Increased Promoter Activity Research Articles

Viral DNA Replication Orientation and hnRNPs Regulate Transcription of the Human Papillomavirus 18 Late Promoter.

ABSTRACTThe life cycle of human papillomaviruses (HPVs) is tightly linked to keratinocyte differentiation. Although expression of viral early genes is initiated immediately upon virus infection of undifferentiated basal cells, viral DNA amplification and late gene expression occur only in the mid to upper strata of the keratinocytes undergoing terminal differentiation. In this report, we show that the relative activity of HPV18 TATA-less late promoter P811 depends on its orientation relative to that of the origin (Ori) of viral DNA replication and is sensitive to the eukaryotic DNA polymerase inhibitor aphidicolin. Additionally, transfected 70-nucleotide (nt)-long single-strand DNA oligonucleotides that are homologous to the region near Ori induce late promoter activity. We also found that promoter activation in raft cultures leads to production of the late promoter-associated, sense-strand transcription initiation RNAs (tiRNAs) and splice-site small RNAs (spliRNAs). Finally, a cis-acting AAGTATGCA core element that functions as a repressor to the promoter was identified. This element interacts with hnRNP D0B and hnRNP A/B factors. Point mutations in the core prevented binding of hnRNPs and increased the promoter activity. Confirming this result, knocking down the expression of both hnRNPs in keratinocytes led to increased promoter activity. Taking the data together, our study revealed the mechanism of how the HPV18 late promoter is regulated by DNA replication and host factors.

Type 2 inositol trisphosphate receptor gene expression in hepatocytes is regulated by cyclic AMP

The type 2 inositol 1,4,5-trisphosphate receptor (IP3R2) is the principal intracellular Ca2+ release channel in hepatocytes, and so is important for bile secretion and other functions. IP3R2 activity is regulated in part by post-translational modifications but little is known about transcriptional regulation of its expression. We found that both IP3R2 mRNA and protein levels in liver were increased during fasting. Treatment of hepatocytes with forskolin or 8-CPT-cAMP also increased IP3R2, and this was reduced by actinomycin D. Analysis of the IP3R2 promoter revealed five CREs, and CREB potently increased promoter activity. Mutation of CRE4 or CRE5 decreased induction by CREB, and ChIP assay showed recruitment of CREB to these sites. Adenylyl cyclase (AC) 6 and 9 were the principal AC isoforms detected in rat hepatocytes, and silencing either one decreased organic anion secretion, which depends on IP3R2. Secretion furthermore was increased by overnight but not acute treatment with forskolin or 8-CPT-cAMP. These findings provide evidence that IP3R2 expression is transcriptionally regulated by cAMP via CREB binding to CRE elements in its promoter. The findings furthermore suggest that this mechanism is relevant for hormonal regulation of bile secretion.

Effect of food-related stress conditions and loss of agr and sigB on seb promoter activity in S. aureus

Staphylococcal enterotoxin B (SEB) causes staphylococcal food poisoning and is produced in up to ten times higher quantities than other major enterotoxins. While Staphylococcus aureus growth is often repressed by competing flora, the organism exhibits a decisive growth advantage under some stress conditions. So far, data on the influence of food-related stressors and regulatory mutations on seb expression is limited and largely based on laboratory strains, which were later reported to harbor mutations. Therefore, the aim of this study was to investigate the influence of stress and regulatory mutations on seb promoter activity. To this end, transcriptional fusions were created in two strains, USA300 and HG003, carrying different seb upstream sequences fused to a blaZ reporter. NaCl, nitrite, and glucose stress led to significantly decreased seb promoter activity, while lactic acid stress resulted in significantly increased seb promoter activity. Loss of agr decreased seb promoter activity and loss of sigB increased promoter activity, with the magnitude of change depending on the strain. These results demonstrate that mild stress conditions encountered during food production and preservation can induce significant changes in seb promoter activity.

AURKA Overexpression Is Driven by FOXM1 and MAPK/ERK Activation in Melanoma Cells Harboring BRAF or NRAS Mutations: Impact on Melanoma Prognosis and Therapy

The cell cycle-related genes AURKA and FOXM1 are overexpressed in melanoma. We show here that AURKA overexpression is associated with poor prognosis in three independent cohorts of melanoma patients and correlates with the presence of genomic amplification of AURKA locus and BRAFV600E mutation. AURKA overexpression may also be driven by increased promoter activation through elements such as ETS and FOXM1 found within the 5' proximal promoter region. Activated MAPK/ERK signaling pathway mediates robust AURKA promoter activation, thereby knockdown of BRAFV600E and ERK inhibition results in reduced AURKA transcription and expression. We show a positive correlation between FOXM1 and AURKA expression in three independent cohorts of melanoma patients. FOXM1 silencing decreases expression of AURKA and late cell cycle genes in melanoma cells. We further found that FOXM1 expression levels are significantly higher in tumors carrying the BRAFV600E mutation compared with the wild-type BRAF (BRAFwt). Accordingly, the knockdown of BRAFV600E also reduces the expression of FOXM1 in BRAFV600E cells. Moreover, Aurora kinase A and FOXM1 inhibition by either genetic knockdown or pharmacologic inhibitors impair melanoma growth and survival both in culture and invivo, underscoring their therapeutic value for melanoma patients who fail to benefit from BRAF/MEK signaling inhibition.

Synthetic introns help identify sequences in the 5' UTR intron of the Glycine max polyubiquitin (Gmubi) promoter that give increased promoter activity.

Specific sequences within the leader intron of a soybean polyubiquitin gene stimulated gene expression when placed either within a synthetic intron or upstream of a core promoter. The intron in the 5' untranslated region of the soybean polyubiquitin promoter, Gmubi, seems to contribute to the high activity of this promoter. To identify the stimulatory sequences within the intron, ten different sequential intronic sequences of 40 nt were isolated, cloned as tetrameric repeats and placed upstream of a minimal cauliflower mosaic virus 35S (35S) core promoter, which was used to control expression of the green fluorescent protein. Intron fragment tetramers were also cloned within a modified, native intron, creating a Synthetic INtron Cassette (SINC), which was then placed downstream of Gmubi and 35S core promoters. Intron fragment tetramers and SINC constructs were evaluated using transient expression in lima bean cotyledons and stable expression in soybean hairy roots. Intron fragments, used as tetramers upstream of the 35S core promoter, yielded up to 80 times higher expression than the core promoter in transient expression analyses and ten times higher expression in stably transformed hairy roots. Tetrameric intronic fragments, cloned downstream of the Gmubi and 35S core promoters and within the synthetic intron, also yielded increased transient and stable GFP expression that was up to 4 times higher than Gmubi alone and up to 40 times higher than the 35S core promoter alone. These intron fragments contain sequences that seem to act as promoter regulatory elements and may contribute to the increased expression observed with this native strong promoter. Intron regulatory elements and synthetic introns may provide additional tools for increasing transgene expression in plants.

Confirmation of the OVOL2 Promoter Mutation c.-307T>C in Posterior Polymorphous Corneal Dystrophy 1.

PurposeTo identify the genetic basis of posterior polymorphous corneal dystrophy (PPCD) in families mapped to the PPCD1 locus and in affected individuals without ZEB1 coding region mutations.MethodsThe promoter, 5’ UTR, and coding regions of OVOL2 was screened in the PPCD family in which linkage analysis established the PPCD1 locus and in 26 PPCD probands who did not harbor a ZEB1 mutation. Copy number variation (CNV) analysis in the PPCD1 and PPCD3 intervals was performed on DNA samples from eight probands using aCGH. Luciferase reporter assays were performed in human corneal endothelial cells to determine the impact of the identified potentially pathogenic variants on OVOL2 promoter activity.ResultsOVOL2 mutation analysis in the first PPCD1-linked family demonstrated segregation of the c.-307T>C variant with the affected phenotype. In the other 26 probands screened, one heterozygous coding region variant and five promoter region heterozygous variants were identified, though none are likely pathogenic based on allele frequency. Array CGH in the PPCD1 and PPCD3 loci excluded the presence of CNV involving either OVOL2 or ZEB1, respectively. The c.-307T>C variant demonstrated increased promoter activity in corneal endothelial cells when compared to the wild-type sequence as has been demonstrated previously in another cell type.ConclusionsPreviously identified as the cause of PPCD1, the OVOL2 promoter variant c.-307T>C was herein identified in the original family that established the PPCD1 locus. However, the failure to identify presumed pathogenic coding or non-coding OVOL2 or ZEB1 variants, or CNV involving the PPCD1 and PPCD3 loci in 26 other PPCD probands suggests that other genetic loci may be involved in the pathogenesis of PPCD.

Effect of the fourth nucleotide at the 3' end of neuraminidase and matrix viral genomic RNA on the pathogenicity of influenza virus A/PR/8/34.

Twelve nucleotides located at the 3′ end of viral genomic RNA (vRNA) are conserved among influenza A viruses (IAV) and have a promoter function. Hoffmann's 8-plasmid reverse genetics vector system introduced mutations at position 4, C nucleotide (C4) to U nucleotide (U4), of the 3′ ends of neuraminidase (NA) and matrix (M) vRNAs of wild-type A/PR/8/34 (PR8). This resulted in a constellation of C4 and U4 vRNAs coding for low (polymerases) and relatively high (all others) copy number proteins, respectively. U4 has been reported to increase promoter activity in comparison to C4, but the constellation effect on the replication efficiency and pathogenicity of reverse genetics PR8 (rgPR8) has not been fully elucidated. In the present study, we generated 3 recombinant viruses with C4 in the NA and/or M vRNAs and rgPR8 by using reverse genetics and compared their pathobiological traits. The mutant viruses showed lower replication efficiency than rgPR8 due to the low transcription levels of NA and/or M genes. Furthermore, C4 in the NA and/or M vRNAs induced lower PR8 virus pathogenicity in BALB/c mice. The results suggest that the constellation of C4 and U4 among vRNAs may be one of the multigenic determinants of IAV pathogenicity.

Serum Amyloid A3 Gene Expression in Adipocytes is an Indicator of the Interaction with Macrophages.

The infiltration of macrophages into adipose tissue and their interaction with adipocytes are essential for the chronic low-grade inflammation of obese adipose tissue. In this study, we identified the serum amyloid A3 (Saa3) gene as a key adipocyte-derived factor that is affected by interaction with macrophages. We showed that the Saa3 promoter in adipocytes actually responds to activated macrophages in a co-culture system. Decreasing C/EBPβ abundance in 3T3-L1 adipocytes or point mutation of C/EBPβ elements suppressed the increased promoter activity in response to activated macrophages, suggesting an essential role of C/EBPβ in Saa3 promoter activation. Bioluminescence based on Saa3 promoter activity in Saa3-luc mice was promoted in obese adipose tissue, showing that Saa3 promoter activity is most likely related to macrophage infiltration. This study suggests that the level of expression of the Saa3 gene could be utilized for the number of infiltrated macrophages in obese adipose tissue.

TATA Box Insertion Provides a Selection Mechanism Underpinning Adaptations to Fe Deficiency.

Intraspecific genetic variation is essential for the responses and adaption of plants to evolutionary challenges, such as changing environmental conditions. The development of the Earth's aerobic atmosphere has increased the demand for iron (Fe) in organisms, and Fe deficiency has become a limiting environmental factor for plant growth. Here, we demonstrate that genus Malus adapt to Fe deficiency through modification of the Iron-Regulated Transporter1 (IRT1) promoter. Specifically, an IRT1 mutant allele with a TATA-box insertion in the promoter region upstream of the coding region exhibited increased IRT1 expression. The altered IRT1 promoter is responsible for enhancing Fe uptake. Increasing the number of synthetic repeat TATA-boxes correlates with increased promoter activity. Furthermore, we demonstrate that the insertion of the TATA-box correlates with an increase in transcriptional activation via specific binding of the transcription factor IID (MDP0000939369). Taken together, these results indicate that an allelic insertion of a TATA-box in a gene promoter has allowed apple to adapt to the selective pressure posed by Fe deficiency. More broadly, this study reveals a new mechanism for enhancing gene expression to help plants adapt to different environments, providing new insights into molecular genetic divergence in plants.

Screening for Mutations in the TBX1 Gene on Chromosome 22q11.2 in Schizophrenia.

A higher-than-expected frequency of schizophrenia in patients with 22q11.2 deletion syndrome suggests that chromosome 22q11.2 harbors the responsive genes related to the pathophysiology of schizophrenia. The TBX1 gene, which maps to the region on chromosome 22q11.2, plays a vital role in neuronal functions. Haploinsufficiency of the TBX1 gene is associated with schizophrenia endophenotype. This study aimed to investigate whether the TBX1 gene is associated with schizophrenia. We searched for mutations in the TBX1 gene in 652 patients with schizophrenia and 567 control subjects using a re-sequencing method and conducted a reporter gene assay. We identified six SNPs and 25 rare mutations with no association with schizophrenia from Taiwan. Notably, we identified two rare schizophrenia-specific mutations (c.-123G>C and c.-11delC) located at 5′ UTR of the TBX1 gene. The reporter gene assay showed that c.-123C significantly decreased promoter activity, while c.-11delC increased promoter activity compared with the wild-type. Our findings suggest that the TBX1 gene is unlikely a major susceptible gene for schizophrenia in an ethnic Chinese population for Taiwan, but a few rare mutations in the TBX1 gene may contribute to the pathogenesis of schizophrenia in some patients.

RETRACTED: Role of HCP5-miR-139-RUNX1 Feedback Loop in Regulating Malignant Behavior of Glioma Cells

Aberrant expression of long noncoding RNAs has recently been reported in tumorigenesis and plays a pivotal role in regulating malignant behavior of cancers. In this study, we confirmed that the long noncoding RNAs human histocompatibility leukocyte antigen (HLA) complex P5 (HCP5) was up-regulated in glioma tissues as well as in U87 and U251 cells. Knockdown of HCP5 inhibited the malignant biological behavior of glioma cells by reducing proliferation, migration and invasion, and inducing apoptosis. HCP5 regulated the malignant behavior of glioma cells by binding to microRNA-139, which functions as a tumor suppressor. Moreover, knockdown of HCP5 down-regulated Runt-related transcription factor 1, a direct and functional downstream target of microRNA-139 that is involved in microRNA-139-mediated tumor-suppressive effects in glioma cells. Runt-related transcription factor 1 increased promoter activities and upregulated expression of the oncogenic gene astrocyte elevated gene-1 (AEG-1). Runt-related transcription factor 1 also increased the promoter activities and expression of HCP5, which showed a positive feedback loop in regulating the malignant behavior of glioma cells. In conclusion, this study demonstrated that the HCP5-microRNA-139- Runt-related transcription factor 1 feedback loop plays a pivotal role in regulating the malignant behavior of glioma cells, which may provide a potential therapeutic strategy for treating glioma.

Cytosolic phospholipase A2α increases proliferation and de-differentiation of human renal tubular epithelial cells

The group IVA calcium-dependent cytosolic phospholipase A2 (cPLA2α) enzyme controls the release of arachidonic acid from membrane bound phospholipids and is the rate-limiting step in production of eicosanoids. A variety of different kidney injuries activate cPLA2α, therefore we hypothesized that cPLA2α activity would regulate pathologic processes in HK-2 cells, a human renal tubular epithelial cell line, by regulating cell phenotype and proliferation. In two lentiviral cPLA2α-silenced knockdowns, we observed decreased proliferation and increased apoptosis compared to control HK-2 cells. cPLA2α-silenced cells also demonstrated an altered morphology, had increased expression E-cadherin, and decreased expression of Ncadherin. Increased levels of E-cadherin were associated with increased promoter activity and decreased levels of SNAIL1, SNAIL2, and ZEB1, transcriptional repressors of E-cadherin expression. Addition of exogenous arachidonic acid, but not PGE2, reversed the phenotypic changes in cPLA2α-silenced cells. These data suggest that cPLA2α may play a key role in renal repair after injury through a PGE2-independent mechanism.

Hepatocyte Nuclear Factor 4α (HNF4α) Is a Transcription Factor of Vertebrate Fatty Acyl Desaturase Gene as Identified in Marine Teleost Siganus canaliculatus.

Rabbitfish Siganus canaliculatus was the first marine teleost demonstrated to have the capability of biosynthesizing long-chain polyunsaturated fatty acids (LC-PUFA) from C18 precursors, and to possess a Δ4 fatty acyl desaturase (Δ4 Fad) which was the first report in vertebrates, and is a good model for studying the regulatory mechanisms of LC-PUFA biosynthesis in teleosts. In order to understand regulatory mechanisms of transcription of Δ4 Fad, the gene promoter was cloned and characterized in the present study. An upstream sequence of 1859 bp from the initiation codon ATG was cloned as the promoter candidate. On the basis of bioinformatic analysis, several binding sites of transcription factors (TF) including GATA binding protein 2 (GATA-2), CCAAT enhancer binding protein (C/EBP), nuclear factor 1 (NF-1), nuclear factor Y (NF-Y), hepatocyte nuclear factor 4α (HNF4α) and sterol regulatory element (SRE), were identified in the promoter by site-directed mutation and functional assays. HNF4α and NF-1 were confirmed to interact with the core promoter of Δ4 Fad by gel shift assay and mass spectrometry. Moreover, over-expression of HNF4α increased promoter activity in HEK 293T cells and mRNA level of Δ4 Fad in rabbitfish primary hepatocytes, respectively. The results indicated that HNF4α is a TF of rabbitfish Δ4 Fad. To our knowledge, this is the first report on promoter structure of a Δ4 Fad, and also the first demonstration of HNF4α as a TF of vertebrate Fad gene involved in transcription regulation of LC-PUFA biosynthesis.

Inflammation-Induced Expression and Secretion of MicroRNA 122 Leads to Reduced Blood Levels of Kidney-Derived Erythropoietin and Anemia

Anemia is associated commonly with acute and chronic inflammation, but the mechanisms of their interaction are not clear. We investigated whether microRNA 122 (MIR122), which is generated in the liver and is secreted into the blood, is involved in the development of anemia associated with inflammation. We characterized the primary transcript of the human liver-specific MIR122 using Northern blot, quantitative real-time polymerase chain reaction, and 3' and 5' rapid amplification of cDNA ends analyses. We studied regulation of MIR122 in human hepatocellular carcinoma cell lines (Huh7 and HepG2) as well as in C57BL/6 and mice with disruption of the tumor necrosis factor (Tnf) gene. Liver tissues were collected and analyzed by bioluminescence imaging or immunofluorescence. Inflammation in mice was induced by lipopolysaccharide (LPS) or by cerulein injections. Mice were given 4 successive injections of LPS, leading to inflammation-induced anemia. Steatohepatitis was induced with a choline-deficient, high-fat diet. Hemolytic anemia was stimulated by phenylhydrazine injection. MIR122 was inhibited in mice by tail-vein injection of an oligonucleotide antagonist of MIR122. MicroRNA and messenger RNA levels were determined by quantitative real-time polymerase chain reaction. The primary transcript of MIR122 spanned 5 kb, comprising 3 exons; the third encodes MIR122. Within the MIR122 promoter region we identified a nuclear factor-κB binding site and showed that RELA (NF-κB p65 subunit), as well as activators of NF-κB (TNF and LPS), increased promoter activity of MIR122. Administration of LPS to mice induced secretion of MIR122 into blood, which required TNF. Secreted MIR122 reached the kidney and reduced expression of erythropoietin (Epo), which we identified as a MIR122 target gene. Injection of mice with an oligonucleotide antagonist of MIR122 increased blood levels of EPO, reticulocytes, and hemoglobin. We found an inverse relationship between blood levels of MIR122 and EPO in mice with acute pancreatitis or steatohepatitis, and also in patients with acute inflammation. In mice, we found that LPS-induced inflammation increases blood levels of MIR122, which reduces expression of Epo in the kidney; this is a mechanism of inflammation-induced anemia. Strategies to block MIR122 in patients with inflammation could reduce the development or progression of anemia.

ATRA transcriptionally induces nSMase2 through CBP/p300-mediated histone acetylation

Neutral sphingomyelinase-2 (nSMase2) is a key ceramide-producing enzyme in cellular stress responses. While many posttranslational regulators of nSMase2 are known, emerging evidence suggests a more protracted regulation of nSMase2 at the transcriptional level. Previously, we reported that nSMase2 is induced by all-trans retinoic acid (ATRA) in MCF7 cells and implicated nSMase2 in ATRA-induced growth arrest. Here, we further investigated how ATRA regulates nSMase2. We find that ATRA regulates nSMase2 transcriptionally through the retinoic acid receptor-α, but this is independent of previously identified transcriptional regulators of nSMase2 (Sp1, Sp3, Runx2) and is not through increased promoter activity. Epigenetically, the nSMase2 gene is not repressively methylated in MCF7 cells. However, inhibition of histone deacetylases (HDACs) with trichostatin A (TSA) induced nSMase2 comparably to ATRA; furthermore, combined ATRA and TSA treatment was not additive, suggesting ATRA regulates nSMase2 through direct modulation of histone acetylation. Confirming this, the histone acetyltransferases CREB-binding protein and p300 were required for ATRA induction of nSMase2. Finally, use of class-specific HDAC inhibitors suggested that HDAC4 and/or HDAC5 are negative regulators of nSMase2 expression. Collectively, these results identify a novel pathway of nSMase2 regulation and suggest that physiological or pharmacological modulation of histone acetylation can directly affect nSMase2 levels.

A novel type 2 diabetes risk allele increases the promoter activity of the muscle-specific small ankyrin 1 gene

Genome-wide association studies have identified Ankyrin-1 (ANK1) as a common type 2 diabetes (T2D) susceptibility locus. However, the underlying causal variants and functional mechanisms remain unknown. We screened for 8 tag single nucleotide polymorphisms (SNPs) in ANK1 between 2 case-control studies. Genotype analysis revealed significant associations of 3 SNPs, rs508419 (first identified here), rs515071, and rs516946 with T2D (P < 0.001). These SNPs were in linkage disequilibrium (r2 > 0.80); subsequent analysis indicated that the CCC haplotype associated with increased T2D susceptibility (OR 1.447, P < 0.001). Further mapping showed that rs508419 resides in the muscle-specific ANK1 gene promoter. Allele-specific mRNA and protein level measurements confirmed association of the C allele with increased small ANK1 (sAnk1) expression in human skeletal muscle (P = 0.018 and P < 0.001, respectively). Luciferase assays showed increased rs508419-C allele transcriptional activity in murine skeletal muscle C2C12 myoblasts, and electrophoretic mobility-shift assays demonstrated altered rs508419 DNA-protein complex formation. Glucose uptake was decreased with excess sAnk1 expression upon insulin stimulation. Thus, the ANK1 rs508419-C T2D-risk allele alters DNA-protein complex binding leading to increased promoter activity and sAnk1 expression; thus, increased sAnk1 expression in skeletal muscle might contribute to T2D susceptibility.

Assessment of Urinary Inhibitor or Promoter Activity in Uric Acid Nephrolithiasis

We assessed decreased inhibitor activity or increased promoter activity in the urine of idiopathic uric acid stone formers compared to nonstone formers independent of urinary pH. A total of 30 idiopathic uric acid stone formers, and 9 obese and 12 lean nonstone formers collected 24-hour urine while on a metabolic diet. Three urine aliquots per subject were used to assess spontaneous nucleation (de novo crystal formation), crystal growth using a 0.1 mg/ml anhydrous uric acid seed and steady-state uric acid solubility (the maximum amount of uric acid dissolvable in urine) using a 5 mg/ml uric acid seed. All experiments were performed for 6 hours at a constant pH of 5.0. Uric acid concentration was measured in filtered aliquots at 0, 3 and 6 hours. At baseline 24-hour urinary pH was significantly lower and uric acid saturation was significantly higher in idiopathic uric acid stone formers. No significant spontaneous nucleation developed and similar uric acid steady-state solubility was reached in the 3 groups. Idiopathic uric acid stone formers and lean nonstone formers showed a similar decrease in uric acid concentration during crystal growth. Obese nonstone formers started with a higher uric acid concentration and consequently demonstrated a greater decrease in the uric acid concentration for crystal growth. This study suggests that there is no significant difference between idiopathic uric acid stone formers and nonstone formers in promoter or inhibitor activity in whole urine against uric acid stone formation when urine pH is maintained constant. The findings suggest that uric acid stone formation is dictated by high urinary saturation with respect to uric acid, which is driven primarily by low urine pH.

FOXP1 functions as an oncogene in promoting cancer stem cell-like characteristics in ovarian cancer cells.

Ovarian cancer has the highest mortality rate of all gynecological cancers with a high recurrence rate. It is important to understand the nature of recurring cancer cells to terminally eliminate ovarian cancer. The winged helix transcription factor Forkhead box P1 (FOXP1) has been reported to function as either oncogene or tumor-suppressor in various cancers. In the current study, we show that FOXP1 promotes cancer stem cell-like characteristics in ovarian cancer cells. Knockdown of FOXP1 expression in A2780 or SKOV3 ovarian cancer cells decreased spheroid formation, expression of stemness-related genes and epithelial to mesenchymal transition-related genes, cell migration, and resistance to Paclitaxel or Cisplatin treatment, whereas overexpression of FOXP1 in A2780 or SKOV3 ovarian cancer cells increased spheroid formation, expression of stemness-related genes and epithelial to mesenchymal transition-related genes, cell migration, and resistance to Paclitaxel or Cisplatin treatment. In addition, overexpression of FOXP1 increased promoter activity of ABCG2, OCT4, NANOG, and SOX2, among which the increases in ABCG2, OCT4, and SOX2 promoter activity were dependent on the presence of FOXP1-binding site. In xenotransplantation of A2780 ovarian cancer cells into nude mice, knockdown of FOXP1 expression significantly decreased tumor size. These results strongly suggest FOXP1 functions as an oncogene by promoting cancer stem cell-like characteristics in ovarian cancer cells. Targeting FOXP1 may provide a novel therapeutic opportunity for developing a relapse-free treatment for ovarian cancer patients.

In Vivo Deletion of the Cebpa +37 Kb Enhancer Markedly Reduces Cebpa mRNA in Myeloid Progenitors but Not in Non-Hematopoietic Tissues to Impair Granulopoiesis

The C/EBPα transcription factor is a key mediator of normal myeloid differentiation, and its reduced expression or activity is central to myeloid transformation, as recently reviewed (Friedman, Int. J. Hematol. 2015). The murine Cebpa gene contains a conserved, 450 bp, +37 kb regulatory element that displays the enhancer specific H3K4me1 and the activating H3K27Ac histone modifications at increasing levels as LT-HSC progress to ST-HSC, CMP, and GMP, increases promoter activity 6-fold when introduced into 32Dcl3 myeloid cells linked to a luciferase reporter, directs murine hCD4 transgene expression preferentially to GMP, myeloid CFUs, and functional LT-HSC, and is bound and activated by RUNX1, PU.1, SCL, GATA-2, C/EBPα, and c-Myb (Guo et al, Blood 2012; Guo et al, J. Leuk. Biol., 2014; Cooper et al, PLoS One 2015). In addition, CRISPR/Cas9 mediated enhancer mutation markedly reduces Cebpa transcription in 32Dcl3 cells (Cooper et al, 2015), and in AML samples harboring t(8;21) the RUNX1-ETO oncoprotein interacts with the human CEBPA locus specifically at the homologous +42 kb enhancer. We have now characterized mice harboring floxed Cebpa +37 kb enhancer alleles. Utilizing a B6 BAC encompassing the Cebpa locus, we assembled a targeting vector containing a 5.5 kb 5' homology arm, the +37 kb enhancer flanked by lox P sites, a PGK-Neo cassette flanked by frt sites, and a 0.9 kb 3' homology arm. After introduction into a B6 mESC line, several G418-resistant subclones were isolated, and homologous, heterozygous targeting was confirmed by 3' PCR and 5' and 3' Southern blotting. Two ESC lines were utilized to develop germ line chimeras, and upon breeding with B6 albino mice, one yielded several offspring with germ line transmission of the targeted allele, which were then bred to homozygosity. In an initial set of experiments, marrow from wild-type (WT) and Enh(f/f) mice exposed to 5-FU was transduced with pBabePuro or pBabePuro-Cre followed by puromycin selection and in vitro analysis. Cebpa mRNA was reduced 10-fold in the lineage-negative subset upon Enh(f/f) marrow transduction with Cre. CFU-G were reduced 8-fold, whereas CFU-M and CFU-GM were unaffected, and enhancer deletion enabled serial replating of myeloid CFUs for >16 generations, though these remained dependent on IL-3. Morphologically, enhancer deletion led to depletion of mature granulocytes and accumulation of CD11b- Gr-1- blasts after four days of culture in IL-3, IL-6, and SCF. We next generated Enh(f/f);Mx1-Cre mice and subjected these and Enh(f/f) littermates to pIpC injections, followed by analysis four weeks after the last injection. Cebpa mRNA was reduced 12-fold in GMP, 13-fold in CMP, and >20-fold in the LSK marrow subset. Peripheral blood and marrow neutrophils were reduced 3- to 8-fold, whereas monocytes, RBCs, platelets, and B or T lymphocytes were unaffected. GMP as a percentage of Lin- Sca-1- c-kit+ progenitors were reduced 3-fold, CMP were increased 1.5-fold, and MEP and CLP were unaffected. CFU-G were reduced 3-fold, CFU-M were increased 2-fold, and BFU-E were increased 4-fold. LSK as a percentage of Lin- cells were increased 3-fold, and MPP and ST-HSC as a percentage of LSK were unaffected, whereas LSK/SLAM were reduced 10-fold, and enumeration of functional LT-HSC by competitive transplantation is in progress. We also generated Enh(f/f);Vav-Cre mice; these demonstrated similarly impaired granulopoiesis, but were obtained at only 12% of the Mendelian ratio, suggestive of embryonic lethality as early death was not observed. In addition, we used FLPo-Cre to delete the Neo cassette; homozygous deletion did not alter myeloid CFU numbers or FACS-defined progenitors. Finally, we utilized CMV-Cre to develop germ line enhancer-deleted mice. As with Vav-Cre, homozygous offspring were obtained far below the predicted numbers. Nevertheless, these expressed Cebpa mRNA at levels equivalent to WT controls in liver, lung, adipose, kidney, small intestine, skeletal muscle, and cardiac tissues, and these organs displayed normal cellular architecture upon hematoxylin-eosin staining. In summary, conditional deletion of the +37 kb Cebpa enhancer in adult mice demonstrates that it acts as a critical, hematopoietic specific enhancer of Cebpa gene expression and that marked Cebpa mRNA down-modulation upon enhancer deletion impairs granulopoiesis and enables serial, cytokine-dependent myeloid CFU replating, a pre-leukemic phenotype. DisclosuresNo relevant conflicts of interest to declare.

Expression of angiopoietin-1 in hypoxic pericytes: Regulation by hypoxia-inducible factor-2α and participation in endothelial cell migration and tube formation

We previously reported that hypoxia increases angiopoietin-1 (Ang1), but not Ang2, mRNA expression in bovine retinal pericytes (BRP). However, the mechanism underlying Ang1 expression is unknown. Here, we report that Ang1 protein expression increased in hypoxic BRP in a dose- and time-dependent manner. This increase was accompanied by an increase in hypoxia-inducible factor-2α (HIF2α) expression. Transfection with an antisense oligonucleotide for HIF2α partially inhibited the hypoxia-induced increase in Ang1 expression. HIF2α overexpression further potentiated hypoxia-stimulated Ang1 expression, suggesting that HIF2α plays an important role in Ang1 regulation in BRP. When fused the Ang1 promoter (−3040 to +199) with the luciferase reporter gene, we found that hypoxia significantly increased promoter activity by 4.02 ± 1.68 fold. However, progressive 5′-deletions from −3040 to −1799, which deleted two putative hypoxia response elements (HRE), abolished the hypoxia-induced increase in promoter activity. An electrophoretic mobility shift assay revealed that HIF2α was predominantly bound to a HRE site, located specifically at nucleotides −2715 to −2712. Finally, treatment with conditioned medium obtained from hypoxic pericytes stimulated endothelial cell migration and tube formation, which was completely blocked by co-treatment with anti-Ang1 antibody. This study is the first to demonstrate that hypoxia upregulates Ang1 expression via HIF2α-mediated transcriptional activation in pericytes, which plays a key role in angiogenesis.