeobox 1 (PBX1) gene in two other unrelated case-control cohorts. The functional role of PBX1 in bone metabolism was examined in vitro using human bone-derived cells (HBDC) and murine MC3T3-E1 pre-osteoblasts. PBX1 mRNA was constitutively expressed in both HBDC and MC3T3-E1 cells. Immunostaining revealed that PBX1 is localized in the nucleus compartment. Silencing of PBX1 by RNAi in MC3T3-E1 cells decreased the expression of Runx2 and Osterix, the critical transcription factors for osteogenesis, but accelerated cell proliferation and bone nodule formation. Overall, our data suggest a genetic and functional association of PBX1 with BMD.