Sp1 Mutations Research Articles

Analysis of Lin28B Promoter Activity and Screening of Related Transcription Factors in Dolang Sheep.

The Lin28B gene is involved in the initiation of puberty, but its regulatory mechanisms remain unclear. Therefore, in this study, we aimed to study the regulatory mechanism of the Lin28B promoter by cloning the Lin28B proximal promoter for bioinformatic analysis. Next, a series of deletion vectors were constructed based on the bioinformatic analysis results for dual-fluorescein activity detection. The transcriptional regulation mechanism of the Lin28B promoter region was analyzed by detecting mutations in transcription factor-binding sites and overexpression of transcription factors. The dual-luciferase assay showed that the Lin28B promoter region -837 to -338 bp had the highest transcriptional activity, and the transcriptional activity of the Lin28B transcriptional regulatory region decreased significantly after Egr1 and SP1 mutations. Overexpression of the Egr1 transcription factor significantly enhanced the transcription of Lin28B, and the results indicated that Egr1 and SP1 play important roles in regulating Lin28B. These results provide a theoretical basis for further research on the transcriptional regulation of sheep Lin28B during puberty initiation.

DNA damage-induced sumoylation of Sp1 induces its interaction with RNF4 and degradation in S phase to remove 53BP1 from DSBs and permit HR

The factors involved in DNA damage recognition and repair are tightly regulated to ensure proper repair pathway choice. The mechanism(s) that determines the cell cycle-dependent turnover of these DNA damage repair factors remains unclear. Here, we show that Sp1, which regulates double-strand break (DSB) repair pathway choice through localization of 53BP1, is sumoylated at Lys16 following DNA damage; Sp1 sumoylation is required for its degradation and the removal of both Sp1 and 53BP1 from DSB sites. Induction of DNA DSBs induces Sp1 phosphorylation at DSBs by ATM, which is necessary for the subsequent sumoylation of Sp1. In addition to this damage-induced ATM-dependent phosphorylation and sumoylation, phosphorylation of Sp1 at Ser59 by Cyclin A/cdk2 upon entry into S phase is necessary for recognition, ubiquitination and degradation by the SUMO-targeted E3 ubiquitin ligase, RNF4. Eliminating Sp1 sumoylation by mutation of Sp1 at Lys16 (K16R) precluded removal of both Sp1 and 53BP1 from DSBs in S phase, resulting in decreased BRCA1 recruitment and defective homologous recombination (HR). Like BRCA1 deficient cells, cells expressing Sp1K16R are sensitive to PARP inhibition due to failure to degrade Sp1 and recruit BRCA1 resulting in defective HR that is rescued by knockdown of 53BP1. These results reveal the dynamic regulation of Sp1 and its role in the assembly and disassembly of DNA repair factors at DSBs.

DNA damage-induced degradation of Sp1 promotes cellular senescence

Persistent DNA damage (genotoxic stress) triggers signaling cascades that drive cells into apoptosis or senescence to avoid replicating a damaged genome. Sp1 has been found to play a role in double strand break (DSB) repair, and a link between Sp1 and aging has also been established, where Sp1 protein, but not RNA, levels decrease with age. Interestingly, inhibition ATM reverses the age-related degradation of Sp1, suggesting that DNA damage signaling is involved in senescence-related degradation of Sp1. Proteasomal degradation of Sp1 in senescent cells is mediated via sumoylation, where sumoylation of Sp1 on lysine 16 is increased in senescent cells. Taking into consideration our previous findings that Sp1 is phosphorylated by ATM in response to DNA damage and that proteasomal degradation of Sp1 at DSBs is also mediated by its sumoylation and subsequent interaction with RNF4, we investigated the potential contribution of Sp1’s role as a DSB repair factor in mediating cellular senescence. We report here that Sp1 expression is decreased with a concomitant increase in senescence markers in response to DNA damage. Mutation of Sp1 at serine 101 to create an ATM phospho-null mutant, or mutation of lysine 16 to create a sumo-null mutant, prevents the sumoylation and subsequent proteasomal degradation of Sp1 and results in a decrease in senescence. Conversely, depletion of Sp1 or mutation of Sp1 to create an ATM phosphomimetic results in premature degradation of Sp1 and an increase in senescence markers. These data link a loss of genomic stability with senescence through the action of a DNA damage repair factor.

Characteristics of the BMP7 Promoter in Hu Sheep.

Simple SummaryBone morphogenetic protein 7 (BMP7) is one of the largest secretory signal conductive molecules and is in the TGF-β superfamily. It plays an important role in the growth and development of hair follicles. We cloned the proximal promoter of the BMP7 gene for bioinformatics analysis. Dual-luciferase reporter system and overexpression were used to analyze the key regions and transcription factor binding sites. There was high activity between −758 bp and −545 bp in the core region of the gene and a possible binding site for transcription factors SP1 and EGR1.The BMP7 gene is involved in the growth and development of hair follicles but its regulation mechanism is unclear. We studied the regulation mechanism of the BMP7 promoter by cloning the proximal promoter of BMP7 for bioinformatics analysis. A series of missing vectors was then constructed for dual-fluorescein activity detection based on the bioinformatics analysis results. We tested transcription-factor binding-site mutations and transcription factor over-expression to analyze the transcriptional regulation principle of the BMP7 promoter region. The upstream transcriptional regulatory region of the BMP7 gene proximal promoter was predicted by bioinformatics. There were −1216 bp to −1166 bp and −632 bp to −582 bp transcription initiation sites in the upstream transcriptional regulatory region of the BMP7 gene proximal promoter. The CpG islands’ distribution showed that there were many CpG islands at −549 bp to 1 bp. A dual-luciferase assay revealed high activity between −758 bp and −545 bp in the core region and a possible binding site for transcription factors SP1 and EGR1. The transcriptional activity of BMP7 was significantly decreased in the transcriptional regulatory region of the BMP7 after EGR1 and SP1 mutation. Transcription was significantly enhanced by over expression of the EGR1 transcription factor, which strongly suggests that EGR1 and SP1 play important roles in BMP7 regulation.

Transcriptional regulation of human cyclic GMP-AMP synthase gene

Cyclic GMP-AMP synthase (cGAS, cGAMP synthase) plays crucial roles in autoimmune disease, anti-tumor response, anti-senescence and anti-inflammatory response. Many studies have focused on cGAS-mediated signaling pathway. However, transcriptional mechanisms of cGAS gene have remained largely unknown. Here, we cloned the cGAS promoter region and characterized the molecular mechanisms controlling the cGAS transcriptional activity. By a series of 5′ deletion and promoter constructions, we showed that the region (−414 to +76 relatives to the transcription start site) was sufficient for promoter activity. Mutation of Sp1 and CREB binding sites in this promoter region led to an apparent reduction of the cGAS promoter activity. Overexpression of Sp1 and CREB could obviously enhance promoter activity, whereas knocking-down of endogenous Sp1 and CREB markedly restrained the cGAS promoter activity. Sp1 and CREB binding to the cGAS promoter region in vivo was verified by Chromatin immunoprecipitation assay. These results pointed out that transcription factors Sp1 and CREB regulate the transcription of the cGAS gene.

Quenching protein dynamics interferes with HIV capsid maturation

Maturation of HIV-1 particles encompasses a complex morphological transformation of Gag via an orchestrated series of proteolytic cleavage events. A longstanding question concerns the structure of the C-terminal region of CA and the peptide SP1 (CA–SP1), which represents an intermediate during maturation of the HIV-1 virus. By integrating NMR, cryo-EM, and molecular dynamics simulations, we show that in CA–SP1 tubes assembled in vitro, which represent the features of an intermediate assembly state during maturation, the SP1 peptide exists in a dynamic helix–coil equilibrium, and that the addition of the maturation inhibitors Bevirimat and DFH-055 causes stabilization of a helical form of SP1. Moreover, the maturation-arresting SP1 mutation T8I also induces helical structure in SP1 and further global dynamical and conformational changes in CA. Overall, our results show that dynamics of CA and SP1 are critical for orderly HIV-1 maturation and that small molecules can inhibit maturation by perturbing molecular motions.

Quercetin increases claudin-4 expression through multiple transcription factors in intestinal Caco-2 cells

We previously reported that quercetin increases tight junction (TJ) barrier integrity in intestinal Caco-2 cells. This study was aimed at determining the molecular mechanism underlying the quercetin-mediated effect with a particular focus on the transcriptional regulation of claudin-4. Quercetin aglycone, but not its glycosides, enhanced TJ barrier integrity of Caco-2 cells, indicated by transepithelial electrical resistance. Immunoblot analysis, qPCR, and luciferase promoter assay revealed that quercetin transcriptionally increased the claudin-4 expression. Narrowing the claudin-4 promoter sequences from 1866 bp to 200 bp did not affect quercetin-mediated promoter activation. Site-directed mutation of SP1, AP-1 and GATA binding sites within the 200 bp sequence partially suppressed the effects of quercetin on claudin-4 promoter activity. Taken together, quercetin enhances the TJ barrier integrity in intestinal Caco-2 cells, with the transcription factors, SP1, AP-1, and GATA, involved in the quercetin-mediated transcriptional regulation of claudin-4.

Genetic Selection for Context-Dependent Stochastic Phenotypes: Sp1 and TATA Mutations Increase Phenotypic Noise in HIV-1 Gene Expression

The sequence of a promoter within a genome does not uniquely determine gene expression levels and their variability; rather, promoter sequence can additionally interact with its location in the genome, or genomic context, to shape eukaryotic gene expression. Retroviruses, such as human immunodeficiency virus-1 (HIV), integrate their genomes into those of their host and thereby provide a biomedically-relevant model system to quantitatively explore the relationship between promoter sequence, genomic context, and noise-driven variability on viral gene expression. Using an in vitro model of the HIV Tat-mediated positive-feedback loop, we previously demonstrated that fluctuations in viral Tat-transactivating protein levels generate integration-site-dependent, stochastically-driven phenotypes, in which infected cells randomly ‘switch’ between high and low expressing states in a manner that may be related to viral latency. Here we extended this model and designed a forward genetic screen to systematically identify genetic elements in the HIV LTR promoter that modulate the fraction of genomic integrations that specify ‘Switching’ phenotypes. Our screen identified mutations in core promoter regions, including Sp1 and TATA transcription factor binding sites, which increased the Switching fraction several fold. By integrating single-cell experiments with computational modeling, we further investigated the mechanism of Switching-fraction enhancement for a selected Sp1 mutation. Our experimental observations demonstrated that the Sp1 mutation both impaired Tat-transactivated expression and also altered basal expression in the absence of Tat. Computational analysis demonstrated that the observed change in basal expression could contribute significantly to the observed increase in viral integrations that specify a Switching phenotype, provided that the selected mutation affected Tat-mediated noise amplification differentially across genomic contexts. Our study thus demonstrates a methodology to identify and characterize promoter elements that affect the distribution of stochastic phenotypes over genomic contexts, and advances our understanding of how promoter mutations may control the frequency of latent HIV infection.

Promoter Regulation of the Visinin-like Subfamily of Neuronal Calcium Sensor Proteins by Nuclear Respiratory Factor-1

VILIP-1 (gene name VSNL1), a member of the neuronal Ca(2+) sensor protein family, acts as a tumor suppressor gene by inhibiting cell proliferation, adhesion, and invasiveness. VILIP-1 expression is down-regulated in several types of human cancer. In human non-small cell lung cancer, we found that down-regulation was due to epigenetic changes. Consequently, in this study we analyzed the VSNL1 promoter and its regulation. Serial truncation of the proximal 2-kb VSNL1 promoter (VP-1998) from its 5' terminus disclosed that the last 3' terminal 100-bp promoter fragment maintained similar promoter activity as compared with VP-1998 and therefore was referred to as VSNL1 minimal promoter. When the 5' terminal 50 bp were deleted from the minimal promoter, the activity was dramatically decreased, suggesting that the deleted 50 bp contained a potential cis-acting element crucial for promoter activity. Deletion and site-directed mutagenesis combined with in silico transcription factor binding analysis of VSNL1 promoter identified nuclear respiratory factor (NRF)-1/alpha-PAL as a major player in regulating VSNL1 minimal promoter activity. The function of NRF-1 was further confirmed using dominant-negative NRF-1 overexpression and NRF-1 small interfering RNA knockdown. Electrophoretic mobility shift assay and chromatin immunoprecipitation provided evidence for direct NRF-1 binding to the VSNL1 promoter. Methylation of the NRF-1-binding site was found to be able to regulate VSNL1 promoter activity. Our results further indicated that NRF-1 could be a regulatory factor for gene expression of the other visinin-like subfamily members including HPCAL4, HPCAL1, HPCA, and NCALD.

Oxidative Stress Promotes Transcriptional Up-regulation of Fyn in BCR-ABL1-expressing Cells

Signaling initiated by the BCR-ABL1 kinase causes chronic myelogenous leukemia (CML). Recently, we reported that expression of Fyn, a Src kinase, is heightened in CML cells and patient specimens and confers in vitro and in vivo proliferative advantages. Fyn is regulated by redox, and because BCR-ABL1 raises intracellular oxidant levels, which have been implicated in CML progression, we explored the molecular regulation of Fyn. Here we identify the transcription factors that drive redox- and BCR-ABL1-dependent Fyn expression. Promoter deletion analysis in 293T, BaF3, BaF3-p210, and K562 cells identified the region essential for basal transcriptional activity. Mutation of Sp1 and Egr1 binding sites within the essential region diminished Fyn promoter activity and identified Egr1 as conferring redox sensitivity. Gel shift and chromatin immunoprecipitation assays confirmed the binding of Sp1 and Egr1 to the promoter fragments. Importantly, knockdown of Sp1 or Egr1 with small interference RNA or inhibition of Sp1 binding by mithramycin A repressed Fyn protein expression. Our work is the first to define transcription factors that are responsible for endogenous, oxidative stress-dependent and BCR-ABL1-dependent Fyn expression.

O.111 Intranasal curcumin to improve olfactory CSF pathways – Preliminary findings

We previously reported that quercetin increases tight junction (TJ) barrier integrity in intestinal Caco-2 cells. This study was aimed at determining the molecular mechanism underlying the quercetin-mediated effect with a particular focus on the transcriptional regulation of claudin-4. Quercetin aglycone, but not its glycosides, enhanced TJ barrier integrity of Caco-2 cells, indicated by transepithelial electrical resistance. Immunoblot analysis, qPCR, and luciferase promoter assay revealed that quercetin transcriptionally increased the claudin-4 expression. Narrowing the claudin-4 promoter sequences from 1866 bp to 200 bp did not affect quercetin-mediated promoter activation. Site-directed mutation of SP1, AP-1 and GATA binding sites within the 200 bp sequence partially suppressed the effects of quercetin on claudin-4 promoter activity. Taken together, quercetin enhances the TJ barrier integrity in intestinal Caco-2 cells, with the transcription factors, SP1, AP-1, and GATA, involved in the quercetin-mediated transcriptional regulation of claudin-4.

A Novel Estradiol/Estrogen Receptor α-dependent Transcriptional Mechanism Controls Expression of the Human Prolactin Receptor

Prolactin exerts diverse functions in target tissues through its membrane receptors, and is a potent mitogen in normal and neoplastic breast cells. Estradiol (E(2)) induces human prolactin receptor (hPRLR) gene expression through stimulation of its generic promoter (PIII). This study identifies a novel E(2)-regulated non-estrogen responsive element-dependent transcriptional mechanism that mediates E(2)-induced hPRLR expression. E(2) stimulated transcriptional activity in MCF7A(2) cells transfected with PIII lacking an estrogen responsive element, and increased hPRLR mRNA and protein. The abolition of the E(2) effect by mutation of Sp1 or C/EBP elements that bind Sp1/Sp3 and C/EBPbeta within PIII indicated the cooperation of these transfactors in E(2)-induced transcription of the hPRLR. DNA affinity protein assay showed that E(2) induced estrogen receptor alpha (ERalpha) binding to Sp1/Sp3 and C/EBPbeta DNA-protein complexes. The ligand-binding domain of ERalpha was essential for its physical interaction with C/EBPbeta, and E(2) promoted this association, and its DNA binding domain was required for transactivation of PIII. Co-immunoprecipitation studies revealed tethering of C/EBPbeta to Sp1 by E(2)-activated ERalpha. Chromatin immunoprecipitation analysis showed that E(2) induced recruitment of C/EBPbeta, ERalpha, SRC1, p300, pCAF, TFIIB, and Pol II, with no change in Sp1/Sp3. E(2) also induced promoter-associated acetylation of H3 and H4. These findings demonstrate that an E(2)/ERalpha, Sp1, and C/EBPbeta complex with recruitment of coactivators and TFIIB and Pol II are required for E(2)-activated transcriptional expression of the hPRLR through PIII. Estradiol produced in breast stroma and adipose tissue, which are major sources of estrogen in post-menopausal women, could up-regulate hPRLR gene expression and stimulate breast tumor growth.

Histone Deacetylase Inhibitors Down-Regulate bcl-2 Expression and Induce Apoptosis in t(14;18) Lymphomas

Histone deacetylase (HDAC) inhibitors are promising antitumor agents, but they have not been extensively explored in B-cell lymphomas. Many of these lymphomas have the t(14;18) translocation, which results in increased bcl-2 expression and resistance to apoptosis. In this study, we examined the effects of two structurally different HDAC inhibitors, trichostatin A (TSA) and sodium butyrate (NaB), on the cell cycle, apoptosis, and bcl-2 expression in t(14;18) lymphoma cells. We found that in addition to potent cell cycle arrest, TSA and NaB also dramatically induced apoptosis and down-regulated bcl-2 expression, and overexpression of bcl-2 inhibited TSA-induced apoptosis. The repression of bcl-2 by TSA occurred at the transcriptional level. Western blot analysis and quantitative chromatin immunoprecipitation (ChIP) assay showed that even though HDAC inhibitors increased overall acetylation of histones, localized histone H3 deacetylation occurred at both bcl-2 promoters. TSA treatment increased the acetylation of the transcription factors Sp1 and C/EBPalpha and decreased their binding as well as the binding of CBP and HDAC2 to the bcl-2 promoters. Mutation of Sp1 and C/EBPalpha binding sites reduced the TSA-induced repression of bcl-2 promoter activity. This study provides a mechanistic rationale for the use of HDAC inhibitors in the treatment of human t(14;18) lymphomas.

Alternative initiation of transcription of the human presenilin 1 gene in SH‐SY5Y and SK‐N‐SH cells

We have identified DNA sequences required for the expression of the presenilin 1 (PS1) gene. A promoter region has been mapped in SK-N-SH cells and includes sequences between -118 and +178 flanking the major initiation site (+1). The PS1 gene is also efficiently transcribed in the SH-SY5Y subclone of SK-N-SH cells. However the promoter appears to be utilized in alternative ways in both cell types. Sequences both upstream as well as downstream from the initiation site mapped in SK-N-SH cells were shown by 5'- and 3'-deletion analysis to play a crucial role in both cell lines. However, in SH-SY5Y cells either upstream or downstream sequences are sufficient to direct transcription, whereas in SK-N-SH cells 5'-deletions past the +1 site eliminate over 95% of transcription. Several Ets motifs (GGAA) as well as Sp1 motifs [(G/T)GGCGGRRY] are juxtaposed both upstream and downstream from +1. To understand how the promoter may be utilized alternatively in different cell types we have examined the effect of point mutations in these elements. Altering an Ets motif at -10 eliminates 80% of transcription in SK-N-SH cells whereas the same mutation has only a minor effect in SH-SY5Y cells. Conversely, mutation of the Ets element at +90, which eliminates 70% of transcription in SH-SY5Y cells, has a lesser effect in SK-N-SH cells. In both cell types a promoter including mutations at both -10 and +90 sites loses over 90% transcription activity indicating the crucial importance of these two Ets motifs. The effect of Sp1 mutations appears to be similar in both cell types. Hence the differential expression in each cell type may be at least partially determined by Ets factors and the -10/+90 sites. We have identified several Ets factors that recognize specifically the -10 Ets motif by the yeast one-hybrid selection including avian erythroblastosis virus E26 oncogene homologue 2, Ets-like gene 1, Ets translocation variant 1 and Ets related molecule (ERM). We show here that ERM specifically recognizes Ets motifs on the PS1 promoter located at -10 as well as downstream at +90, +129 and +165 and activates PS1 transcription with promoter fragments containing or not the -10 Ets site.

Molecular cloning and characterization of human acid sensing ion channel (ASIC)2 gene promoter

Acid sensing ion channel (ASIC)2 belongs to the amiloride-sensitive Na +-channel/ degenerin family. Our previous studies suggested that differential regulation of ASIC2 expression occurs between high-grade glial-derived tumor cells and normal astrocytes. To investigate the mechanisms involved in the regulation of ASIC2 gene expression, the human ASIC2 promoter region (−1551 to +117) was cloned and characterized. The ASIC2 promoter lacked a canonical TATA box, but contained one putative CCAAT box. Nucleotide sequencing of the promoter revealed the presence of a number of transcription factor-binding sites and a 404 bp CpG island upstream the transcription start site. Nested deletion mutants and transfection results showed that the construct between −133 and +117 base pairs conferred basal transcription specific activity. Mutation of Sp1 and CP2 sites in this region resulted in a 70 and 95% decrease in promoter activity, respectively. Gel shift assays demonstrated the existence of specific protein binding to the SP1 and CP2 elements. There was no mutation in the CpG island in six glioma cell lines, but methylation-specific PCR showed methylation in some of glioma cell lines and tumor tissues, and treatment with the methylation inhibitor 5-Aza-2′-deoxycytidine could partially restore ASIC2 expression in cell lines, suggesting that epigenetic mechanisms may contribute to dysregulated ASIC2 expression.

Androgen Induction of Cyclin-Dependent Kinase Inhibitor p21 Gene: Role of Androgen Receptor and Transcription Factor Sp1 Complex

Previous studies have shown that androgen up-regulates expression of the p21 (WAF1, CIP1, SDI1, CAP20) gene, which contains a canonical androgen response element (ARE) in its proximal promoter region. We undertook the current studies to determine whether elements in the p21 promoter other than the ARE mediate androgen action. We found that deletion of the ARE did not completely abolish the promoter responsiveness to androgen, suggesting that additional cis-regulatory elements within the p21 core promoter may also be involved in androgen responsiveness. The p21 core promoter is GC-rich and contains six binding sites for transcription factor Sp1. We determined whether one or more of these Sp1 sites mediate androgen responsiveness of the p21 promoter. To do so, we used a transient transfection assay with p21 promoter-luciferase reporter constructs. The reporter activity of a construct lacking the ARE but containing all six Sp1 sites was induced approximately 3-fold by androgen. Mutation of Sp1–3 nearly eliminated basal promoter activity as well as androgen responsiveness, whereas deletion of Sp1–1 and Sp1–2 sites and mutation of Sp1–4, Sp1–5, and Sp1–6 sites had relatively little effect. We also used the mammalian one-hybrid assay and coimmunoprecipitation assay to show that androgen receptor (AR) and transcription factor Sp1 interact with one another. The current studies suggest a model in which AR and transcription factor Sp1 not only bind to their respective consensus sites within the p21 promoter, but also complex with one another, thereby recruiting coactivators and general transcription factors and inducing p21 transcription.

AP-2 (activating protein 2) and Sp1 (selective promoter factor 1) regulatory elements play distinct roles in the control of basal activity and cyclic adenosine 3',5'-monophosphate responsiveness of the human chorionic gonadotropin-beta promoter.

The CG beta-subunit gene (CGbeta) arose evolutionarily from the LH beta-subunit gene (LHbeta) through gene duplication. Although the promoter sequences of the CGbeta and human (h) hLHbeta genes are greater than 90% homologous, their expression patterns are distinct. LHbeta is expressed in pituitary gonadotrope cells and CGbeta is expressed in placental trophoblast cells. The placental specific and cAMP-inducible region within the CGbeta promoter has been mapped to a complex enhancer element spanning 118 bp (-318 to -200). Transcription factor-binding sites within this enhancer have been partially characterized and include multiple binding sites for AP-2 (activating protein 2) and Sp1 (selective promoter factor 1), which activate basal and cAMP-induced expression. In this study, we performed a detailed analysis of the recognition sites for these transcription factors and examined the functional roles of these elements in the control of CGbeta expression. An upstream Sp1/AP-2 binding site (-318 to -279) preferentially binds Sp1, which occludes AP-2 binding to an adjacent site. In contrast, both Sp1 and AP-2 bind concurrently to a downstream composite Sp1/AP-2 element (-220 to -188). Functionally, mutations in any of the Sp1 or AP-2 binding sites cause a progressive decrease in basal CGbeta expression. However, cAMP stimulation of the CGbeta promoter is reduced by AP-2 mutations, whereas Sp1 mutations enhance cAMP activation. We conclude that multiple AP-2 and Sp1 elements are required to maintain basal CGbeta promoter activity, but these factors have opposing effects on cAMP regulation, which is mediated primarily by AP-2.

Molecular mechanism for cooperation between Sp1 and steroidogenic factor-1 (SF-1) to regulate bovine CYP11A gene expression

Bovine cholesterol side-chain cleavage cytochrome P450 (P450scc; product of the CYP11A gene) gene expression is regulated by gonadotropins via cAMP in the ovary, and by ACTH via cAMP in adrenal cortical cells. Previously, we characterized response elements located at −57/−32 and at −111/−101 bp in the 5′-flanking region of the bovine CYP11A gene required for cAMP-stimulated transcription in both mouse Y-1 adrenal tumor cells and bovine ovarian cells in primary culture, which bind SF-1 (or Ad4-BP) and Sp1, respectively. The role of these transcription factors in CYP11A transcription was further confirmed by deletion and mutation analyses. In addition, results obtained employing a double mutation of the Sp1- and SF-1-binding sites and a mammalian two-hybrid system indicate that Sp1 and SF-1 function cooperatively in the transactivation of the bovine CYP11A promoter in both bovine luteal cells and Y-1 cells. Here we report that SF-1 and Sp1 are able to associate with one another in vitro and in vivo. The NH 2-terminal region of SF-1, especially the DNA-binding domain, is the binding site for Sp1. In addition, as CBP is a common coactivator required for the transcriptional activity of numerous transcription factors including nuclear receptors, we investigated whether CBP functions as a cofactor for the regulation of bovine CYP11A promoter activity. We show here that CBP enhanced the PKA-induced CYP11A promoter activity, while a double mutation of both Sp1 and SF-1 sites within the CYP11A promoter region abolished CBP-induced activity. Furthermore, CBP stimulated Sp1-dependent transactivation, and a CBP/Sp1 complex in vivo was demonstrated by a co-immunoprecipitation assay. Also, CBP potentiated the transcriptional activity of GAL4-SF-1 in the presence of PKA. Thus, the cooperation between SF-1 and Sp1, required for the regulation of bovine CYP11A gene expression, is mediated by a direct protein–protein interaction and/or the common coactivator CBP.

Point mutations in SP1 motifs in the upstream regulatory region of human papillomavirus type 18 isolates from cervical cancers increase promoter activity.

Evidence of the functional significance of two naturally occurring mutations at nt 40 or 41 in the Sp1 motif in the promoter proximal segment of the upstream regulatory region (URR) of human papillomavirus (HPV) type 18 is presented. In electrophoretic mobility shift assays, Sp1 protein bound more efficiently to the Sp1 mutant motifs than to the prototype; while in both HeLa and HT3 cells, luciferase activity controlled by the mutant URRs was upregulated 2- and 3-fold, or 4- and 6-fold, in comparison with the prototype URR or HeLa cell-derived URR respectively. The HeLa URR represents a more appropriate baseline for promoter activity, containing a series of point mutations representative of most HPV-18 cancer isolates, including one in the Yin Yang 1 (YY1) site at the P105 promoter. The effect of the Sp1 mutations was found to be largely maintained in the context of the HeLa URR containing the prototype YY1 site.

Functional Characterization of the Major Late Promoter of Mouse Adenovirus Type 1

During the late phase of adenovirus infection, the major late promoter (MLP) controls the regulated expression of the genes that encode most viral structural proteins. Recently, the region of the genome of mouse adenovirus type 1 (MAV-1), predicted to contain the MLP, was sequenced and compared to that of the human virus MLP. The general organization of the transcriptional elements of the putative MAV-1 MLP is similar to that of the human virus counterpart, with some interesting differences. We wished to investigate the function of the predicted MLP of MAV-1 and to determine the significance of the differences found in the MAV-1 MLP. To test the activity of the predicted MLP of MAV-1, both Northern blot and primer extension analyses were performed on intracellular RNA isolated from cells infected with MAV-1. The results show that late RNA can be detected 48 hr postinfection and increases up to 6 days p.i. Primer extension analysis revealed that the major start sites of transcription are 28 and 31 nt downstream of the first T residue of the predicted TATA box. To analyze the functional significance of the predicted transcriptional elements, a transient transfection system, using the firefly luciferase gene controlled by the MAV-1 MLP sequence, was established. The predicted MLP sequence was capable of directing luciferase gene expression, to a level some 60% of that of the human virus MLP. Mutations were created in the inverted CAAT box, the SP1 site, and the TATA box, either singly or in combination. Each single-element mutation causes a marked reduction in luciferase gene expression, with the SP1 mutation showing the greatest effect. Double mutations were even more deficient, suggesting a level of functional redundancy among the various transcriptional elements. Finally, the putative SP1-binding site was examined by gel mobility shift assay and shown to interact with purified SP1 protein specifically, supporting the functional significance of this transcriptional element. These findings contribute to a better understanding of gene expression in MAV-1 and to its development as an appropriate model for the study of the molecular basis of pathogenesis in a natural host animal.