Basal Promoter Activity Research Articles

Characterization and functional evaluation of goat PDX1 regulatory modules through comparative analysis of conserved interspecies homologs.

PDX1 is a crucial transcription factor in pancreas development and mature β-cell function. However, the regulation of PDX1 expression in larger animals mirroring human pancreas morphogenesis and endocrine maturation remains poorly understood. Therefore, we conducted a comparative analysis to characterize regulatory regions of goat PDX1 gene and assessed their transcriptional activity by transient transfection of several transgenic EGFP constructs in β- and non-β cell lines. We recognized several highly conserved regions encompassing the promoter and cis-regulatory elements (Area I-IV) at 5' flanking sequence of the genes. Within the promoter, we identified that a key E-box and nearby CAAT element synergistically drive transcription, constituting the basal promoter of goat PDX1 gene. Furthermore, each recognized regulatory area separately enhances this basal promoter activity in β-cells compared to non-β cells; however, cooperatively, they exhibit a bifunctional regulatory effect on transcription. Additionally, the intact ~ 3kb upstream region (Area I-III) functions as the most efficient reporter transgene in vitro and shows islet-specific expression in native rat pancreas. Together, our findings suggest that the regulation of goat PDX1 gene is governed by conserved regions similar to other mammals, while both structurally and functionally, these regions exhibit a closer resemblance to those found in humans.

The promoters of two CpMYB106-like paralog genes respond to abiotic stresses and phytohormones and drive differential expression in contrasting cultivars of Cucurbita pepo

Zucchini fruit (Cucurbita pepo L.) is susceptible to chilling injury (CI) during its postharvest life, with this response being cultivar-dependent. A previous transcriptomic analysis comparing cold-tolerant fruit from the cultivar ‘Natura’ with cold-sensitive fruit from the cultivar ‘Sinatra’, revealed the transcription factor (TF) CpMYB106-like as a relevant candidate gene for the acquisition of postharvest low-temperature tolerance. C. pepo has two CpMYB106-like paralogs, which are differentially present in the ‘Natura’ (CpMYB106-likeA) and ‘Sinatra’ (CpMYB106-likeB) cultivars due to gene and promoter deletions. The aim of this study was to investigate the transcriptional regulation of the CpMYB106-like paralogs to unravel their role in the postharvest life of zucchini fruit. For this, gene expression was analyzed and promoter sequences and their activities were studied through reporter β-glucuronidase (GUS) gene analysis in heterologous systems. An expression analysis showed that CpMYB106-like mRNA levels were induced only in the cold-stored fruit of ‘Natura’. The analysis of the promoter sequences showed numerous cis-regulatory elements (CREs), many of which have a differential distribution or frequency between the two paralogs. Promoter basal activity was determined by transient transformation of Nicotiana benthamiana leaves, which showed a higher expression driven by the promoter from the cold-tolerant cultivar ‘Natura’, which could be explained by the presence of three additional copies of the enhancer element EECCRCAH1 in proMYB106A. The tissue-specific expression pattern, and the response to abiotic stresses and phytohormones controlled by CpMYB106-like promoters was analyzed in transgenic Arabidopsis thaliana plants. GUS activity was mainly detected in the vascular system, leaves, and inflorescences, especially in siliques. With respect to abiotic stresses, low temperature decreased GUS expression in Arabidopsis, but increased it in plants carrying the ‘Natura’ promoter after a 24-h acclimation period at 22 °C. Salt treatment induced GUS activity driven by both promoters in Arabidopsis seedlings, but it was higher with the ‘Natura’ promoter. Furthermore, methyl jasmonate (MeJA) also induced a sharp increase of GUS controlled by proMYB106A. These results suggest that the regulation of CpMYB106-like TFs in zucchini is promoter-driven, and reveal the involvement of these genes in the acquisition of cold tolerance in fruit during postharvest stress conditions.

Implications of differential transcription start site selection on chronic myeloid leukemia and prostate cancer cell protein expression.

The relevance of minor transcription start sites in broad promoters is not well understood. We have studied AGAP2 expression in prostate cancer and chronic myeloid leukemia (CML), showing transcription is initiated from alternative transcription start sites (TSSs) within a single TSS cluster, producing cancer-type-specific AGAP2 mRNAs with small differences in their 5' UTR length. Interestingly, in the CML cell lines where the 5' UTR is longer, AGAP2 protein levels are lower. We demonstrate that the selection of an upstream TSS involved the formation of a G quadruplex in the 5' UTR, decreasing polysome formation. After developing a bioinformatics pipeline to query data from the FANTOM project and the NCl-60 human tumor cell lines screen, we found HK1 expression can also be regulated by the same mechanism. Overall, we present compelling data supporting TSS selection within a TSS cluster play a role in protein expression and should not be ignored.

Level of constitutively expressed BMAL1 affects the robustness of circadian oscillations

The circadian rhythm is a biological oscillation of physiological activities with a period of approximately 24 h, that is driven by a cell-autonomous oscillator called the circadian clock. The current model of the mammalian circadian clock is based on a transcriptional-translational negative feedback loop in which the protein products of clock genes accumulate in a circadian manner and repress their own transcription. However, several studies have revealed that constitutively expressed clock genes can maintain circadian oscillations. To understand the underlying mechanism, we expressed Bmal1 in Bmal1-disrupted cells using a doxycycline-inducible promoter and monitored Bmal1 and Per2 promoter activity using luciferase reporters. Although the levels of BMAL1 and other clock proteins, REV-ERBα and CLOCK, showed no obvious rhythmicity, robust circadian oscillation in Bmal1 and Per2 promoter activities with the correct phase relationship was observed, which proceeded in a doxycycline-concentration-dependent manner. We applied transient response analysis to the Bmal1 promoter activity in the presence of various doxycycline concentrations. Based on the obtained transfer functions, we suggest that, at least in our experimental system, BMAL1 is not directly involved in the oscillatory process, but modulates the oscillation robustness by regulating basal clock gene promoter activity.

Noise-Reduction and Sensitivity-Enhancement of a Sleeping Beauty-Based Tet-On System.

Tetracycline-inducible systems are widely used control elements for mammalian gene expression. Despite multiple iterations to improve inducibility, their use is still compromised by basal promoter activity in the absence of tetracyclines. In a mammalian system, we previously showed that the introduction of the G72V mutation in the rtTA-M2 tetracycline activator lowers the basal level expression and increases the fold-induction of multiple genetic elements in a long chimeric antigen receptor construct. In this study, we confirmed that the G72V mutation was effective in minimising background expression in the absence of an inducer, resulting in an increase in fold-expression. Loss of responsiveness due to the G72V mutation was compensated through the incorporation of four sensitivity enhancing (SE) mutations, without compromising promoter tightness. However, SE mutations alone (without G72V) led to undesirable leakiness. Although cryptic splice site removal from rtTA did not alter the inducible control of the luciferase reporter gene in this simplified vector system, this is still recommended as a precaution in more complex multi-gene elements that contain rtTA. The optimized expression construct containing G72V and SE mutations currently provides the best improvement of fold-induction mediated by the rtTA-M2 activator in a mammalian system.

Characterization of the promoter of the human farnesyltransferase beta subunit and the impact of the transcription factor OCT-1 on its expression.

Farnesyltransferase (FTase) enables about 100 proteins to interact with cellular membranes by catalyzing the posttranslational addition of a farnesyl group. Farnesylated proteins provide important functions and inhibitors against the β-subunit of the heterodimer of FTase are intensively studied in clinical and preclinical trials. However, very little is known about the transcriptional regulation of the β-subunit. The examined promoter region of the human FTase β-subunit gene (FNTB) showed significant basal promoter activity in HEK-293 and in HeLa cells. We were able to locate the core promoter at -165 to -74. Ten potential binding sites of the transcription factor OCT-1 were detected. Three could be confirmed using EMSA super shift experiments. OCT-1 overexpression and knockdown confirmed it as an important regulator of FNTB expression. Our results provide a basis for further research on FNTB/OCT-1 regulation, its inhibitors and diseases influenced by both such as colon carcinoma or diabetes mellitus.

Abstract P4-02-11: A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer

Abstract BACKGROUND: Estrogen Receptor (ER) + breast cancers (BC) comprise over 70% of BC cases and can be targeted via ER modulated therapies. Despite this, ER+BC patients can experience recurrence within 20 years and the majority of BC related deaths can be attributed to metastatic ER+BC. These distant metastases commonly become diagnosed as endocrine therapy resistant. Thus, there is an unmet need to identify novel biomarkers for treating ER+ patients with metastasis. We have identified a tumor suppressor, Singleminded 2s (SIM2s), expressed in breast epithelial cells that inhibits EMT and metastasis, and is downregulated in the progression of breast disease. Our previous studies found that loss of SIM2s expression results in downregulation of ESR1 and increased basal markers in the MCF7 (ER+BC) cell line. Likewise, Semaphorin 7a (SEMA7A) expression is associated with decreased overall survival in ER+BC patients. Patients with SEMA7Ahi tumors treated with Tamoxifen exhibit shortened relapse free survival. SEMA7A is a downstream target of both NFKB and PTGS2/COX-2; and our previously published results establish a cross-talk between NFKB and SIM2s to regulate PTGS2 via interactions with the AKT pathway. Moreover, COX-2 inhibition is associated with better prognosis for breast cancer patients. Therefore, we are investigating how SIM2s may repress AKT signaling in SEMA7A driven ER resistance and metastasis. Methods: We performed in silico analysis of the SEMA7A promoter using the ConTra web server. For in vitro assays, we examined estrogen dependent differences in SIM2s, ER, and SP1 binding utilizing luciferase reporter assays using intact promoter and SIM2s site deletion mutants (Invitrogen) in MCF7 cells. Using immunoblotting and qPCR analysis we also assessed expression of SEMA7A, ESR1, PI3K/AKT signaling genes and EMT associated genes in Crispr/Cas9 SIM2s knockout (SIM2KO) MCF7 cells. Treatment of MCF7 cells with exogenous SEMA7A was used to observe possible changes in SIM2s expression and SIM2s promoter activity. For in vivo assays, we used immunohistochemistry (IHC) to assess SIM2s in SEMA7A low and high expressing MCF7 xenografts. To examine the relationship between SIM2s and ER, SEMA7A-/- (KO) mice were crossed with C57/BL6 MMTV-PYMT mice. Results: In silico analysis of the SEMA7A promoter via ConTra revealed four potential SIM2 response elements containing the SIM2s central midline elements (CME) binding motif and highly conserved estrogen response element half-sites adjacent to two specificity protein 1 consensus bindings sites. Luciferase reporter assays in MCF7 cells confirmed SIM2s represses basal and estrogen induced-SEMA7A promoter activity. Moreover, SIM2s was unable to repress the SEMA7A promoter activity with CME mutation. MCF7 SIM2KO cells exhibit increased SEMA7A promoter activity, SEMA7A, AKT signaling, EMT signatures and decreased ESR1, CDH1, and PTEN protein and mRNA expression. Both SIM2s expression and SIM2s promoter activity are significantly decreased with the addition of exogenous SEMA7A. Moreover, MCF7 xenografts reveal reciprocal expression of SIM2s and SEMA7A. Additionally in MMTV-PyMT mice, SIM2s expression is lost as tumors transition from pre-malignant to invasive phenotypes, yet we observe maintenance of SIM2s and ER in SEMA7A-/-;PyMT mice. Conclusion: These findings establish a regulatory relationship between SIM2s and SEMA7A in ER+BC. SIM2s functions to downregulate SEMA7A mediated pro-tumor signaling and maintain ER expression, but can be turned off to allow for resistance in ER+BC. Given the observed reciprocal expression, our study suggests a SIM2s/SEMA7A switch which may confer resistance in ER+BC via AKT signaling through downregulation of PTEN. Thus, a SIM2s/SEMA7A switch may act as a prognostic indicator to provide therapeutic advantages in resistant ER+BC metastasis. Citation Format: Garhett L Wyatt, Lyndsey S Crump, Traci R Lyons, Weston W Porter. A SIM2s/SEMA7A switch drives therapeutic resistance in ER+ breast cancer [abstract]. In: Proceedings of the 2021 San Antonio Breast Cancer Symposium; 2021 Dec 7-10; San Antonio, TX. Philadelphia (PA): AACR; Cancer Res 2022;82(4 Suppl):Abstract nr P4-02-11.

A mutation in the SAA1 promoter causes hereditary amyloid A amyloidosis

Amyloid A amyloidosis is a serious clinical condition resulting from the systemic deposition of amyloid A originating from serum amyloid A proteins with the kidneys being the most commonly and earliest affected organ. Previously described amyloid A amyloidosis is linked to increased production and deposition of serum amyloid A proteins secondary to inflammatory conditions arising from infectious, metabolic, or genetic causes. Here we describe a family with primary amyloid A amyloidosis due to a chr11:18287683 T>C (human genome version19) mutation in the SAA1 promoter linked to the amyloidogenic SAA1.1 haplotype. This condition leads to a doubling of the basal SAA1 promoter activity and sustained elevation of serum amyloid A levels that segregated in an autosomal dominant pattern in 12 genetically affected and in none of six genetically unaffected relatives, yielding a statistically significant logarithm of odds (LOD) score over 5. Affected individuals developed proteinuria, chronic kidney disease and systemic deposition of amyloid composed specifically of the SAA1.1 isoform. Tocilizumab (a monoclonal antibody against the interleukin-6 receptor) had a beneficial effect when prescribed early in the disease course. Idiopathic forms represent a significant and increasing proportion (15-20%) of all diagnosed cases of amyloid A amyloidosis. Thus, genetic screening of the SAA1 promoter should be pursued in individuals with amyloid A amyloidosis and no systemic inflammation, especially if there is a positive family history.

Transcriptional downregulation of FAM3C/ILEI in the Alzheimer's brain.

Amyloid-β (Aβ) accumulation in the brain triggers the pathogenic cascade for Alzheimer's disease (AD) development. The secretory protein FAM3C (also named ILEI) is a candidate for an endogenous suppressor of Aβ production. In this study, we found that FAM3C expression was transcriptionally downregulated in the AD brain. To determine the transcriptional mechanism of the human FAM3C gene, we delineated the minimal 5'-flanking sequence required for basal promoter activity. From a database search for DNA-binding motifs, expression analysis using cultured cells, and promoter DNA-binding assays, we identified SP1 and EBF1 as candidate basal transcription factors for FAM3C, and found that SMAD1 was a putative inducible transcription factor and KLF6 was a transcription repressor for FAM3C. Genomic deletion of the basal promoter sequence from HEK293 and Neuro-2a cells markedly reduced endogenous expression of FAM3C and abrogated SP1- or EBF1-mediated induction of FAM3C. Nuclear protein extracts from AD brains contained lower levels of SP1 and EBF1 than did those from control brains, although the relative mRNA levels of these factors did not differ significantly between the groups. Additionally, the ability of nuclear SP1 and EBF1 in AD brains to bind with the basal promoter sequence-containing DNA probe was reduced compared with the binding ability of these factors in control brains. Thus, the transcriptional downregulation of FAM3C in the AD brain is attributable to the reduced nuclear levels and genomic DNA binding of SP1 and EBF1. An expressional decline in FAM3C may be a risk factor for Aβ accumulation and eventually AD development.

Regulation of Lysyl Oxidase, a Key Regulator of Bone Marrow Fibrosis, in Myelofibrosis

Primary myelofibrosis (PMF) is a myeloproliferative neoplasm (MPN) that arises from clonal proliferation of hematopoietic stem cells, which leads to expansion of immature megakaryocytes (MKs), progressive bone marrow (BM) fibrosis and cytopenias. Our previous work showed that lysyl oxidase (LOX), a matrix cross-linking enzyme highly expressed by MKs in PMF, contributes to disease progression. GATA1 is a transcription factor that is important for MK development and has been found to be downregulated in MKs of patients with PMF and in mouse models of PMF. We observed an inverse correlation between GATA-1 and LOX levels in MKs of PMF mice. GATA1 can act as an activator as well as a repressor, and our chromatin precipitation study showed that it binds to the Lox gene. To determine whether GATA-1 suppresses LOX expression in MKs, BM cells induced to differentiate to MKs were transfected with a retrovirus containing GATA-1 with GFP reporter or empty virus with GFP. MKs were isolated using a BSA gradient and RNA was extracted for LOX mRNA expression via qRT-PCR. GATA-1 transduced MKs suppressed LOX mRNA levels. Further, to assess the effect of GATA1 on Lox gene promoter activity, we transfected a murine pluripotent mesenchymal cell line (C3H10T1/2 cells) with the murine Lox gene promoter linked to a reporter gene encoding luciferase. Co-transfection with a vector expressing mouse GATA-1 down-regulated basal LOX promoter activity compared to empty vector. Given that PMF MKs express high levels of TGF-β, and TGF-β has been shown to stimulate LOX expression, we next examined the ability of GATA-1 to suppress TGF-β-mediated LOX expression. Indeed, transfection of GATA-1 suppressed LOX expression even in the presence of TGF-β compared to empty vector. Taken together, we identified a key role for GATA-1 in LOX regulation, which has significant relevance to fibrosis in PMF. DisclosuresNo relevant conflicts of interest to declare.

Hepatic Expression of the Mouse Angptl8 Gene Is Regulated by Insulin Through Increases in Hepatocyte Nuclear Factor-1

Angiopoietin-like proteins (ANGPTLs) are a family of proteins structurally similar to angiopoietins. ANGPTL8 is an important regulator of circulating triglyceride (TG) levels in mammals. Increasing evidence revealed an association between ANGPTL8 expression and serum lipid profiles, especially in subjects with metabolic syndrome. Several mice studies demonstrated that Angptl8 is suppressed by fasting and induced by long term refeeding, however the detailed mechanism is still unclear. In humans, ANGPTL8 is mainly expressed in the liver. Therefore, this study aims to investigate the mechanisms that control the refeeding induced increase in the hepatic Angptl8 gene expression. Methods and Results: Twenty-week-old male C57/BL6 mice were used in this study. Mice were fasted for 12h during the dark cycle and re-fed for 30, 60, 120, 240 and 360 minutes during the light cycle. Mice were euthanized after each refeeding time course and tissues were collected. We found even short refeeding times (~60 min) enhanced the expression levels of hepatic Angptl8 in mice. We cloned the mouse Angptl8 gene promoter region. Promoter deletion analyses showed that the basal promoter activity was significantly attenuated by a deletion of -309/-60 region in hepatocytes. A computational motif search revealed the presence of a potential binding motif for hepatocyte nuclear factor 1α/1β (HNF-1α/β) at -84/-68 bp of the promoter. Mutations of the HNF-1 binging site significantly decreased the promoter activity in mouse hepatoma cells (Hepa1-6) and mouse primary hepatocytes, and the promoter carrying the mutated HNF-1 site was not transactivated by co-transfected HNF-1 in a non-hepatic cell line. These findings indicated that HNF-1 was essential and critical factor for the basal expression of Angptl8 in murine liver. In fact, knockdown of Hnf-1 using siRNA method in mouse Hepa1-6 and mouse primary hepatocytes reduced Angptl8 protein levels. We also performed Electrophoretic mobility-shift assays and confirmed the direct binding of Hnf-1 to its Angptl8 promoter binding motif. To elucidate whether refeeding could enhance HNF-1, we checked the expression levels of Hnf-1 in mouse liver. Hnf-1 expression levels of both mRNA and protein were increased after short-term refeeding, paralleling the enhanced expression of the Angptl8. Moreover, insulin-stimulated primary hepatocytes showed increased expression of Angptl8 protein, but knockdown of Hnf-1 completely abolished this enhancement by insulin. Chromatin immunoprecipitation (ChIP) analyses confirmed the recruitment of endogenous Hnf-1 to the Angptl8 promoter region and it was strongly induced by insulin. Conclusion: HNF-1 plays essential role in hepatocyte-specific and refeeding-induced rapid increases in Angptl8 expression via insulin.

Characterization of G‐quadruplex Structures in Serum Amyloid A Gene as a Novel Approach for Systemic Amyloidosis Therapy

Transcriptional initiation is an important step in the regulation of transcription. When transcriptional initiation is dysfunctional, it will lead to abnormal gene expression. Overexpression of Serum Amyloid A (SAA) and self-assembly into fibrils have been associated with chronic inflammatory conditions and certain neoplasms, which lead to the deposition of AA amyloid in organs such as the liver, spleen, and kidney (systemic amyloidosis). For this reason, SAA is a highly significant molecular target. Insight into the structural biology of the promoter element that determines the combinatorial control of gene expression by different transcriptional factors is essential for understanding the dysfunction of SAA gene expression. The highly GC-rich region of the human SAA promoter is crucial for basal promoter activity and likely to assume a DNA secondary structure, such as G-quadruplex (GQ). GQs often serve as transcriptional silencer elements. Stabilization of GQ is possible with small molecule therapeutics to modulate gene expression. In the present study, G-quadruplex formation has been isolated from the GC-rich region of the 5’-untranslated region (5’UTR) of SAA2. An electrophoretic mobility shift and thioflavin T fluorescence assays were used to confirm the formation of a biologically relevant intramolecular structure. The structure of the G-quadruplex is currently under investigation by circular dichroism and DMS footprinting. These data characterizing the formation of a unique secondary structure in SAA gene are promising to develop novel anti-amyloid therapy for the treatment of systemic amyloidosis.

Mutant glucocorticoid receptor binding elements on the interleukin-6 promoter regulate dexamethasone effects

BackgroundGlucocorticoids (GCs) have been extensively used as essential modulators in clinical infectious and inflammatory diseases. The GC receptor (GR) is a transcription factor belonging to the nuclear receptor family that regulates anti-inflammatory processes and releases pro-inflammatory cytokines, such as interleukin (IL)-6.ResultsFive putative GR binding sites and other transcriptional factor binding sites were identified on theIL-6 promoter, and dexamethasone (DEX) was noted to reduce the lipopolysaccharide (LPS)-induced IL-6 production. Among mutant transcriptional factor binding sites, nuclear factor-kappa B (NF-κB), activator protein (AP)-1, and specificity protein (Sp)1–2 sites reduced basal and LPS-induced IL-6 promoter activities through various responses. The second GR binding site (GR2) was noted to play a crucial role in both basal and inducible promoter activities in LPS-induced inflammation.ConclusionsWe concluded that selective GR2 modulator might exert agonistic and antagonistic effects and could activate crucial signaling pathways during the LPS-stimulated inflammatory process.

Effect of 5-azacytidine (5-aza) on UCP2 expression in human liver and colon cancer cells.

The function of the uncoupling protein 2 (UCP2) is different for each cancer cell. However, the mechanism of expression is still unclear. DNA methylation affects protein expression and is one factor that transforms normal cells into cancer cells. In this study, the hepatocellular carcinoma Hep3B and HepG2 cells and colorectal cancer HT-29 cells were treated with 5-azacytidine (5-aza), a DNA demethylation agent, to observe the modification of UCP2 expression and the methylation degree in the UCP2 promoter region. Promoter basal activity and degree of UCP2 expression were measured in Hep3B, HepG2, and HT-29 cells. In addition, methylation-specific PCR (MSP) was performed to investigate the degree of methylation in the UCP2 promoter region. The methylation region in the UCP2 promoter was confirmed based on bisulfite sequencing. In Hep3B cells in which UCP2 mRNA was not transcribed, the promoter basal activity was significantly higher than in HT-29 or HepG2 cells in which UCP2 mRNA was transcribed. Treatment with 5-aza increased UCP2 expression in Hep3B and HT-29 cells; however, the expression in HepG2 cells was unchanged. The UCP2 promoter in Hep3B cells has numerous methylated regions compared with HT-29 and HepG2 cells. The results of the present study revealed that inhibition of UCP2 expression in Hep3B cells was due to methylation of the promoter region. Investigating the mechanism that induces UCP2 expression in cancer cells is important to understand the function of UCP2, which could aid in cancer treatment.

Фактор роста эпидермиса участвует в повышении экспрессии онкогена URGCP в клетках гепатомы человека

Hepatocellular Carcinoma (HCC) is the fourth leading cause of cancer-related death in the World. Epidermal Growth Factor Receptor (EGFR) pathway plays an important role in HCC tumorigenesis. In the tumor microenvironment of HCC, the expression of EGF is aberrant. Here we describe the EGF-dependent regulation of URGCP gene in Human Hepatoma cancer cells (Hep3B). The effect of EGF cytokine on Hep3B proliferation was shown using MTT method. EGF-mediated URGCP expression was determined at mRNA and protein level with qRT-PCR analyses and Western blotting method, respectively. Different lengths of URGCP promoter constructs were transient transfected in to Hep3B cells and the basal promoter activities were determined in the presence of EGF. In addition, some pathway analyses were performed to find out the mechanism of EGF mediated up-regulation of the URGCP gene. In the presence of EGF, URGCP expression increases in concentration and time dependent manner. EGF mediated URGCP up-regulation might depend on a cis-acting element located between -344 and -482 positions in its promoter.

An optimized method for plasma extracellular vesicles isolation to exclude the copresence of biological drugs and plasma proteins which impairs their biological characterization.

Extracellular vesicles (EVs) are cell membrane-derived phospholipid bilayer nanostructures that contain bioactive proteins, enzymes, lipids and polymers of nucleotides. They play a role in intercellular communication and are present in body fluids. EVs can be isolated by methods like ultracentrifugation (UC), polyethylene-glycol-precipitation (PEG) or size exclusion chromatography (SEC). The co-presence of immunoglobulins (Ig) in EV samples isolated from plasma (pEVs) is often reported and this may influence the assessment of the biological function and phenotype of EVs in bio- and immunoassay. Here, we studied the presence of an Ig-based therapeutic (etanercept) in pEV samples isolated from rheumatoid arthritis (RA) patients and improved the isolation method to obtain purer pEVs. From plasma of etanercept (Tumor-necrosis-factor (TNF)-α antibodies)-treated RA patients pEVs were isolated by either UC, PEG or SEC. SEC isolated pEVs showed the highest particle-to-protein ratio. Strong TNF-α inhibition determined in a TNF-α sensitive reporter assay was observed by pEVs isolated by UC and PEG, and to a lesser extent by SEC, suggesting the presence of functional etanercept. SEC isolation of etanercept or labelled immunoglobulin G (IgG) showed co-isolation of these antibodies in the pEV fraction in the presence of plasma or a high protein (albumin) concentration. To minimize the presence of etanercept or immunoglobulins, we extended SEC (eSEC) column length from 56mm to 222mm (total stacking volume unchanged). No effect on the amount of isolated pEVs was observed while protein and IgG content were markedly reduced (90%). Next, from six etanercept- treated RA patients, pEVs were isolated on a eSEC or standard SEC column, in parallel. TNF-α inhibition was again observed in pEVs isolated by conventional SEC but not by eSEC. To confirm the purer pEVs isolated by eSEC the basal IL-8 promoter activation in human monocytes was determined and in 4 out of 5 SEC isolated pEVs activation was observed while eSEC isolated pEVs did not. This study shows that extended SEC columns yielded pEVs without detectable biologicals and with low protein and IgG levels. This isolation method will improve the characterization of pEVs as potential biomarkers and mediators of disease.

Transcriptional Regulation of the Angptl8 Gene by Hepatocyte Nuclear Factor-1 in the Murine Liver

Brief refeeding times (~60 min) enhanced hepatic Angptl8 expression in fasted mice. We cloned the mouse Angptl8 promoter region to characterise this rapid refeeding-induced increase in hepatic Angptl8 expression. Deletion of the −309/−60 promoter region significantly attenuated basal promoter activity in hepatocytes. A computational motif search revealed a potential binding motif for hepatocyte nuclear factor 1α/1β (HNF-1α/β) at −84/−68 bp of the promoter. Mutation of the HNF-1 binding site significantly decreased the promoter activity in hepatocytes, and the promoter carrying the mutated HNF-1 site was not transactivated by co-transfection of HNF-1 in a non-hepatic cell line. Silencing Hnf-1 in hepatoma cells and mouse primary hepatocytes reduced Angptl8 protein levels. Electrophoretic mobility-shift assays confirmed direct binding of Hnf-1 to its Angptl8 promoter binding motif. Hnf-1α expression levels increased after short-term refeeding, paralleling the enhanced in vivo expression of the Angptl8 protein. Chromatin immunoprecipitation (ChIP) confirmed the recruitment of endogenous Hnf-1 to the Angptl8 promoter region. Insulin-treated primary hepatocytes showed increased expression of Angptl8 protein, but knockdown of Hnf-1 completely abolished this enhancement. HNF-1 appears to play essential roles in the rapid refeeding-induced increases in Angptl8 expression. HNF-1α may therefore represent a primary medical target for ANGPTL8-related metabolic abnormalities. The study revealed the transcriptional regulation of the mouse hepatic Angptl8 gene by HNF-1.

Hepatitis B Virus-X Downregulates Expression of Selenium Binding Protein 1.

Selenium binding protein 1 (SELENBP1) has been known to be reduced in various types cancer, and epigenetic change is shown to be likely to account for the reduction of SELNEBP1 expression. With cDNA microarray comparative analysis, we found that SELENBP1 is markedly decreased in hepatitis B virus-X (HBx)-expressing cells. To clarify the effect of HBx on SELENBP1 expression, we compared the expression levels of SELENBP1 mRNA and protein by semi-quantitative RT-PCR, Northern blot, and Western blot. As expected, SELENBP1 expression was shown to be reduced in cells expressing HBx, and reporter gene analysis showed that the SELENBP1 promoter is repressed by HBx. In addition, the stepwise deletion of 5′ flanking promoter sequences resulted in a gradual decrease in basal promoter activity and inhibition of SELENBP1 expression by HBx. Moreover, immunohistochemistry on tissue microarrays containing 60 pairs of human liver tissue showed decreased intensity of SELENBP1 in tumor tissues as compared with their matched non-tumor liver tissues. Taken together, our findings suggest that inhibition of SELENBP1 expression by HBx might act as one of the causes in the development of hepatocellular carcinoma caused by HBV infection.

Identification of a Novel Methylation Site (CpG 388) in the Promoter/Enhancer Region of Cytochrome P450 (CYP)1B1: Possible Role of Promoter Methylation in Attenuation of CYP1B1 expression and DNA adducts by Omega 3‐ Fatty Acids in Mouse Lung In Vivo

3‐Methylcholanthrene (MC) and benzo[a]pyrene (BP) are polycyclic aromatic hydrocarbons (PAHs) carcinogens. Cytochrome P450 (CYP)1B1 enzymes plays a key role in the activation of PAHs to carcinogenic metabolites, which initiate carcinogenesis by binding covalently to DNA, and the adducts, if not repaired, could lead to tumorigenesis. In this study we tested the hypothesis that pre‐treatment of mice with omega‐3‐fatty acids, i.e. [eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) will lead to attenuation of PAH‐mediated pulmonary DNA adduct formation in part by inhibiting CYP1B1. Twelve week old male and female A/J mice received EPA (60 mg/kg) and DHA (40 mg/kg) from day 1 to day 24. Control mice were treated with vehicle corn oil. On day 3, mice were treated with BP (40 μmol/kg) or 3‐methylcholanthrene (MC, 40 μmol/kg) by i.p. In the short‐term experiment (DNA adduct studies), 3–5 mice from each group were terminated at day 10 (7 days after BP or MC treatment). EPA/DHA significantly suppressed formation of BP‐DNA and MC‐DNA adducts in lung and liver of both male and female mice. CYP1B1 expression in lung at protein and mRNA levels was induced significantly by PAHs, but was suppressed by about 70% in the lungs of EPA/DHA‐treated mice. We tested the hypothesis that PAHs in part induce CYP1B1 by methylating specific CpG sites on the negative regulatory elements (NREs) of the promoter. Mouse lung DNA samples were subjected to bisulfite deamination, Methylation‐Specific PCR (MSP), and a sequencing primer‐flanking nested PCR. ClustalW multiple sequence alignment determined the methylation state of a total of 103 putative CpG sites on the CYP1B1 promoter/enhancer region, which included 3 putative CpG islands. We found that mice treated with BP resulted in partially methylated CpG 388 (83%) in lung DNAs, while co‐administration with EPA/DHA significantly suppressed the methylation of CpG 388 (to 33%). These results suggest that PAHs induce CYP1B1 in part by inducing methylation of CpG 388, which might be putative NREs of CYP1B1. We hypothesize that suppression of methylation by EPA/DHA at CpG 388 leads to restoration of negative regulation, leading to repression of CYP1B1 expression, which in turn leads to inhibition of PAH carcinogenesis. We further verified the role of CpG 388 by mutating it in a luficerase reporter plasmid containing mouse CYP1B1 promoter/enhancer. Preliminary results in transiently transfected H441 lung cell line indicated that both wild type and mutant CYP1B1 promoters were activated about 2.5 fold by BP, and mutation of CpG 384 decreased both basal and inducible promoter activities. Further studies could lead to CYP1B1 as an important molecular target for the prevention and/or treatment of lung carcinogenesis by PAHs in humans.Support or Funding InformationThis work was supported in part by NIEHS grant R01ES029382 to BM.

Transcription Factor Elf3 Modulates Vasopressin-Induced Aquaporin-2 Gene Expression in Kidney Collecting Duct Cells.

Aquaporin-2 (AQP2) is a molecular water channel protein responsible for water reabsorption by the kidney collecting ducts. Many water balance disorders are associated with defects in AQP2 gene expression regulated by the peptide hormone vasopressin. Here, we studied roles of Elf3 (E26 transformation-specific (Ets)-related transcription factor 3) in AQP2 gene expression in the collecting duct cells (mpkCCD). Vasopressin increased AQP2 mRNA and protein levels without affecting AQP2 mRNA degradation, indicative of transcriptional regulation. Elf3 knockdown and overexpression, respectively, reduced and increased AQP2 gene expression under basal and vasopressin-stimulated conditions. However, the vasopressin-to-basal ratios of AQP2 gene expression levels remained constant, indicating that Elf3 does not directly mediate vasopressin response but modulates the level of AQP2 gene expression inducible by vasopressin. The Elf3-modulated AQP2 gene expression was associated with AQP2 promoter activity, in line with Elf3’s ability to bind an Ets element in the AQP2 promoter. Mutation in the Ets element reduced both basal and vasopressin-stimulated AQP2 promoter activity, again without affecting vasopressin-to-basal ratios of the AQP2 promoter activity. Lithium chloride reduced both Elf3 and AQP2 mRNA in the mpkCCD cells as well as in mouse kidney inner medulla. We conclude that Elf3 modulates AQP2 promoter activity thereby gauging vasopressin-inducible AQP2 gene expression levels. Our data provide a potential explanation to lithium-induced nephrogenic diabetes insipidus where lithium reduces Elf3 and hence AQP2 abundance.