GT Box Research Articles

BANet: Small and multi-object detection with a bidirectional attention network for traffic scenes

Improving the detection accuracy and speed for small and multi-object detection is a hot issue in traffic environments. Despite the substantial advances in object detection algorithms based on deep neural networks, addressing the inaccuracy and low efficiency of small and multi-object detection remains challenging. In this paper, we propose a bidirectional attention network called BANet, which includes multichannel attention (MCA) blocks, alpha-effective intersection-over-union (α-EIoU) loss, and a multiple attention fusion (MAF) module. Each MCA block consists of low-layer, medium-layer, and high-layer features to provide rich base information for feature fusion at the neck module. We introduce MAF to alleviate the spatial location loss and poor semantic performance resulting from the continuous downsampling of the path aggregation feature pyramid network (PAFPNet). Finally, α-EIoU is our regression loss module, which calculates the difference between the predicted box and the ground truth (gt) box. Our study further demonstrates that these strategies yield significant improvements in performance over some existing YOLO detectors. Compared with the performance of YOLOX, BANet demonstrates 0.39%–0.52% mAP@0.5 improvement on the PASCAL VOC 2007 (VOC 07) dataset and 0.55%–2.93% mAP@0.5 improvement on the PASCAL VOC 2012 (VOC 12) dataset. Additionally, 0.3%–1.01% improvement in the mAP@0.5 is achieved on the MS COCO 2017 (COCO 17) dataset, indicating that BANet has a significant effect on multi-object detection. Experiments to determine the approximate number of parameters with YOLOX, show that our strategy not only improves by 7.5 frames per second (FPS) but also reduces the Average forward time by 0.97 ms.

Myogenic differentiation 1 and transcription factor 12 activate the gene expression of mouse taste receptor type 1 member 1

ObjectivesMyogenic differentiation 1 (Myod1) is involved in the expression of taste receptor type 1 member 1 (Tas1r1) during myogenic differentiation. Further, the target genes of Myod1 participate in transcriptional control, muscle development, and synaptic function. We examined, for the first time, the function of Myod1 in the transcriptional regulation of Tas1r1. MethodsENCODE chromatin immunoprecipitation and sequencing (ChIP-seq) data of myogenically differentiated C2C12 cells were analyzed to identify the Myod1 and transcription factor 12 (Tcf12) binding sites in the Tas1r1 promoter region. Luciferase reporter assays, DNA affinity precipitation assays, and co-immunoprecipitation assays were also performed to identify the functions of Myod1, Tcf12, and Krüppel-like factor 5 (Klf5). ResultsBased on ENCODE ChIP-seq, Myod1 bound to the Tas1r1 promoter region containing E-boxes 1–3. Luciferase reporter assays revealed that site-directed E-box1 mutations significantly reduced promoter activation induced by Myod1 overexpression. According to the DNA affinity precipitation assay and co-immunoprecipitation assay, Myod1 formed a heterodimer with Tcf12 and bound to E-box1. Further, Klf5 bound to the GT box near E-box1, activating Tas1r1 expression. ConclusionsDuring myogenic differentiation, the Myod1/Tcf12 heterodimer, in collaboration with Klf5, binds to E-box1 and activates Tas1r1 expression.

Detecting dense text in natural images

Most existing text detection methods are mainly motivated by deep learning‐based object detection approaches, which may result in serious overlapping between detected text lines, especially in dense text scenarios. It is because text boxes are not commonly overlapped, as different from general objects in natural scenes. Moreover, text detection requires higher localisation accuracy than object detection. To tackle these problems, the authors propose a novel dense text detection network (DTDN) to localise tighter text lines without overlapping. Their main novelties are: (i) propose an intersection‐over‐union overlap loss, which considers correlations between one anchor and GT boxes and measures how many text areas one anchor contains, (ii) propose a novel anchor sample selection strategy, named CMax‐OMin, to select tighter positive samples for training. CMax‐OMin strategy not only considers whether an anchor has the largest overlap with its corresponding GT box (CMax), but also ensures the overlapping between one anchor and other GT boxes as little as possible (OMin). Besides, they train a bounding‐box regressor as post‐processing to further improve text localisation performance. Experiments on scene text benchmark datasets and their proposed dense text dataset demonstrate that the proposed DTDN achieves competitive performance, especially for dense text scenarios.

Apolipoprotein L1 is transcriptionally regulated by SP1, IRF1 and IRF2 in hepatoma cells

Apolipoprotein L1 (APOL1) participates in lipid metabolism. Here, we investigate the mechanisms regulating APOL1 gene expression in hepatoma cells. We demonstrate that the -80-nt to +31-nt region of the APOL1 promoter, which contains one SP transcription factor binding GT box and an interferon regulatory factor (IRF) binding ISRE element, maintains the maximum activity. Mutation of the GT box and ISRE element dramatically reduces APOL1 promoter activity. EMSA and chromatin immunoprecipitation assay reveal that the transcription factors Sp1, IRF1 and IRF2 could interact with their cognate binding sites on the APOL1 promoter. Overexpression of Sp1, IRF1 and IRF2 increases promoter activity, leading to increased APOL1 mRNA and protein levels, while knockdown of Sp1, IRF1 and IRF2 has the opposite effects. These results demonstrate that the APOL1 gene could be regulated by Sp1, IRF1 and IRF2 in hepatoma cells.

Krüppel-like factor 5 (Klf5) regulates expression of mouse T1R1 amino acid receptor gene (Tas1r1) in C2C12 myoblast cells.

T1R1 and T1R3 are receptors expressed in taste buds that detect L-amino acids. These receptors are also expressed throughout diverse organ systems, such as the digestive system and muscle tissue, and are thought to function as amino acid sensors. The mechanism of transcriptional regulation of the mouse T1R1 gene (Tas1r1) has not been determined; therefore, in this study, we examined the function of Tas1r1 promoter in the mouse myoblast cell line, C2C12. Luciferase reporter assays showed that a 148-bp region upstream of the ATG start codon of Tas1r1 had a promoter activity. The GT box in the Tas1r1 promoter was conserved in the dog, human, mouse, and pig. Site-directed mutagenesis of this GT box significantly reduced the promoter activation. The GT box in promoters is a recurring motif for Sp/KLF family members. RNAi-mediated depletion of Sp4 and Klf5 decreased Tas1r1 expression, while overexpression of Klf5, but not Sp4, significantly increased Tas1r1 expression. The ENCODE data of chromatin immunoprecipitation and sequencing (ChIP-seq) showed that Klf5 bound to the GT box during the myogenic differentiation. Furthermore, the Klf5 knockout cell lines led to a considerable decrease in the levels of Tas1r1 expression. Collectively, these results showed that Klf5 binds to the GT box in the Tas1r1 promoter and regulates Tas1r1 expression in C2C12 cells.

Kruppel-like factors and vascular inflammation: implications for atherosclerosis.

Kruppel-like factors and vascular inflammation: implications for atherosclerosis.

Inhibition of Transcription Factor Specificity Protein 1 Alters the Gene Expression Profile of Keratinocytes Leading to Upregulation of Kallikrein-Related Peptidases and Thymic Stromal Lymphopoietin

Transcription factor specificity protein 1 (Sp1) is involved in diverse cellular functions. We recently found that Sp1 was significantly decreased in skin biopsies from patients with atopic dermatitis (AD) and had an even greater reduction in AD patients with a history of eczema herpeticum. In the current study, we sought to better understand the role of Sp1 in skin biological processes by using a small interfering RNA (siRNA) technique to knock down Sp1 gene expression in normal human keratinocytes (NHK) and investigated the genome-wide gene expression profiling of Sp1- silenced NHK. The gene arrays revealed that 53 genes had more than three-fold changes in expression in Sp1-silenced NHK as compared to scrambled siRNA silenced cells. Strikingly, six kallikrein-related peptidase genes, KLK5, KLK6, KLK7, KLK8, KLK10, and KLK12 were up-regulated in NHK following Sp1 silencing. Functionally, protease activity was significantly enhanced in Sp1-silenced keratinocytes as compared to scrambled siRNA silenced keratinocytes. Moreover, thymic stromal lymphopoietin (TSLP), an epithelial derived TH2 promoting cytokine, was induced in Sp1-silenced keratinocytes due to elevated kallikrein activity. These results indicate that Sp1 expression deficiency leads to abnormally increased kallikrein protease activity in keratinocytes and may contribute to TH2 immune responses in the skin by inducing TSLP.

Pro-oncogene Pokemon promotes breast cancer progression by upregulating survivin expression

IntroductionPokemon is an oncogenic transcription factor involved in cell growth, differentiation and oncogenesis, but little is known about its role in human breast cancer. In this study, we aimed to reveal the role of Pokemon in breast cancer progression and patient survival and to understand its underlying mechanisms.MethodsTissue microarray analysis of breast cancer tissues from patients with complete clinicopathological data and more than 20 years of follow-up were used to evaluate Pokemon expression and its correlation with the progression and prognosis of the disease. DNA microarray analysis of MCF-7 cells that overexpress Pokemon was used to identify Pokemon target genes. Chromatin immunoprecipitation (ChIP) and site-directed mutagenesis were utilized to determine how Pokemon regulates survivin expression, a target gene.ResultsPokemon was found to be overexpressed in 158 (86.8%) of 182 breast cancer tissues, and its expression was correlated with tumor size (P = 0.0148) and lymph node metastasis (P = 0.0014). Pokemon expression led to worse overall (n = 175, P = 0.01) and disease-related (n = 79, P = 0.0134) patient survival. DNA microarray analyses revealed that in MCF-7 breast cancer cells, Pokemon regulates the expression of at least 121 genes involved in several signaling and metabolic pathways, including anti-apoptotic survivin. In clinical specimens, Pokemon and survivin expression were highly correlated (n = 49, r = 0.6799, P < 0.0001). ChIP and site-directed mutagenesis indicated that Pokemon induces survivin expression by binding to the GT boxes in its promoter.ConclusionsPokemon promotes breast cancer progression by upregulating survivin expression and thus may be a potential target for the treatment of this malignancy.

Identification and characterization of human PCDH10 gene promoter

Recent studies have suggested roles for PCDH10 as a novel tumor suppressor gene. In our previous work, we located the core promoter of PCDH10 to a 462-bp segment of 5′-flanking region characterized by a high GC content. Here we further identified and characterized the promoter for PCDH10. Transient transfection of PC3 and LNCaP cells with a series of deleted promoter constructs indicated that the minimal promoter region was between nucleotides −144 and −99. This segment contained a CAAT box, a GT box, and a putative transcription factor binding site for AP-4. Mutational analysis identified that the CAAT box and GT box are necessary for promoter activity. Ectopic expression of NF-Ys increased reporter gene activity, whereas expression of a dominant-negative NF-YA decreased reporter gene activity. Co-transfection of Sp1/Sp3 expression plasmids enhanced reporter gene activity in a dose-dependent manner. Mithramycin A, an inhibitor of Sp–DNA interaction, reduced PCDH10 promoter activity. Electrophoretic mobility shift assays and chromatin immunoprecipitation demonstrated binding of transcription factors Sp1/Sp3 to the promoter region in vitro and in vivo. Our data show that Sp1/Sp3 and CBF/NF-Y transcription factors play a crucial role in the basal expression of the human PCDH10 gene.

The activity of interleukin-4 receptor α-chain promoter is regulated by a GT box element

Interleukin-4 receptor (IL-4R) is the cell surface complex through which interleukin-4 (IL-4) signals exert its critical biological effects. The α-chain of IL-4R is responsible for the high affinity binding of IL-4. In this report, is characterized, the 5′ untranslated flanking region of murine IL-4Rα gene in the Th2 clone D10.G4.1. We have analyzed a DNA fragment spanning from −995 to +84 relative to the transcription start point. Mutagenesis analysis shows that, neither the previously described Stat6 (–395) nor the NFAT (−266) and NFκB (+25) sequences localized here, are involved in the IL-4Rα promoter activity. Reporter assays demonstrate that maximum transcriptional activity is achieved by the −89 to +84 sequence and this activity is independent of a TATA-like box located at −25. We have identified a GT box located at −45 as the critical element for the IL-4Rα promoter activity. Experiments in SL2 cells, which lack endogenous Sp proteins, show that IL-4Rα minimal promoter is transactivated by proteins of Sp family.

Characterization of a Downstream Positive Element Involved in Transcriptional Control of the Human CD3γ Gene Promoter

Experimental data from our laboratory has shown that TCR/CD3 surface receptors are downmodulated after HIV-1 and HIV-2 infection of CD4+ T cells due to a specific defect in CD3γ gene transcripts. In an effort to better understand the mechanism(s) involved, our laboratory has been investigating the critical elements responsible for regulating this gene. We have shown that the CD3 γ gene is transcribed from an independent but weak, lymphoidspecific TATA-less promoter and demonstrated that a cluster of transcription initiation sites is present in the vicinity of the principal core promoter with the major start site situated in a classical initiator sequence. A GT box upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these contiguous elements enhanced by a second Sp binding GC box ten nucleotides further upstream. We found that two previously identified NFAT motifs positively (NFAT γ 1) or negatively (NFAT γ 1 and NFAT γ 2) regulate expression of the CD3 γ gene by their differential binding of NFATc1 plus NF-κB p50 or NFATc2 containing complexes, respectively. Analysis of various mutant and deletion CD3 γ promoter constructs in a transient reporter assay revealed that a 53 bp region downstream from the major transcription start site is critical for positive gene expression. Deletion of ten nucleotides in this region results in a 50% decrease in promoter activity, while deletion of 39 nucleotides completely eliminates promoter activity. EMSA experiments using DNA or RNA probes covering the 53 bp region demonstrate that this element functions through an RNA rather than a DNA intermediate. At least three specific nuclear protein complexes bind to the RNA probe. Deletion of the U at position +9 and the U at +37 completely abrogate binding and promoter activity. Experiments are currently underway to determine whether the composition of the transcription factor(s) complexes bound to the CD3γ element contain components of P-TEFb, which binds to HIV TAR. from 2005 International Meeting of The Institute of Human Virology Baltimore, USA, 29 August – 2 September 2005

The binding of the ubiquitous transcription factor Sp1 at the locus control region represses the expression of β-like globin genes

To investigate the function of transcription factor Sp1 in beta-like globin gene activation, we analyzed the recruitment of Sp1, fetal Krüppel-like factor 2 (FKLF2), and related factors at the human beta-globin locus in a human fetal liver and mouse erythroleukemia hybrid cell (A181gamma cell) that contains a single copy of human chromosome 11. Sp1 binds at the GT boxes of the cis-elements throughout the beta-locus, but it is phosphorylated and lost over DNase I hypersensitive site (HS)2, HS3, HS4, and the human beta-globin gene promoter after A181gamma cell differentiation. The binding of FKLF2 at HS2 and HS3 was unchanged. Histone deacetylase 1, which could be recruited by Sp1, is also lost over HS2 and HS3 after differentiation, resulting in the acetylation of histones 3 and 4 across the human beta-globin locus. We previously detected in vivo GT footprints over the beta-globin locus after A181gamma differentiation. Here, we report that after differentiation, the p300/CREB-binding protein-associated factor is recruited by FKLF2 to the locus control region to acetylate histones 3 and 4 at the human beta-globin gene locus. Our results suggest that Sp1 is an inhibitor of beta-like globin gene transcription during erythroid terminal differentiation. Its phosphorylation and release allow the erythroid-specific FKLF2 or erythroid Krüppel-like factor to interact with other erythroid-specific transcription factors to initiate the transcription of beta-like globin genes.

Transcriptional Regulation of the HumanCD3γ Gene: The TATA-LessCD3γ Promoter Functions via an Initiator and Contiguous Sp-Binding Elements

Growing evidence that the CD3gamma gene is specifically targeted in some T cell diseases focused our attention on the need to identify and characterize the elusive elements involved in CD3gamma transcriptional control. In this study, we show that while the human CD3gamma and CD3delta genes are oriented head-to-head and separated by only 1.6 kb, the CD3gamma gene is transcribed from an independent but weak, lymphoid-specific TATA-less proximal promoter. Using RNA ligase-mediated rapid amplification of cDNA ends, we demonstrate that a cluster of transcription initiation sites is present in the vicinity of the primary core promoter, and the major start site is situated in a classical initiator sequence. A GT box immediately upstream of the initiator binds Sp family proteins and the general transcription machinery, with the activity of these adjacent elements enhanced by the presence of a second GC box 10 nt further upstream. The primary core promoter is limited to a sequence that extends upstream to -15 and contains the initiator and GT box. An identical GT box located approximately 50 nt from the initiator functions as a weak secondary core promoter and likely generates transcripts originating upstream from the +1. Finally, we show that two previously identified NFAT motifs in the proximal promoter positively (NFATgamma(1)) or negatively (NFATgamma(1) and NFATgamma(2)) regulate expression of the human CD3gamma gene by their differential binding of NFATc1 plus NF-kappaB p50 or NFATc2 containing complexes, respectively. These data elucidate some of the mechanisms controlling expression of the CD3gamma gene as a step toward furthering our understanding of how its transcription is targeted in human disease.

Distal enhancer of the mouse FGF‐4 gene and its human counterpart exhibit differential activity: Critical role of a GT box

Previous studies have shown that there is a strict requirement for fibroblast growth factor-4 (FGF-4) during mammalian embryogenesis, and that FGF-4 expression in embryonic stem (ES) cells and embryonal carcinoma (EC) cells are controlled by a powerful downstream distal enhancer. More recently, mouse ES cells were shown to express significantly more FGF-4 mRNA than human ES cells. In the work reported here, we demonstrate that mouse EC cells also express far more FGF-4 mRNA than human EC cells. Using a panel of FGF-4 promoter/reporter gene constructs, we demonstrate that the enhancer of the mouse FGF-4 gene is approximately tenfold more active than its human counterpart. Moreover, we demonstrate that the critical difference between the mouse and the human FGF-4 enhancer is a 4 bp difference in the sequence of an essential GT box. Importantly, we demonstrate that changing 4 bp in the human enhancer to match the sequence of the mouse GT box elevates the activity of the human FGF-4 enhancer to the same level as that of the mouse enhancer. We extended these studies by examining the roles of Sp1 and Sp3 in FGF-4 expression. Although we demonstrate that Sp3, but not Sp1, can activate the FGF-4 promoter when artificially tethered to the FGF-4 enhancer, we show that Sp3 is not essential for expression of FGF-4 mRNA in mouse ES cells. Finally, our studies with human EC cells suggest that the factor responsible for mediating the effect of the mouse GT box is unlikely to be Sp1 or Sp3, and this factor is either not expressed in human EC cells or it is not sufficiently active in these cells.

Functional analysis of the mouse Fcgrt 5′ proximal promoter

The Brambell receptor (FcRB, FcRn) mediates transmission of immunity from mother to young perinatally and plays a central role in IgG protection and homeostasis throughout life. Developmental and tissue specific expression is via transcriptional regulation of the gene for the receptor heavy chain, Fcgrt. In neonatal mouse intestine, expression is high to absorb IgG from mother's milk, but then nearly absent in adult intestine and in most other tissues. In this initial functional characterization, reporter gene assays revealed at least two upstream promoter regions (−372/−140 and −105/−1), each with repressor and activator functions, and a downstream activator domain (+1/+78). The gene carries no upstream TATA element or Inr sequence, and an apparent downstream promoter feature (DPE) lacks typical context for an active element, rendering uncertain the nature of the organizing feature for the transcription initiation complex. Electrophoretic mobility shift analysis of the proximal upstream region identified transcription factor (TF) binding to motifs for NF1, Sp1 (GT box) and Ets. Binding to the GT box and Ets motif was observed only with mature cell sources, and not with neonatal enterocyte extracts. Site-directed mutagenesis confirmed that TF binding to the GT box up-regulates promoter activity in adult (NIH3T3) cells, whereas binding to the Ets motif represses activity. Binding of NF1 protein to the Fcgrt promoter was confirmed in nuclear extracts of NIH3T3 cells and in adult mouse enterocytes, whereas an apparently different TF bound uniquely to the NF1 site in neonatal enterocyte extracts as the sole identified candidate for an expected developmental-specific high-level activator in this tissue. These data indicate regions of potential importance in the Fcgrt proximal promoter and additionally suggest that the selective temporal and spatial availability of specific TFs may contribute to the developmental and tissue-specific regulation of Fcgrt expression.

ZEC, a zinc finger protein with novel binding specificity and transcription regulatory activity

A novel 114-kDa zinc finger protein, ZEC, has been found by cDNA cloning and characterized. ZEC was strongly expressed in the testis, liver and kidney, and also in embryonic stem cells. Epitope-tagged experiments indicated nuclear localization of ZEC. ZEC contained 18 C2H2 zinc fingers which were organized in two clusters. A ZEC binding DNA sequence, C/GA/TA/TGGTTGGTTGC, which we have designated the GT box, was identified by random oligonucleotide binding selection assay. The GT box did not contain binding sites for other previously characterized transcription factors and thus represented a potentially novel DNA target sequence. Electrophoretic mobility shift assay (EMSA) showed that both clusters of zinc fingers bound to the same DNA sequence. Site-directed mutagenesis revealed that the core sequence TTGGTT within the GT box was essential to ZEC binding, while DNA sequences outside of the core sequence enhanced this interaction. Furthermore, co-transfection assays demonstrated that ZEC could activate a reporter luciferase gene driven by this DNA sequence.

Methylation in the Core-promoter Region of the Chondromodulin-I Gene Determines the Cell-specific Expression by Regulating the Binding of Transcriptional Activator Sp3

Transcriptional regulation of cell- and stage-specific genes is a crucial process in the development of mesenchymal tissues. Here we have investigated the regulatory mechanism of the expression of the chondromodulin-I (ChM-I) gene, one of the chondrocyte-specific genes, in osteogenic cells using osteosarcoma (OS) cells as a model. Methylation-specific sequence analyses revealed that the extent of methylation in the core-promoter region of the ChM-I gene was correlated inversely with the expression of the ChM-I gene in OS primary tumors and cell lines. 5-Aza-deoxycytidine treatment induced the expression of the ChM-I gene in ChM-I-negative OS cell lines, and the induction of expression was associated tightly with the demethylation of cytosine at -52 (C(-52)) in the middle of an Sp1/3 binding site to which the Sp3, but not Sp1, bound. The replacement of C(-52) with methyl-cytosine or thymine abrogated Sp3 binding and also the transcription activity of the genomic fragment including C(-52). The inhibition of Sp3 expression by small interfering RNA reduced the expression of the ChM-I gene in ChM-I-positive normal chondrocytes, indicating Sp3 as a physiological transcriptional activator of the ChM-I gene. These results suggest that the methylation status of the core-promoter region is one of the mechanisms to determine the cell-specific expression of the ChM-I gene through the regulation of the binding of Sp3.

A novel retinoic acid-responsive element regulates retinoic acid-induced BLR1 expression.

The mechanism of action of retinoic acid (RA) is of broad relevance to cell and developmental biology, nutrition, and cancer chemotherapy. RA is known to induce expression of the Burkitt's lymphoma receptor 1 (BLR1) gene which propels RA-induced cell cycle arrest and differentiation of HL-60 human myeloblastic leukemia cells, motivating the present analysis of transcriptional regulation of blr1 expression by RA. The RA-treated HL-60 cells used here expressed all RA receptor (RAR) and retinoid X receptor (RXR) subtypes (as detected by Northern analysis) except RXRgamma. Treatment with RAR- and RXR-selective ligands showed that RARalpha synergized with RXRalpha to transcriptionally activate blr1 expression. A 5'-flanking region capable of supporting RA-induced blr1 activation in HL-60 cells was found to contain a 205-bp sequence in the distal portion that was necessary for transcriptional activation by RA. Within this sequence DNase I footprinting revealed that RA induced binding of a nuclear protein complex to an element containing two GT boxes. Electromobility shift assays (EMSAs) and supershift assays showed that this element bound recombinant RARalpha and RXRalpha. Without RA there was neither complex binding nor transcriptional activation. Both GT boxes were needed for binding the complex, and mutation of either GT box caused the loss of transcriptional activation by RA. The ability of this cis-acting RAR-RXR binding element to activate transcription in response to RA also depended on downstream sequences where an octamer transcription factor 1 (Oct1) site and a nuclear factor of activated T cells (NFATc) site between this element and the transcriptional start, as well as a cyclic AMP response element binding factor (CREB) site between the transcriptional start and first exon of the blr1 gene, were necessary. Each of these sites bound its corresponding transcription factor. A transcription factor-transcription factor binding array analysis of nuclear lysate from RA-treated cells indicated several prominent RARalpha binding partners; among these, Oct1, NFATc3, and CREB2 were identified by competition EMSA and supershift and chromatin immunoprecipitation assays as components of the complex. RA upregulated expression of these three factors. In sum the results of the present study indicate that RA-induced expression of blr1 expression depends on a novel RA response element. This cis-acting element approximately 1 kb upstream of the transcriptional start consists of two GT boxes that bind RAR and RXR in a nuclear protein complex that also contains Oct1, NFATc3, and CREB2 bound to their cognate downstream consensus binding sites.

Identification of Transcription Factor KLF8 as a Downstream Target of Focal Adhesion Kinase in Its Regulation of Cyclin D1 and Cell Cycle Progression

Focal adhesion kinase (FAK) is an important mediator of integrin signaling in the regulation of cell adhesion, migration, survival, and proliferation. Here we report the identification of the transcription factor KLF8 as a target of FAK in cell cycle regulation. KLF8 is induced by FAK and decreased by FAK dominant-negative mutant ΔC14. Overexpression of KLF8 increases cell cycle progression, whereas inhibition of endogenous KLF8 by siRNA reduces it. Cyclin D1 promoter is identified as a target of KLF8, which is activated both directly by KLF8 binding to the GT box A and by an indirect mechanism through its repression of a potential inhibitory regulator of cyclin D1. Transcription activation of cyclin D1 by FAK requires both Ets family and KLF8 factors in a temporally differential manner. Together, our data provide further insights into molecular mechanism for FAK to regulate cell cycle progression.

Transcriptional Regulation of the Epiregulin Gene in the Rat Ovary

Ovarian follicular development is initiated by FSH secreted from the pituitary gland. The FSH-induced follicular development involves granulosa cell proliferation and differentiation. We demonstrated that a growth factor of epidermal growth factor (EGF) family epiregulin was rapidly induced in the primary culture of rat ovarian granulosa cells by FSH within 1 h. Epiregulin gene expression was also observed in granulosa cells of antral ovarian follicles from pregnant mare's serum gonadotropin-primed rats in vivo. To analyze the regulation of gene expression of epiregulin, we isolated and characterized the rat epiregulin gene of 22.1 kb, including 3.8 kb of 5'-upstream region as well as all five exons and four introns. We determined the transcriptional start site of rat epiregulin gene by primer extension analysis and then characterized the upstream promoter region of the gene. By using a luciferase reporter system, deletion and mutation analyses of rat epiregulin gene promoter region revealed that 125 bp upstream of transcriptional start site was essential, and that two CT boxes and one GT box within this region were important for the gene expression. We also demonstrated by EMSAs that Sp1/Sp3 proteins were involved in the epiregulin gene expression via the upstream sequence. Involvement of Sp1/Sp3 was also demonstrated that transfection of Sp1 or Sp3 expression plasmids dramatically increased the epiregulin gene promoter activities about 90- or 7.9-fold, respectively, in Drosophila SL2 cells that lack endogenous Sp family proteins. Such an increase in the promoter activity was also observed in mammalian cells when NIH-3T3 cells were used. In conclusion, we demonstrated here for the first time that EGF-type growth factor epiregulin is rapidly and strongly induced in the ovarian granulosa cells by FSH stimulation, and that two CT boxes and one GT box present in the upstream region are essential for the promoter activity of rat epiregulin. We also demonstrated that Sp family members play crucial roles in the epiregulin promoter activity through the CT boxes. The restricted and hormonally regulated expression of epiregulin in the rat ovarian granulosa cells may correspond to the physiological relevance of this peptide growth factor to the FSH-induced ovarian follicular growth and maturation.