Hua-Guo Xu

Direct repression of the human IRF-3 promoter by E2F1

Interferon regulatory factor 3 (IRF-3) plays an important role in virus and double-stranded RNA-mediated induction of type I interferon and RANTES, DNA damage signaling, tumor suppression, and virus-induced apoptosis. However, cis elements or trans factors responsible for regulating IRF-3 expression remain largely unknown. Here we report that the transcription factor E2F1 negatively regulates the basal transcriptional activity of IRF-3 and deregulates IRF-3 expression at mRNA level. By transient transfection analysis, we demonstrate that the mutation of E2F-binding site results in a profound promotion of IRF-3 promoter activity. Overexpression of E2F1, but not a mutant E2F1, represses the IRF-3 promoter activity in reporter gene assays while knocking down of endogenous E2F1 by shRNA strategy results in enhanced IRF-3 promoter activity. Electrophoretic gel mobility shift assays and antibody competition assays confirm that E2F1 protein binds to the E2F consensus binding site in the IRF-3 promoter. Chromatin immunoprecipitation assays demonstrate that E2F1 interacts with the IRF-3 promoter in vivo. These results suggest that E2F1 negatively regulates IRF-3 transcription through binding to the E2F consensus binding site.

Promoter characterization and role of cAMP/PKA/CREB in the basal transcription of the mouse ORMDL3 gene.

Orosomucoid 1-like 3 (ORMDL3) gene was strongly linked with the development of asthma in genetic association studies, and its expression could be significantly induced by allergen in airway epithelial cells of mice. However, the expression mechanism of ORMDL3 was still unclear. Here we have identified and characterized the mouse ORMDL3 gene promoter. Deletion constructs of the 5′ flanking region were fused to a luciferase reporter gene. After transient transfection in mouse fibroblast cell line NIH3T3, a CRE (−27/−20) binding CREB was identified in the core promoter region. Deletion or mutation of the CRE consensus sequence resulted in a significant loss of the promoter activity. EMSA and ChIP assays demonstrated the binding of CREB to the core promoter. Knocking down endogenous CREB led to a reduction in ORMDL3 expression. Conversely, overexpression of CREB up-regulated ORMDL3 expression. Moreover, forskolin, a PKA activator, could facilitate the phosphorylation of CREB, which in turn heightens ORMDL3 expression. H-89, a PKA-specific inhibitor, could significantly inhibit ORMDL3 expression. This study delineates the characterization of mouse ORMDL3 gene promoter and shows signaling pathway cAMP/PKA/CREB plays an important role in regulating ORMDL3 expression, which will be helpful for future animal model studies regarding the regulation or function of ORMDL3 gene.

Transcriptional control of human CD2AP expression: the role of Sp1 and Sp3

The CD2 associated protein (CD2AP) is characterized as a T-lymphocyte CD2 adapter protein and is found to be related to glomerulosclerosis, and CD2AP knockout mice develop a rapid onset nephrotic syndrome and die of renal failure. Here we report that the transcription factor Sp1 and Sp3 up-regulate the basal transcriptional activity of CD2AP and increase CD2AP expression at mRNA level. We show by Chromatin immunoprecipitation (ChIP) assay that Sp1 and Sp3 interact with the CD2AP promoter region in vivo. By transient transfection analysis we also demonstrate the mutations of Sp1/3 binding sites result in a profound reduction of CD2AP promoter activity. Overexpression of Sp1 and Sp3 transactivates the CD2AP promoter, whereas small interfering RNA-mediated (siRNA) blockage of Sp1 and Sp3 genes expressions inhibits markedly its activity. These results suggest that Sp1 and Sp3 play an important role in regulating CD2AP transcription through binding to the Sp1/3 binding sites.

Characterization of a spliced variant of human IRF-3 promoter and its regulation by the transcription factor Sp1

Interferon regulatory factor 3 (IRF-3), an essential transcriptional regulator of the interferon genes, plays an important role in host defense against viral and microbial infection as well as in cell growth regulation. Promoter plays a crucial role in gene transcription. We have reported the characterization of the wide type of human IRF-3 promoter, but the characterization of the spliced variant of human IRF-3 Int2V1 promoter has not been systematically analyzed. To observe the spliced variant of human IRF-3 promoter, we have cloned the human IRF-3 gene promoter region containing 300 nucleotides upstream the transcription start site (TSS). Transient transfection of 5′ deleted promoter-reporter constructs and luciferase assay illustrated the region −159/−100 relative to the TSS is sufficient for full promoter activity. This region contains GATA1 and specific protein-1 (Sp1) transcription factor binding sites. Interestingly, mutation of this Sp1 site reduced the promoter activity by 50%. However, overexpression of Sp1 increased the transcription activity by 2.4-fold. These results indicated that the spliced variant of human IRF-3 gene core promoter was located within the region −159/−100 relative to the TSS. Sp1 transcription factor upregulates the spliced variant of human IRF-3 gene promoter.

Transcriptional Activation of the Human CD2AP Promoter by E2F1

CD2-associated protein (CD2AP) is an adaptor molecule involved in T cell receptor signaling and podocyte homeostasis. CD2AP-deficient mice develop nephritic syndrome and renal failure caused by glomerulosclerosis. Transcription factor E2F1 is a key regulator of cell proliferation and apoptosis. Here we report that E2F1 up-regulates the human CD2AP promoter and further increases the mRNA and protein levels of the human CD2AP in human embryonic kidney (HEK) 293 cells. By semi-quantitative RT-PCR and Western blot analysis we demonstrate that ectopic expression of E2F1 elevates the mRNA and protein levels of CD2AP. Consistently, transient transfection assays prove that overexpression of E2F1 transactivates the CD2AP promoter while knocking-down of endogenous E2F1 by a shRNA strategy results in reduction of the CD2AP promoter activity. Toward understanding the underlying mechanism of this regulation, we performed chromatin immunoprecipitation and mutations of the putative Sp1 binding sites, demonstrating that E2F1 can bind to Sp1 binding site and overexpression of E2F1 is capable of increasing the binding of E2F1 and decreasing the binding of Sp1 to Sp1 binding sites.

Mechanisms of transcriptional activation of the stimulator of interferon genes by transcription factors CREB and c-Myc

Stimulator of interferon genes (STING) plays an important role in host defense, autoimmune disease, osteoclast differentiation and anti-tumor response. Although many downstream targets have been studied in depth, the regulation of STING gene expression remains largely unknown. Here we demonstrate that transcription factors CREB and c-Myc maintain the transcriptional activity of STING. By 5′-rapid amplification of cDNA ends analysis, we identified the transcriptional start site (TSS) of STING. We illustrated that the region -124/+1 relative to TSS was sufficient for full promoter activity by a series of 5′ deletion promoter constructs. Transcriptional activity of the STING minimal promoter was dependent on CREB and c-Myc binding motifs and was abolished after mutation of these two DNA elements. Chromatin immunoprecipitation assays demonstrated that transcription factors CREB and c-Myc bind to STING promoter in vivo. Overexpression of CREB and c-Myc increased the STING promoter activity. Meanwhile, knocking-down of CREB and c-Myc by a small interfering RNA (siRNA) strategy markedly reduced endogenous STING expression. In summary, these results demonstrated that transcription factors CREB and c-Myc are involved in the regulation of STING transcription.

High Uric Acid (UA) Negatively Affects Serum Tartrate-Resistant Acid Phosphatase 5b (TRACP 5b) Immunoassay

Background Bone metastases often occur in the majority of patients with advanced cancer, such as prostate cancer, lung cancer and breast cancer. Serum tartrate-resistant acid phosphatase 5b (TRACP 5b), a novel bone resorption marker, has been used gradually in the clinics as a specific and sensitive marker of bone resorption for the early diagnosis of cancer patients with bone metastasis. Here, we reported that high concentrations of uric acid (UA) lead to decrease of TRACP 5b levels and determined whether TRACP 5b level was associated with UA in interference experiment. Methods A total of 77 patients with high concentrations of UA and 77 healthy subjects were tested to evaluate the differences in their TRACP 5b levels. Serial dilutions of UA were respectively spiked with a known concentration of TRACP 5b standard sample, then Serum TRACP 5b was detected by using bone TRAP® Assay. A correction equation was set to eliminate UA-derived TRACP 5b false-decrease. The effect of this correction was evaluated in high-UA individuals. Results The average TRACP level of the high-UA individuals (1.47± 0.62 U/L) was significantly lower than that of the healthy subjects (2.62 ± 0.63 U/L) (t-test, p<0.0001). The UA correction equation derived: ΔTRACP 5b = -1.9751lgΔUA + 3.7365 with an R2 = 0.98899. Application of the UA correction equation resulted in a statistically non-significant difference in TRACP 5b values between the healthy subjects and high-UA individuals (p = 0.24). Conclusions High UA concentrations can falsely decrease TRACP 5b levels due to a method-related systematic error. To avoid misdiagnoses or inappropriate therapeutic decisions, increased attention should be paid to UA interference, when TRACP 5b is used for early diagnosis of cancer patients with bone metastasis, evaluation of the aggressiveness of osteosarcoma or prediction of survival in prostate cancer and breast cancer with bone metastases.

High uric acid (UA) downregulates bone alkaline phosphatase (BALP) expression through inhibition of its promoter activity

Bone metastases often occur in prostate cancers, lung cancers and breast cancers. Bone alkaline phosphatase (BALP) is one of the most commonly used serological markers for clinical evaluation of bone metabolism. Here, we reported that high concentrations of uric acid (UA) caused decrease of BALP levels and revealed that the effect of high concentrations of UA on the BALP expression was through inhibition of its promoter activity. Our results suggested physicians to think about serum UA status of patients with advanced cancer to avoid misdiagnosis when BALP was used to diagnose or assess the extent of bone metastases.

Involvement of GATA1 and Sp3 in the activation of the murine STING gene promoter in NIH3T3 cells

Stimulator of Interferon Gene (STING) is a key mediator of innate immune signaling. STING plays a pivotal role in the pathogenesis of many diseases including infectious diseases, auto-immune diseases and cancer. Many studies have been carried out recently in the field of STING-regulated pathway, however, rarely of transcriptional mechanisms. To characterize the murine STING (mSTING) promoter, we cloned a series of different nucleotide sequences of the 5′-flanking region of the mSTING gene. Transient transfection of promoter-reporter recombinant plasmids and luciferase assay illustrated the region (−77/+177) relative to the transcription start site (TSS) of the mSTING gene was sufficient for full promoter activity. This region contains GATA1, IK2, Sp1/Sp3 and STAT putative transcription factor binding sites. Mutation of GATA1 or Sp1/Sp3 sites led to obvious decrease of the mSTING promoter activity. Overexpression of GATA1 and Sp3 enhanced the mSTING promoter activity, whereas knockdown of GATA1 and Sp3 by a siRNA strategy significantly reduced the transcription activity. Chromatin immunoprecipitation assays demonstrated that GATA1 and Sp3 interact with the mSTING promoter in vivo. These results provided the first analysis of mSTING promoter and demonstrated that transcription factor GATA1 and Sp3 positively regulate the basal transcription of the mSTING gene.

All-trans retinoic acid up-regulates the human CD2AP gene expression through Sp1/Sp3 binding sites

Abstract All-trans retinoic acid (ATRA), an active metabolite of vitamin A, plays an important role in regulating cell differentiation, proliferation, and apoptosis. It was reported that ATRA could cause an up-regulation of protein expression of CD2AP in nephrotic animals. However, the mechanism of ATRA-mediated up-regulation is not well understood. In the present study, deletion analysis and luciferase assays demonstrated that ATRA caused a marked increase in the activity of the CD2AP promoter, and the region between nt −599 and −328 from the transcription start site, where there are two clusters of Sp1/3 binding sites, was indispensable for ATRA-mediated up-regulation. Chromatin immunoprecipitation assays revealed that ATRA activated the CD2AP transcription through enhancing the DNA-binding activity of Sp1 and Sp3 with the CD2AP promoter. Taken together, this study provided evidence for the first time showing the stimulating effect of ATRA on CD2AP and new therapeutic strategies for the treatment of nephritic syndrome and other associated diseases of CD2AP deficiency.