GC Motifs Research Articles

Engineered cytosine base editor enabling broad-scope and high-fidelity gene editing in Streptomyces

Base editing (BE) faces protospacer adjacent motif (PAM) constraints and off-target effects in both eukaryotes and prokaryotes. For Streptomyces, renowned as one of the most prolific bacterial producers of antibiotics, the challenges are more pronounced due to its diverse genomic content and high GC content. Here, we develop a base editor named eSCBE3-NG-Hypa, tailored with both high efficiency and -fidelity for Streptomyces. Of note, eSCBE3-NG-Hypa recognizes NG PAM and exhibits high activity at challenging sites with high GC content or GC motifs, while displaying minimal off-target effects. To illustrate its practicability, we employ eSCBE3-NG-Hypa to achieve precise key amino acid conversion of the dehydratase (DH) domains within the modular polyketide synthase (PKS) responsible for the insecticide avermectins biosynthesis, achieving domains inactivation. The resulting DH-inactivated mutants, while ceasing avermectins production, produce a high yield of oligomycin, indicating competitive relationships among multiple biosynthetic gene clusters (BGCs) in Streptomyces avermitilis. Leveraging this insight, we use eSCBE3-NG-Hypa to introduce premature stop codons into competitor gene cluster of ave in an industrial S. avermitilis, with the mutant Δolm exhibiting the highest 4.45-fold increase in avermectin B1a compared to the control. This work provides a potent tool for modifying biosynthetic pathways and advancing metabolic engineering in Streptomyces.

Predictive modeling of oocyte maternal mRNA features for five mammalian species reveals potential shared and species-restricted regulators during maturation.

Oocyte maturation is accompanied by changes in abundances of thousands of mRNAs, many degraded and many preferentially stabilized. mRNA stability can be regulated by diverse features including GC content, codon bias, and motifs within the 3'-untranslated region (UTR) interacting with RNA binding proteins (RBPs) and miRNAs. Many studies have identified factors participating in mRNA splicing, bulk mRNA storage, and translational recruitment in mammalian oocytes, but the roles of potentially hundreds of expressed factors, how they regulate cohorts of thousands of mRNAs, and to what extent their functions are conserved across species has not been determined. We performed an extensive in silico cross-species analysis of features associated with mRNAs of different stability classes during oocyte maturation (stable, moderately degraded, and highly degraded) for five mammalian species. Using publicly available RNA sequencing data for germinal vesicle (GV) and MII oocyte transcriptomes, we determined that 3'-UTR length and synonymous codon usage are positively associated with stability, while greater GC content is negatively associated with stability. By applying machine learning and feature selection strategies, we identified RBPs and miRNAs that are predictive of mRNA stability, including some across multiple species and others more species-restricted. The results provide new insight into the mechanisms regulating maternal mRNA stabilization or degradation.NEW & NOTEWORTHY Conservation across species of mRNA features regulating maternal mRNA stability during mammalian oocyte maturation was analyzed. 3'-Untranslated region length and synonymous codon usage are positively associated with stability, while GC content is negatively associated. Just three RNA binding protein motifs were predicted to regulate mRNA stability across all five species examined, but associated pathways and functions are shared, indicating oocytes of different species arrive at comparable physiological destinations via different routes.

UDP-glycosyltransferase gene SlUGT73C1 from Solanum lycopersicum regulates salt and drought tolerance in Arabidopsis thaliana L.

Among abiotic stresses, plants are the most vulnerable to salt and drought stresses. These stresses affect plant growth and development. Glycosyltransferases are involved in the responses of plants to abiotic stresses. In this study, a UDP-glycosyltransferase gene (SlUGT73C1) from Solanum lycopersicum was isolated and identified, which exhibited induction under salt or drought stress. The full length of SlUGT73C1 was 1485bp, encoding 494 amino acids. Stress-related cis-acting elements were present in the promoter sequence of SlUGT73C1, such as ARE, LTR, and GC motifs. Compared with the wild-type plants, Arabidopsis thaliana overexpressing SlUGT73C1 exhibited increased seed germination rate and SOD and POD activities, decreased MDA content, and increased expression levels of osmotic stress regulators genes, rate-limiting enzymes genes in the proline synthesis pathway, Na+/K+ reverse transporter genes, and rate-limiting genes in the ABA biosynthesis pathway under salt or drought stress. These results indicated that SlUGT73C1 plays an important role in regulating salt and drought tolerance in plants.

Strand-preferred base editing of organellar and nuclear genomes using CyDENT.

Transcription-activator-like effector (TALE)-based tools for base editing of nuclear and organellar DNA rely on double-stranded DNA deaminases, which edit substrate bases on both strands of DNA, reducing editing precision. Here, we present CyDENT base editing, a CRISPR-free, strand-selective, modular base editor. CyDENT comprises a pair of TALEs fused with a FokI nickase, a single-strand-specific cytidine deaminase and an exonuclease to generate a single-stranded DNA substrate for deamination. We demonstrate effective base editing in nuclear, mitochondrial and chloroplast genomes. At certain mitochondrial sites, we show editing efficiencies of 14% and strand specificity of 95%. Furthermore, by exchanging the CyDENT deaminase with one that prefers editing GC motifs, we demonstrate up to 20% mitochondrial base editing at sites that are otherwise inaccessible to editing by other methods. The modular nature of CyDENT enables a suite of bespoke base editors for various applications.

Identification of novel SSR markers for predicting the geographic origin of fungus Schizophyllum commune Fr

The fungus Schizophyllum commune is a cosmopolitan basidiomycete, which is popular as an edible, medical mushroom. It causes wood rot and often used as a model object in research. In this study, we analyzed thirty-two genomes of S. commune strains from the NCBI database and designed forty-seven unique SSR DNA markers. The detailed analysis revealed the enrichment of the S. commune genome for CG, GC, CTC, GAG, and TCG motifs. Principal components analysis confirmed the effectiveness of novel SSR DNA markers that preserve the initial heterogeneity of populations. The construction of a network between strains showed single one at a maximum similarity of 38%, and increasing the similarity to 55% breaks the linkage between large groups while separating two new groups containing strains of the population Ru and test cultures S. commune. The amplicons' presence was identified as a sufficient sign of relation of the culture to a specific population. Testing the novel SSR markers allowed to establish a clear delimitation of all groups by geographic location and to differentiate the H4-8 (GCA_000143185.1) strain from the USA population. This research is the basis for the further analysis of S. commune populations and improvement of approaches to determine its genetic diversity.

Identification of genes involved in the regulation of TERT in hepatocellular carcinoma.

Telomerase reverse transcriptase (TERT) promotes immortalization by protecting telomeres in cancer cells. Mutation of the TERT promoter is one of the most common genetic alterations in hepatocellular carcinoma (HCC), indicating that TERT upregulation is a critical event in hepatocarcinogenesis. Regulators of TERT transcription are, therefore, predicted to be plausible targets for HCC treatment. We undertook a genome‐wide shRNA library screen and identified C15orf55 and C7orf43 as regulators of TERT expression in HepG2 cells. Promoter assays showed that C15orf55‐ and C7orf43‐responsive sites exist between base pairs −58 and +36 and −169 and −59 in the TERT promoter, respectively. C15orf55 upregulates TERT expression by binding to two GC motifs in the SP1 binding site of the TERT promoter. C7orf43 upregulates TERT expression through Yes‐associated protein 1. The expression levels of C15orf55 and C7orf43 also correlated with that of TERT, and were significantly increased in both HCC tissues and their adjacent non‐tumor tissues, compared to normal liver tissues from non‐HCC patients. Analysis of 377 HCC patients in The Cancer Genome Atlas dataset showed that overall survival of patients with low levels of C15orf55 and C7orf43 expression in tumor tissues was better compared with patients with high levels of C15orf55 and/or high C7orf43 expression. These results indicate that C15orf55 and C7orf43 are involved in the incidence and progression of HCC by upregulating TERT. In conclusion, we identified C15orf55 and C7orf43 as positive regulators of TERT expression in HCC tissues. These genes are promising targets for HCC treatment.

Structural basis for genome wide recognition of 5-bp GC motifs by SMAD transcription factors

Smad transcription factors activated by TGF-β or by BMP receptors form trimeric complexes with Smad4 to target specific genes for cell fate regulation. The CAGAC motif has been considered as the main binding element for Smad2/3/4, whereas Smad1/5/8 have been thought to preferentially bind GC-rich elements. However, chromatin immunoprecipitation analysis in embryonic stem cells showed extensive binding of Smad2/3/4 to GC-rich cis-regulatory elements. Here, we present the structural basis for specific binding of Smad3 and Smad4 to GC-rich motifs in the goosecoid promoter, a nodal-regulated differentiation gene. The structures revealed a 5-bp consensus sequence GGC(GC)|(CG) as the binding site for both TGF-β and BMP-activated Smads and for Smad4. These 5GC motifs are highly represented as clusters in Smad-bound regions genome-wide. Our results provide a basis for understanding the functional adaptability of Smads in different cellular contexts, and their dependence on lineage-determining transcription factors to target specific genes in TGF-β and BMP pathways.

The dehydrin wzy2 promoter from wheat defines its contribution to stress tolerance

Dehydrins (DHNs), which are stress-related proteins, are important for plant survival under various abiotic and biotic stresses. To elucidate the regulatory mechanisms of wheat-derived DHNs under these stresses, we characterized the DHN wzy2 promoter of the wheat cultivar Zhengyin 1 and studied its contribution to stress tolerance. Sequence analysis indicated that the wzy2 gene contains one 109-bp intron inserted in the nucleotide sequence encoding the S-motif and characterized by a GT-AG border. The wzy2 promoter was revealed to contain several potential stress-related cis-acting regulatory elements, including elements responsive to abscisic acid (ABA; ABREs), anoxia (GC motifs), low temperature (LTREs), auxin (TGA elements), methyl jasmonate (MeJA; TGACG motifs), and gibberellin (TATC boxes). Quantitative real-time PCR analysis showed that transcript accumulation occurred in response to low temperature, anoxia, indoleacetic acid, MeJA, ABA, and gibberellin (GA) treatments. Histochemical analysis of GUS expression demonstrated that wzy2 promoter activity could be upregulated by low temperature, anoxia, ABA, and GA treatments. Interestingly, wzy2 promoter element-driven β-glucuronidase expression was first observed in meristemoids rather than calli of wheat seeds subjected to anoxia. Taken together, these results indicate that YSK2-type wzy2 can be induced directly by ABA, low temperature, anoxia, and GA treatments and indirectly by drought, implying that different cis-acting elements interact in stress response cross talk.

The Krüppel-like factor 4 controls biosynthesis of thyrotropin-releasing hormone during hypothalamus development

Embryonic neurogenesis is controlled by the activation of specific genetic programs. In the hypothalamus, neuronal thyrotropin-releasing hormone (TRH) populations control important physiological process, including energy homeostasis and autonomic function; however, the genetic program leading to the TRH expression is poorly understood. Here, we show that the Klf4 gene, encoding the transcription factor Krüppel-like factor 4 (Klf4), was expressed in the rat hypothalamus during development and regulated Trh expression. In rat fetal hypothalamic cells Klf4 regulated Trh promoter activity through CACCC and GC motifs present on the Trh gene promoter. Accordingly, hypothalamic Trh expression was down-regulated at embryonic day 15 in the Klf4 −/− mice resulting in diminished bioactive peptide levels. Although at the neonatal stage the Trh transcript levels of the Klf4 −/− mice were normal, the reduction in peptide levels persisted. Thus, our data indicate that Klf4 plays a key role in the maturation of TRH expression in hypothalamic neurons.

MBNL1 binds GC motifs embedded in pyrimidines to regulate alternative splicing

Muscleblind-like 1 (MBNL1) regulates alternative splicing and is a key player in the disease mechanism of myotonic dystrophy (DM). In DM, MBNL1 becomes sequestered to expanded CUG/CCUG repeat RNAs resulting in splicing defects, which lead to disease symptoms. In order to understand MBNL1’s role in both the disease mechanism of DM and alternative splicing regulation, we sought to identify its RNA-binding motif. A doped SELEX was performed on a known MBNL1-binding site. After five rounds of SELEX, MBNL1 selected pyrimidine-rich RNAs containing YGCY motifs. Insertion of multiple YGCY motifs into a normally MBNL1-independent splicing reporter was sufficient to promote regulation by MBNL1. MBNL1 was also shown to regulate the splicing of exon 22 in the ATP2A1 pre-mRNA, an exon mis-spliced in DM, via YGCY motifs. A search for YGCY motifs in 24 pre-mRNA transcripts that are mis-spliced in DM1 patients revealed an interesting pattern relative to the regulated exon. The intronic regions upstream of exons that are excluded in normal tissues relative to DM1, are enriched in YGCY motifs. Meanwhile, the intronic regions downstream of exons that are included in normal tissues relative to DM1, are enriched in YGCY motifs.

Unexpected dissociation energetics of the Na+ counterion from GC motifs in DNA hole-migration

We present here a theoretical investigation of the electronic and energetic properties of Na(+)GC, a DNA motif bound to a sodium ion (Na(+)) at the N(7) and O(6) sites of guanine (G), and its hole-trapped derivative [Na(+)GC](+) using density functional theory calculations. Normally, Na(+)GC has positive dissociation energies along various dissociation channels. However, hole-trapping of the Na(+)GC motif can lead to an unusual energetic phenomenon. Hole-trapping can reduce not only the dissociation barrier by destabilizing the Na(+)GC motif to a metastable state, but also the dissociation energy of the Na(+)N(7)/O(6) bond with an unexpected change from a positive to a negative value (61.51 versus-16.18 kcal mol(-1)). This unexpected negative dissociation energy phenomenon implies that this motif can store energy (∼16 kcal mol(-1)) in the Na(+)N(7)/O(6) bond zone due to hole-trapping. The topological properties of electron densities and the Laplacian values at the bond critical points indicate that this energetic phenomenon mainly originates from additional electrostatic repulsions between two moieties linked via a high-energy bond (Na(+)N(7)/O(6)). Proton transfer from G induced by hole-trapping can expand the negative dissociation energy zone to both Na(+)N(7)/O(6) and Watson-Crick (WC) H-bond zones. Similar phenomena can be observed for the Na(+) binding at the minor groove. Solvation of the hole-trapped Na(+)GC motif can change the negative dissociation energies by varying degrees, depending on the solvent-binding sites and the polarity of the solvents.

Activation and Repression Domains Within the Promoter of the Rat Cathepsin L Gene Orchestrate Sertoli Cell-Specific and Stage-Specific Gene Transcription in Transgenic Mice1

In murine testes, only Sertoli cells express the cathepsin L (Ctsl) gene, and this expression is restricted to stages V-VIII of the cycle. Our previous transgenic analysis of Tg (-2065/+977) demonstrated that this expression is regulated by a approximately 2-kb promoter. To begin to elucidate this regulation, we analyzed the in vivo expression of two new transgenes, Tg (-935/+977) and Tg (-451/+977). Tg (-935/+977) was expressed by Sertoli cells but, in contrast to Tg (-2065/+977), was expressed at all stages of the cycle, by spermatocytes, by the vascular endothelium, and by seven other organs. Tg (-451/+977) was not expressed by Sertoli cells but by spermatogenic cells and by the brain. Lack of expression of Tg (-451/+977) by Sertoli cells was not due to a lack of essential cis-acting elements. Transient transfection analysis of primary cultures of mature rat Sertoli cells demonstrated that in mature Sertoli cells, most of the activity of the Ctsl promoter is accounted for by one of two redundant upstream GC motifs and an Initiator that are within 100 bp of the transcription start site. We conclude that transcriptional repressors upstream from nucleotide -935 of the rat Ctsl gene restrict testicular expression of this gene to Sertoli cells at stages V-VIII. At these stages, transcriptional activators located between nucleotides -935 and -452 promote access of the transcriptional machinery to the two GC boxes and to the Initiator. Thus, upstream repressors and activators as well as cis-acting elements near the transcription start site control stage-specific Ctsl transcription by Sertoli cells.

KLF5 enhances SREBP‐1 action in androgen‐dependent induction of FASN in prostate cancer cell line

The Kruppel like factor 5 (KLF5) is a zinc‐finger transcription factor that plays important roles in regulating all aspects of cellular signaling involved in cell proliferation and oncogenesis. In the present study, KLF5 interacts with sterol regulatory element‐binding protein‐1 (SREBP‐1) in regulating the expression of lipogenic protein, fatty acid synthase (FASN), which plays important roles in androgen‐dependent prostate cancer cells. Genes involved in lipid metabolism, including FASN, are reported to be up‐regulated by androgens in prostate cancer cells through a coordinated cascade controlled by the activation of SREBP‐1. The promoter and intron 1 region of FASN gene contains multiple GC‐boxes that might show response to KLF5. Deletion and mutation analyses indicated that KLF5 binds to the FASN gene and strongly induces its activity. Furthermore, KLF5 protein is able to bind to SREBP‐1 and enhance the SREBP‐1 regulated activation of FASN. Transfection of KLF5 into prostate cancer cells enhanced FASN promoter activity, and conversely, silencing of KLF5 by siRNA down‐regulated FASN expression. The multiple GC motifs are scattered throughout the upstream sequence and first intron of FASN gene whereas these are scarce in the promoters of other lipogenic and cholesterogenic genes such as acetyl‐CoA carboxylase, ATP‐citrate lyase, LDL receptor, HMG‐CoA synthase and HMG‐CoA reductase, which makes FASN gene very unique among them. Taken together, FASN gene could be activated by the synergistic action of KLF5 and SREBP‐1 which is increased by androgen treatment.

Regulation of T-cell receptor Dβ1 promoter by KLF5 through reiterated GC-rich motifs

AbstractRearrangement of T-cell receptor (TCR) and immunoglobulin genes by a common V(D)J recombination machinery is regulated by developmentally specific chromatin changes at the target locus, a process associated with transcription. At the TCRβ locus, the Eβ enhancer and the Dβ1 promoter regulate germline transcription originating near the TCR Dβ1 gene segment. The Dβ1 promoter contains 3 GC-rich motifs that bind a common set of nuclear proteins from pro–T-cell lines. Mutations that diminish the binding of nuclear proteins also diminish the activity of the Dβ1 promoter in transcriptional reporter assays. Using a yeast one-hybrid approach, 3 Krüppel-like factors—KLF3, KLF5, and KLF6—and a novel zinc finger protein were identified in a thymus library, all of which bound the GC-rich motif in a sequence-specific manner. Of these genes, KLF5 mRNA was expressed in a restricted manner in lymphoid cells and tissues, with highest expression in pro–T-cell lines and Rag-deficient thymocytes. Antibody supershift studies and chromatin immunoprecipitation assay confirmed that KLF5 bound the Dβ1 promoter. In reporter gene assays, KLF5 but not KLF6 efficiently transactivated the Dβ1 promoter, whereas a dominant-negative KLF5 construct inhibited reporter expression. These data suggest that reiterated GC motifs contribute to germline TCRβ transcription through binding of KLF5 and other Krüppel family members and that restricted expression of KLF5 may contribute to lineage-specific regulation of germline TCRβ transcription.

Necrosis has orderly DNA fragmentations

Necrosis from freezing manifested an orderly pattern of DNA fragmentations, including the apoptosis signature of 200 bp ladder, in three different cell populations, despite pancaspase suppression by zVAD-fmk. Immediately on thawing, all three populations had 100% dead cell indices and 2.2, 1.6, and 1.1 megabase fragmentations, which marked the point of death. Kilobase and 200 bp DNA ladder fragmentations manifested later together with overt necrotic morphologies. CpG oligodeoxynucleotides (ODNs) complementary to highly conserved GCn x GC motifs inhibited the megabase fragmentations and retarded their electrophoretic mobility (gel shift), indicating ODN–DNA binding, which is known to confer site-specific resistance to cleavage. Cleavage specificity was confirmed using EDTA–CpG ODN conjugates to direct free-radical-producing transitional element, vanadyl(4), to the binding sites to reproduce the megabase fragmentations in normal cells. Specific orderly fragmentations in necrosis suggested a necrosis–apoptosis convergence after death has been committed.

TPA-activated transcription of the human MnSOD gene: role of transcription factors Sp-1 and Egr-1.

Induction of manganese superoxide dismutase (MnSOD) in response to oxidative stress has been well established in animals, tissues, and cell culture. However, the role of the human MnSOD (hMnSOD) promoter in stimulus-dependent activation of transcription is unknown. The hMnSOD promoter lacks both a TATA and a CAAT box but possesses several GC motifs. In a previous study, we showed that the basal promoter contains multiple Sp1 and AP-2 binding sites and that Sp1 is essential for the constitutive expression of the hMnSOD gene. In this study, we identified an Egr-1 binding site in the basal promoter of hMnSOD. We also found that the basal promoter is responsive to 12-O-tetradecanoylphorbol-13-acetate (TPA)-activated hMnSOD transcription in the human hepatocarcinoma cell line HepG2. The contributions of these binding sites and the roles of the transcription factors Egr-1, AP-2, and Sp1 in the activation of hMnSOD transcription by TPA were investigated by site-directed mutation analysis, Western blotting, and overexpression of transcription factors. The results showed that Sp1 plays a positive role for both basal and TPA-activated hMnSOD transcription, whereas overexpression of Egr-1 has a negative role in the basal promoter activity without any effect on TPA-mediated activation of hMnSOD transcription.

Analysis of a genomic clone of hydrophobin ( ssgA) from the entomopathogenic fungus Metarhizium anisopliae

A 909 bp region containing a genomic clone encoding for hydrophobin (ssgA) from the entomopathogenic fungus Metarhizium anisopliae has been sequenced and the regulatory motifs analysed against those recognized in other fungi. The genomic clone was also compared with the open reading frame of the hydrophobin ssgA (starvation stress gene) cDNA sequence. The genomic clone contained a 291 bp coding sequence with one intron of 64 nucleotides. From this sequence primers were established that could be used to amplify the hydrophobin. Restriction fragment polymorphism analysis of hydrophobin amplified by the polymerase chain reaction from 80 isolates of M. anisopliae showed no variability. Analysis of the potential regulatory elements 313 bp upstream from the transcriptional start site revealed typical TATAA and CCAAT boxes. CT or GC motifs were not found. Upstream regulatory elements were also found with sequence homologies to the AREA, CREA, CRE (cAMP response element) and BRLA regions of Aspergillus nidulans as well as the CYS3 and AmyB regions of Aspergillus oryzae. The promoter regions of other fungal hydrophobins were also assessed for the presence of regulatory elements. Upstream regulatory elements are also present for the gene encoding a cuticle-degrading protease (Pr1) from M. anisopliae. We suggest that nutrient levels and cAMP mediation of thigmotropic signals in the entomopathogenic fungus, M. anisopliae, co-ordinate the regulation of the gene products required for morphological development and secretion of ‘penetration’ enzymes.

Multiple Functional Sp1 Domains in the Minimal Promoter Region of the Neuronal Nicotinic Receptor α5 Subunit Gene

The alpha5 subunit is a component of the neuronal nicotinic acetylcholine receptors, which are probably involved in the activation step of the catecholamine secretion process in bovine adrenomedullary chromaffin cells. The promoter of the gene coding for this subunit was isolated, and its proximal region was characterized, revealing several GC boxes located close to the site of transcription initiation (from -111 to -40). Deletion analysis and transient transfections showed that a 266-base pair region (-111 to +155) gave rise to approximately 77 and 100% of the maximal transcriptional activity observed in chromaffin and SHSY-5Y neuroblastoma cells, respectively. Site-directed mutagenesis of five different GC motifs indicated that all of them contribute to the activity of the alpha5 gene, but in a different way, depending on the type of transfected cell. Thus, in SHSY-5Y cells, alteration of the most promoter-proximal of the GC boxes decreased alpha5 promoter activity by approximately 50%, whereas single mutations of the other GC boxes had no effect. In chromaffin cells, by contrast, modification of any of the GC boxes produced a similar decrease in promoter activity (50-69%). In both cell types, however, activity was almost abolished when four GC boxes were suppressed simultaneously. Electrophoretic mobility shift assays using nuclear extracts from either chromaffin or SHSY-5Y cells showed the specific binding of Sp1 protein to fragment -111 to -27. Binding of Sp1 to the GC boxes was also demonstrated by DNase I footprint analysis. This study suggests that the general transcription factor Sp1 plays a dominant role in alpha5 subunit expression, as has also been demonstrated previously for alpha3 and beta4 subunits. Since these three subunits have their genes tightly clustered and are expressed in chromaffin cells, probably as components of the same receptor subtype, we propose that Sp1 constitutes the key factor of a regulatory mechanism common to the three subunits.

Cell-type expression, immunolocalization, and deoxyribonucleic acid-binding activity of basic transcription element binding transcription factor, an Sp-related family member, in porcine endometrium of pregnancy.

Basic transcription element binding (BTEB) protein is a newly identified member of the C2H2 zinc finger family that also includes the transcription factors, Sp1, Sp2, Sp3, and Sp4. This family of proteins binds GC-rich motifs widely distributed in gene promoters, resulting in distinct activation or repression of transcriptional activities. Whereas Sp proteins are ubiquitously expressed, expression of BTEB appears more limited and has not been documented in the female reproductive tract of any mammalian species. This study was designed to identify and characterize the cellular distribution of BTEB in the porcine endometrium and placenta at known stages of pregnancy. Northern analysis of uterine endometrium detected BTEB mRNA that corresponds in size (5 kilobases) to that of the major BTEB transcript in rat brain. The steady-state levels of BTEB mRNA were higher (p < 0.05) in endometrium than placenta at corresponding days of pregnancy, although for each tissue, the levels did not change with pregnancy stage (p > 0.05). Luminal epithelial (LE), glandular epithelial (GE), and stromal (ST) cells isolated from pregnancy endometrium expressed the BTEB gene, but mRNA abundance varied with cell type (LE, GE > ST). Western blot analysis using an antiserum generated against the N-terminal region of a porcine BTEB fusion protein produced in Escherichia coli revealed the presence of BTEB protein only in endometrium, not in placenta. Immunohistochemical studies localized BTEB predominantly to the nuclei of endometrial GE and LE cells. Consistent with the presence of functional BTEB protein, binding to a double-stranded oligonucleotide containing multiple GC motifs was demonstrated in nuclear extracts prepared from endometrium and from endometrial LE and GE, but not ST, cells by electrophoretic mobility shift assay. These results demonstrate the preferential endometrial cell-type expression of BTEB and suggest its regulatory role in pregnancy-associated endometrial epithelial gene expression.

Transcription factor Sp3 antagonizes activation of the ornithine decarboxylase promoter by Sp1.

Ornithine decarboxylase (ODC) expression is important for proliferation and is elevated in many tumor cells. We previously showed that Sp1 is a major positive regulator of ODC transcription. In this paper we have investigated transcriptional regulation of rat ODC by the closely related factor Sp3. While over-expression of Sp1 caused a dramatic activation of the ODC promoter, over-expression of Sp3 caused little or no activation in either Drosophila SL2 cells (lacking endogenous Sp1 or Sp3) or in H35 rat hepatoma cells. Furthermore, co-transfection studies demonstrated that Sp3 abolished trans -activation of the ODC promoter by Sp1. DNase I footprint studies and electrophoretic mobility shift assays demonstrated that both recombinant Sp1 and Sp3 bind specifically to several sites within the ODC promoter also protected by nuclear extracts, including overlapping GC and CT motifs located between -116 and -104. This CT element is a site of negative ODC regulation. Mutation of either element reduced binding, but mutation of both sites was required to eliminate binding of either Sp1 or Sp3. These results demonstrate that ODC is positively regulated by Sp1 and negatively regulated by Sp3, suggesting that the ratio of these transcription factors may be an important determinant of ODC expression during development or transformation.